Effects of the Il-6 Gene Polymorphism -174G/C on Interleukin-6 Production and Endurance Exercise Performance

EFFECTS OF THE IL-6 GENE POLYMORPHISM -174G/C ON INTERLEUKIN-6 PRODUCTION AND ENDURANCE EXERCISE PERFORMANCE

A dissertation submitted to the Kent State University College of Education, Health, and Human Services in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Scott Habowski

August 2018

A dissertation written by

Scott Habowski

B.S., Kent State University, 2008

M.A., Kent State University, 2010

Ph.D., Kent State University, 2018

Approved by

______, Co-director, Doctoral Dissertation Committee J. Derek Kingsley

______, Co-director, Doctoral Dissertation Committee Ellen Glickman

______, Member, Doctoral Dissertation Committee Adam Jajtner

______, Outside Member, Doctoral Dissertation Committee Helen Piontkivska

Accepted by

______, Interim Director, School of Health Sciences Ellen Glickman

______, Dean, College of Education, Health and Human James C. Hannon Services

HABOWSKI, SCOTT M., Ph.D., August 2018

EFFECTS OF THE IL-6 GENE POLYMORPHISM -174G/C ON INTERLEUKIN-6 PRODUCTION AND ENDURANCE EXERCISE PERFORMANCE (129 pp.)

Co-Directors of Dissertation: J. Derek Kingsley, Ph.D. Ellen Glickman, Ph.D.

The purpose of the current study sought to determine the impact of the -174G/C polymorphism on the Interleukin-6 (IL-6) gene may have on IL-6 production and athletic performance. IL-6 has been seen to dramatically increase following endurance exercise events, with events of longer duration resulting in larger increases. This increase is thought to aide in metabolism which allows for sustained energy output during exercise.

Alterations in the genetic sequence on the IL-6 gene at the -174 position within the promoter region have been recognized to result in significant differences in circulating

IL-6 in various medical conditions. It is unknown if this change impacts IL-6 following exercise. Circulating IL-6 and endurance exercise performance were compared based on each participants sequence at the -174G/C polymorphism on the IL-6 gene.

19 trained participants completed a simulated sprint duathlon, all were instructed to complete the events as quickly as possible to mimic athletic competition as closely as possible. At pre-determined distances performance times and assessments of difficulty were recorded. IL-6 and circulating blood glucose were assessed prior to, during and following the simulated sprint duathlon. Analysis of variance was used to compare the

recorded measurements based on participants genetic profile. Findings suggest that a particular genetic profile leads to significant differences in IL-6 production in the later stages of endurance exercise.

ACKNOWLEDGMENTS

I would like to acknowledge the many friends and family members who have allowed me to develop the characteristics to pursue such an endeavor. My parents Ron and Melissa Habowski deserve they type of appreciation and admiration which far exceeds my ability to express. My grandfather Ken Cardinal who from every moment in my recollection was there as universally regarded and respected figure. The type of personal and moral character he displayed through in his personal and professional career serves as an inspiration to the person I still aspire to be. His work as an educator and in

Kent City Schools allowed me to encounter many coaches and teachers which made invaluable contributions to my personal and academic development. Terry Slattery,

Kevin Hockett, and John Nemec all helped to develop my love and understanding of exercise.

The faculty and staff at Kent State University have provided countless contributions to my academic career, which has allowed me to arrive at this point. As I began my collegiate career the concept of pursuing a Ph.D. could not have been further from goals. Dr. Kingsley deserves recognition for his steady and dependable support and advise during this process. He was able to provide sound direct advice while maintaining a personality and patience which if not there would have not made this possible. Dr. Ellen

Glickman also provided an enormous amount of support for this project, and through my career at Kent. From when I began my undergraduate degree to the conclusion of my

Ph.D. she was continually served as a source of knowledge and inspiration to learn more

v in field which has lead me to where I am today. Beyond the academic contributions, her willingness to share her personal and professional insights on all topics beyond the classroom are lessons I will always carry with me.

TABLE OF CONTENTS

Page ACKNOWLEDGMENTS ...... v

LIST OF FIGURES………………………………………………………………...... …...ix CHAPTER I. INTRODUCTION ...... 1

II. LITERATURE REVIEW ...... 5 Interleukin-6 Role in Inflammation ...... 5 Acute Inflammation ...... 5 Chronic Inflammation ...... 7 Interleukin-6 Production in Skeletal Muscle ...... 8 Role of IL-6 during and following Exercise ...... 11 Interleukin-6 Role in Metabolism...... 11 Glycogen metabolism...... 13 Lipid metabolism...... 16 Interleukin-6 Role in Hypertrophy...... 17 Exercise-derived Interleukin-6 as an Anti-Inflammatory Myokine ...... 18 The IL-6 Gene: The -174 G/C Polymorphism ...... 20 The -174 G/C Polymorphism Role in Metabolism ...... 21 The -174 G/C Polymorphism Response to Exercise Intervention ...... 22 The -174 G/C Polymorphism Associations with Athletic Performance ...... 24

III. METHODOLOGY ...... 25 Inclusion/Exclusion Criteria ...... 25 Protocol ...... 25 Laboratory Visit ...... 25 Time Trial ...... 27 Specimen Handling ...... 29 Specimen Analysis ...... 29 Primary Outcome Measures ...... 29 Secondary Outcome Measures ...... 30 Lean Mass ...... 30 Event Times ...... 30 Blood Glucose ...... 31 Data Analysis ...... 31

IV. MANUSCRIPT 1 ...... 33 Introduction ...... 33

vii

Methods...... 36 Subjects ...... 37 Procedures ...... 38 Anthropometric measurements...... 38 Maximal oxygen consumption...... 38 Genetic collection...... 39 Simulated sprint duathlon...... 39 Statistical Analyses ...... 40 Results ...... 40 Discussion ...... 43 Practical Applications ...... 46 Acknowlegments...... 46

V. MANUSCRIPT 2 ...... 47 Introduction ...... 47 Methods...... 51 Subjects ...... 52 Procedures ...... 52 Anthropometric measurements...... 53 Maximal oxygen consumption ...... 53 Genetic collection and processing...... 53 Simulated sprint duathlon...... 54 Blood collection and storage...... 55 Statistical Analyses ...... 55 Results ...... 56 Discussion ...... 60 Conclusion ...... 62

APPENDIXES ...... 65 APPENDIX A. TELEPHONE SCREENING ...... 66 APPENDIX B. IRB APPROVAL ...... 68 APPENDIX C. IL-6 GENE VARIANT STUDY ...... 80 APPENDIX D. EXERCISE RESEARCH STUDY...... 86 APPENDIX E. INFORMED CONSENT ...... 80

REFERENCES ...... 94

viii

List of Figures

Figure Page

1. The Order of Events during the Simulated Sprint Duathlon…………………………..39

2. RPE during 2.5 KM Run……………………………………………………………....42

3. Timeline of Events and Samples during the Simulated Sprint Duathlon……………..55

4. IL-6 Production during the Simulated Sprint Duathlon.…………………………...... 58

5. Change in IL-6 from Middle to Post…………………………………………………..58

6. Lean Mass and IL-6 Post …………………………………………………………...... 59

CHAPTER I

INTRODUCTION

Interleukin-6 (IL-6) is molecule that has an important role as an inflammatory cytokine in the immune response at the detection of injury or infection (Tanka, Narazaki,

Masuda, & Kishimoto, 2013; Moshage, 1997; Heinrish et al., 2003). When secreted in response to exercise sessions, it functions as an anti-inflammatory agent and catalyst to metabolism (Fischer, 2006). IL-6 is one of the initial cytokines that the body releases in response to any signs of infections (Scheller, Chalaris, Schmidt-Arras, & Rose-John,

2011; Tanka & Kishimoto, 2014; Moshage, 1997). The presence of IL-6, as well as other inflammatory agents, serves to begin the acute phase of the inflammatory response

(Pedersen et al., 2005). Beyond the beginning stages of the acute inflammatory response, the presence of IL-6 has been measured during conditions of chronic low-grade inflammation (Ehlers & Kaufmann, 2010). In patients with conditions that display chronic low-grade inflammation, traditional inflammatory cytokines are found in circulation at concentrations at two to three fold greater than healthy counterparts

(Mathur & Pedersen, 2008). Chronic elevated levels of the inflammatory cytokines, such as IL-6 as well as tumor necrosis factor (TNF)-alpha and Interleukin-1β (IL-1β), have been linked to a multitude of medical conditions (Bruunsgaard et al., 1999; Dandona,

Aljada, & Bandyopadhyay, 2004; Tanaka et al., 2014).

In response to exercise sessions, the presence of circulating IL-6 rises dramatically in relation to the intensity (Ostrowski, Schjerling, & Pedersen, 2000),

1 2 duration (Ostrowski et al., 1998; Fischer, 2006), and amount of muscle mass (Fischer,

2006) used during the session. Despite the traditionally recognized role as an inflammatory agent in response to infection (Tanaka et al., 2013; Ehlers et al., 2010) and tissue damage (Sugita et al., 2004), the presence of IL-6 plays a role as anti-inflammatory myokine, a cytokine released from the muscle in response to exercise (Pedersen &

Fischer, 2007; Steensberg et al., 2002). IL-6 release following an exercise session varies based the characteristics of the exercise session; duration of the session has the largest impact and accounts for an estimated 51% of IL-6 production (Fischer, 2006). The exercise-initiated release of IL-6 appears to inhibit the actions of other inflammatory cytokines such as Interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF- α) (Tilg,

Trehu, Atkins, Dinarello, & Mier, 1994). Therefore, when produced from skeletal muscle in response to exercise sessions, IL-6 functions as an anti-inflammatory myokine as opposed to its being traditionally recognized as a marker inflammation along with the other established markers, TNF-α and IL-1β (Pedersen et al., 2007; Fischer, 2006).

The presence of IL-6 has been shown to influence substrate metabolism including glucose (Cox, Pyne, Callister, & Gleeson, 2008; Keller et al., 2001) and lipid metabolism

(Van Hall et al., 2003; Trujillo et al., 2004). The ability to produce IL-6 in response to low-energy states allows for a continued substrate metabolism, which in turn creates the continual energy production necessary for exercise (Nieman et al., 2003). In times of low muscle glycogen content, whether it be in a fasted state or in times during exhaustive exercise, muscle-derived IL-6 production is seen to increase (Keller et al., 2001; Cox et

3 al., 2008). Production of IL-6 has been shown to increase measures of lipolysis in skeletal muscle independently of other hormonal influences (Petersen et al., 2004).

The single nucleotide polymorphism (SNP) in the IL-6 gene of cytosine (C) in place of guanine (G) at the -174 position within the IL-6 gene sequence has resulted in lower levels of circulating IL-6 (Fishman et al., 1998; Terry, Loukaci, & Green, 2000;

Bennermo et al., 2004). The influence of this SNP has been shown to alter glucose tolerance and insulin response (Fernadez-Real et al., 2000a; McKenzie et al., 2004;

Kubaszek et al., 2003), affect metabolic syndrome status (Teixeria, Quinto, Dalboni,

Rodrigues, & Batista, 2015), and influence lipid metabolism (Fernadez-Real, Broch,

Vendrell, Richart, & Richart, 2000b). The influence of this SNP has been established to influence the response to exercise intervention over the course of an 8-week training program (Huuskonen et al., 2009). The evidence has produced conflicting results. Some researchers have suggested that the -174 G/C polymorphism is associated with power events that involve national-level competitors who compete in the events of sprinting, running, and jumping (Ruiz et al., 2009); however, other researchers have failed to replicate the results with different sample populations (Eynon et al., 2010). To date, the presence of C in place of G has been shown to produce more creatine kinase (CK), a measure of muscle breakdown, following acute eccentric arm exercise (Yamin et al.,

2008). Despite the influence of the -174 C/G polymorphism on metabolism and long- term influence on exercise adaptation, it is unclear if the existence of this SNP has immediate influence on IL-6 production and aerobic exercise performance.

For the present study, four hypotheses will be tested:

1. If subjects possess the G/G allele, they will display a higher level of

circulating IL-6 following exhaustive endurance exercise than individuals

with either C/G or C/C allele.

2. Subjects who possess SNP G/G will perform better following endurance

exercise collectively (total course time), and larger differences will be seen in

the later run than the beginning event run when compared to those in

possession of the C allele. The G/G allele will also present lower measures of

exercise difficulty as determined through heart rate and rate of perceived

exertion.

3. Subjects with the G allele will possess favorable phenotypes in traits which

may influence event performance including higher levels of aerobic fitness

and favorable body composition.

4. IL-6 production will be greater with faster performance times, lower levels of

blood glucose, and in larger amounts in subjects with higher measures of lean

body mass.

CHAPTER II

LITERATURE REVIEW

Interleukin-6 Role in Inflammation

IL-6 is a member of the interleukin family that is produced from a variety of cells within the body. IL-6 is recognized as an inflammatory cytokine and, dependent on the source of production, can also function as an anti-inflammatory myokine (Pedersen,

2000). Elevated levels of circulating IL-6 have been associated with a variety of medical conditions that fall under the broad classification of “Low Grade Chronic Inflammation”

(Petersen & Pedersen, 2005). The conditions that have been linked to elevated cytokines, one of which being IL-6, include arthritis, asthma, Crohn’s disease, inflammation- associated cancers (Neurath & Finotto, 2011), type 2 diabetes (Kristiansen & Mandrup-

Poulsen, 2005), atherosclerosis, cardiovascular disease (Abeywardena, Leifert, Warnes,

Varghese, & Head, 2009), depression (Dowlati et al., 2010), and Alzheimer’s disease

(Swardfager et al., 2010).

Acute Inflammation

The cytokine family of proteins are produced acutely in response to tissue injuries or infection. This group of cytokines includes TNF- α, interluekin-6 Beta (IL-6β), IL-6, and IL-1 receptor antagonist (IL1ra). The presence of these inflammatory cytokines also inhibits transcription of anti-inflammatory cytokines including IL-1 and soluble tumor necrosis factor receptors (sTNF-R, which are TNF-α inhibitors). These pro-inflammatory cytokines help increase the number of white blood cells, leukocytes, and lymphocytes

5 6 within the tissue (Petersen, 2005; Ortlepp et al., 2009), which in turn may act to protect the body against foreign substances such as diseases or bacteria. Viral substances have been shown to increase the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), which increases IL-6 mRNA transcription (Tanaka,

Narazaki, Masuda, & Kishimoto, 2013). The presence of IL-6 serves to differentiate the type of B-cells produced (Kishimoto & Hirano, 1988) and also acts to control the initial steps of T-cell proliferation (Van Snick, 1990). Once the inflammatory stimulus is removed, transcription of the IL-6 gene, which is responsible for IL-6 synthesis, ceases

(Tanaka & Kishimoto, 2014).

Beyond the localized inflammatory responses, the body then begins an acute phase reaction in the presence of the influx in the inflammatory cytokines. IL-6 is responsible for several steps in the initial stages of the acute phase response. Initiation of the production of acute phase proteins begins once IL-6 reaches the liver (Van Snick,

1990). Because of this action, IL-6 had previously been referred to as the hepatocyte- stimulating factor. The acute phase reaction triggers the release of several acute phase proteins, including C-reactive protein (CRP), serum amyloid A, fibrinogen, and hepcidin

(Tanka et al., 2014), all of which function within the inflammatory response. The presence of these acute phase proteins serves to aid immune system function by creating a strong localized response at the presence of the inflammatory trigger. The higher the concentration and duration of cytokine levels at the cause of the local inflammatory response, the more dramatic the increase in acute phase protein release will be

(Kilicarslan, Uysal, & Roach, 2013).

IL-6 also serves to trigger prostaglandin E2 (PGE-2) production in the hypothalamus, which initiates the elevation of body temperature in response to infection

(Petersen et al., 2006) and stimulates the release of the adrenocorticotropic (ACTH) hormone from the pituitary gland (Van Snick, 1990). Together, these responses then serve to raise the circulating levels of cortisol and ACTH (Spath-Schwalbe et al., 1994) that collectively act to control the expression of various interleukins, along with helping recruit and differentiate macrophage cells.

Chronic Inflammation

While beneficial in the immediate stages of inflammation, chronically elevated circulating levels of inflammatory cytokines have been linked to a multitude of medical conditions (Neurath et al., 2011) such as type 2 diabetes (Dandona, Aljadam, &

Bandyopadhyay, 2004), cardiovascular events (Ridker, Rifal, Rose, Buring, & Cook,

2002), metabolic disorders (Hotamisligil, 2006), and insulin resistance (Hotamisligil,

2006; Plomgaard & Fischer, 2007). Chronic low-grade systemic inflammation is a term that refers to resting levels of cytokines (TNF-α, IL-1, IL-6, IL-8, and IL-10), cytokine receptors and antagonists (IL-1ra and sTNF-R), and acute phase proteins; for example,

CRP are found at two- to fourfold greater circulating levels than in non-diseased controls

(Petersen et al., 2005; Brandt & Pedersen, 2010). As a person ages, elevated levels of circulating levels of IL-6 and TNF-α have all been linked to cause morbidity and mortality (Harris et al., 1999), frailty (Leng, Xue, Tian, Walston, & Fried, 2007), development of malnutrition (Cederholm et al., 1997), osteoporosis (Khosla, Peterson,

Egan, Jones, & Riggs, 1994), atherosclerosis (Libby, Sukhova, Lee, & Galis, 1995), and development of Alzheimer’s disease (Griffin, 2013).

Interleukin-6 Production in Skeletal Muscle

Beyond serving as an inflammatory cytokine, IL-6 can be marked by up to a 100- fold increase following exhaustive endurance exercise such as a marathon race

(Ostrowski, Rohde, Zacho, Asp, & Pedersen, 1998). This dramatic increase in circulating

IL-6 levels immediately following exercise is most closely related to the intensity and duration of the exercise (Pedersen, Steensberg, & Schjerling, 2001). IL-6 production has been attributed to various cells including myoblasts (De Rossi, Bernasconi, Baggia, Waal

Malefyt, & Mantegazza, 2000), endothelial (Corbel & Melchers, 1984), fibroblasts (De

Rossi et al., 2000), and smooth muscle cells (Klouche, Bhakdi, Hemmes, & Rose-John,

1999). These organelles produce varying levels of IL-6, but the overwhelming majority of the exercise-induced increases in IL-6 have been attributed to the locally contracting myocytes (Febbraio et al., 2003).

IL-6 appears to be a largely local response of the involved exercising muscle groups and the duration and intensity of the activity (Ostrowski et al., 2000; Pedersen et al., 2001). Concentrations of IL-6 have been found in contracting skeletal muscles at levels 5-100-fold greater than circulating levels (Rosendal et al., 2005; Ostrowski et al.,

1998). These concentrations, in turn, may result in a mobilization of energy substrates that will allow for continued energy production. Previous work has demonstrated that using unilateral exercise, the working muscle group accounts for the majority of the circulating IL-6 measured over the course of a 5-hour exercise program (Steensberg et

9 al., 2000). Researchers have come to this conclusion by employing various testing techniques that have served to eliminate alternative theories for post-exercise IL-6 increases.

Previous researchers have used injections of epinephrine matched to measures taken during exercise and achieved only a 4-fold increase compared to the 30-fold increase of IL-6 measured at 1-hour intervals over the course of a 5-hour exercise session, thus eliminating epinephrine as a catalyst of IL-6 production (Steensberg et al.,

2001). Additional work has been done to exclude alternative sites of IL-6 production that would be responsible for post-exercise training increases. IL-6 mRNA remains unchanged following low- and moderate-intensity cycling in monocytes (Ullum, Haahr,

Diamant, Palmo, & Halkjaer-Kristensen, 1994; Moldoveanu, Shepard, & Shek, 2000), and the liver serves to eliminate IL-6 as opposed to produce it in response to exercise

(Febbraio et al., 2003). Finally, analyses of biopsy samples obtained after 3-hour bicycle sessions indicated that IL-6 mRNA content from biopsies peaks immediately following exercise. These maximal IL-6 mRNA levels within the muscle tissue also reflected peak plasma levels. These findings have further indicated that muscle-derived IL-6 is the primary source of the measured increase in circulating IL-6 following high-volume exercise sessions (Penkowa, Keller, Keller, Jauffred, & Pedersen, 2003).

Employing isolated bouts of eccentric and concentric exercise that were matched for intensity based on heart rate, some researchers have suggested that eccentric exercise leads to an increase in IL-6 production (Bruunsgaard et al., 1997). Others have proposed that IL-6 levels were correlated to the amount of muscle damage created (Peake, Nosaka,

& Suzuki, 2005). Subsequent studies that used eccentric-only exercise protocols

(MacIntrye, Sorichter, Mair, Berg, & McKenzie, 2001; Willoughby, McFarlin, & Bois,

2003) found that IL-6 was independently associated with traditional markers of muscle damage such as CK (Ostrowski et al., 1998; Croisier et al., 1999), lactate (Starkie,

Arkinstall, Koukoulas, Hawley, & Febbraio, 2001), and myoglobin (Toft et al., 2002).

Further work using rat models that exercised with either concentric or eccentric exercise reported no difference in levels of IL-6 mRNA within skeletal muscle (Jonsdottir et al.,

2000). This finding suggests that muscle damage resulting from exercise is not the primary source of the rise in circulating IL-6 as traditionally measured; rather, the source is the collective activity of both the concentric and eccentric phases over time that occur over the course of a bout of exercise.

With exercising myocytes identified as the source of origin for the majority of IL-

6 found in circulation, further work has attributed the intensity of the exercise session to impact the amount of IL-6 released. Intensity is typically a measure of the volume of oxygen consumed and amount of effort exerted in the exercise session. It is measured by the volume of oxygen consumed (VO2) as compared to some subjects’ previously measured maximal VO2 (Fischer, 2006; Ostrowski et al., 2000). It has been suggested that 51% of the increased production of IL-6 is attributable to the duration of the exercise session (Fischer, 2006). When matched for duration, exercising in an unfed state saw an average 52-fold increase in circulating IL-6 in response to running, compared to an average 24-fold increase following a cycling session. Nieman et al. (1998) suggested that this difference occurs due to the larger amounts of muscle mass required to support

11 posture during running. Levels of IL-6 have also been closely linked to duration of activity with IL-6 production beginning to rise, showing on average a 4-fold increase 30 minutes into exercise and peaking at the end of the exercise session, showing an average increase of 25-fold (Ostrowski et al., 1998).

Role of IL-6 during and following Exercise

IL-6 production in response to exercise maintains its typical role of a cytokine by attaching to cells and initiating a response, but when released, IL-6 produced over the course of an exercise session has multiple systemic effects that include roles in inflammation, metabolism, protein synthesis, and hypertrophy.

Interleukin-6 Role in Metabolism.

IL-6 has a variety of roles in the overall regulation of metabolism and energy balance. One of the key roles of IL-6 is its action on the hypothalamus, which serves to regulate energy expenditure via stimulation of the hypothalamo-pituitary-adrenocortical axis (Wang & Dunn, 1999). IL-6 has been shown to increase energy metabolism and energy expenditure in mice when they are directly injected with IL-6 directly into the brain; however, injections into the intraperitoneal failed to elicit the same response

(Wallenius et al., 2002). In healthy human subjects, IL-6 appears to have a dose response in stimulating resting metabolic rates by increasing levels of ACTH, cortisol, growth- hormone, and thyroid-stimulating hormone (Tsigos et al., 1997). In human subjects suffering from cachexia-anorexia, elevated brain levels of cytokines, including IL-6, have been suggested as a contributing factor to weight loss due to an increase in measured energy expenditure. Cytokines are found to work both locally by influencing

12 metabolism, and centrally by influencing the brain’s mechanisms through appetite (Plata-

Salaman, 2000).

The lack of IL-6 production has been shown to lower lipid metabolism, increasing the accumulation of adipose tissue (Van Hall et al., 2003; Wallenius et al., 2002), while at the same time increasing leptin production (Trujillo et al., 2004). Elevated levels of adipose tissue will serve to increase circulating of levels of leptin (Berggren, Hulver, &

Houmard, 2005). The increased circulating leptin results in an insensitivity within the hypothalamus, requiring a larger amount of leptin to trigger feelings of satiety (Meier &

Gressner, 2004).

Leptin has been shown to be a key hormone in the regulation of energy balance by acting to stimulate physical activity, regulate fat accumulation, and decrease food intake (Klok, Jakobsdottir, & Drent, 2006). Mice deficient in the IL-6 gene have been shown to develop obesity due to disrupted carbohydrates through lower glucose tolerance and lipid metabolism. These mice also showed increased circulating leptin levels and a decreased response to leptin dosing when compared to their wild-type counterparts

(Wallenius et al., 2002).

IL-6 administration has also been shown to stimulate activity within the sympathetic branch of the Autonomic Nervous System. Administration of IL-6 increases levels of norepinephrine, and consequently heart rate, which were seen in both healthy and diseased populations (Torpy et al., 2000). This activation of sympathetic drive is supported by the fact that the sympathetic neurons express IL-6, display sIL-6R (an IL-6 receptor), and respond to circulating IL-6 (Marz et al., 1998). IL-6 has also been found in

13 neural tissue of mice to play several important roles including: to protect against neural damage (Hirota, Kiyama, Kishimoto, & Taga, 1996), promote neural differentiation

(Marz et al., 1998), promote neural survival (Ikeda, Iwasaki, Shiojima, & Kinoshita,

1996), and decreased perceived levels of pain (Sainoh et al., 2015; Svensson, 2010). The nervous system has been shown to influence metabolism hormonal actions of epinephrine and norepinephrine as well as to regulate caloric intake (Nonogaki, 2000).

Glycogen metabolism. One of the primary roles of muscle-derived IL-6 appears to be energy production and regulation. The AMP-activated protein kinase (AMPK) is an enzyme that increases with a rise in the amount of Adenosine Monophosphate (AMP) and a lowering of Adenosine Trisphosphate (ATP) within muscle, liver, and adipose cells. When activated, AMPK begins fatty acid oxidation, glucose transport, and glycolysis in cardiac tissue and leukocytes. MacDonald, Jorgen, Pedersen, Kiens, and

Richter (2005) suggested that in response to exercise, IL-6 serves to activate AMPK.

Further, in mouse models, a dependent relationship has been established between IL-6 levels and AMPK activity. When circulating IL-6 levels are high, AMPK activity increases (Kelly et al., 2004). This finding suggests that, at least partially, IL-6 helps to catalyze the AMPK reaction that helps to provide new sources of energy levels

(Adenosine Triphoshpate, ATP) to exercising muscles via fat oxidation and glucose uptake (Pedersen & Febbraio, 2007).

Several studies (Keller et al., 2003; Steensberget et al., 2001; Cox, Pyne, Callister,

& Gleeson, 2008) have shown that muscle glycogen content has an inverse relationship with IL-6 production. Comparing fasting and fed conditions, IL-6 production has been

14 shown to increase earlier and to a greater extent in the fasted conditions (Nieman et al.,

2005). Ingestion of carbohydrates during exercise has also been shown to blunt circulating measures of IL-6 (Nieman et al., 2003). Using pre-exhaustive single-leg exercises, subjects have been shown to produce more IL-6 in a previously exercised muscle that has been depleted of glycogen content than compared to the alternate non- pre-exhausted limb (Steensberg et al., 2001). This increased production of IL-6 in a glycogen-depleted muscle appears to align with its increased production in response to duration-intensive exercise sessions. With the previously established metabolic influences on lipid and glucose metabolism, one may conclude that an increase in circulating IL-6 levels would lead to an increase in sports performance through metabolic energy production.

Physiologically, IL-6 increases hepatic glucose production. It has also been shown to increase glycogen phosphorylase, a rate-limiting enzyme in glycogenolysis, and inhibit glycogen synthesis (Kanemaki et al., 1998). Administering IL-6 has been shown to increase fasting circulating glucose measures in a dose-dependent manner. This increase in glucose was attributed to the rise in glucagon, a hormone released when low levels of glucose are detected in the blood (Tsigos et al., 1997). IL-6 production appears to be dependent on pre-existing levels of glycogen content within the muscle, as muscles depleted of glycogen produce larger amounts of IL-6 than those in a fed state

(Steensberg, Fischer, Keller, Moller, & Pedersen, 2003).

Further evidence has shown that IL-6 has the ability to increase insulin sensitivity when released in response to exercise (Ellingsgaard et al., 2011), though it appears that it

15 serves to increase resistance with elevated exposure during resting conditions (Pedersen et al., 2007; Senn, Klover, Nowak, & Mooney, 2002). When matched based on Body

Mass Index (BMI), obese subjects’ resting IL-6 levels were shown to be related to their level of insulin resistance (Kern, Ranganathan, Li, Wood, & Ranganathan, 2001). This fact suggests that the dual relationship IL-6 has when related to inflammation is also present with respect to insulin sensitivity.

IL-6 has been shown to act directly on alpha cells within the pancreas by triggering their proliferation and inhibiting their death. When treated with high levels of glucose and the free fatty acid palmitate over a 12-hour period, the presence of IL-6 has been shown to prevent glucolipotoxicity-induced apoptosis when compared to cells left untreated. Treating rodent muscles with IL-6 has been shown to increase transcription of the Myc protein. The Myc protein is the transcription factor that helps in alpha-cell proliferation. Although it encourages proliferation, at the same time, treatment of rodent cells with IL-6 has been shown to decrease p27, a cell cycle inhibitor (Ellingsgaard et al.,

2008). The presence of p21 and p27 within a cell serves to block the actions of kinase, preventing any catalytic activity and leading to longer cell life (Coqueret, 2003). The collective actions suggest that the presence of IL-6 allows for the preservation of the alpha cells of the pancreas.

Alpha cells within the pancreas release glucagon, which helps elevate the levels of circulating glucose when a hypoglycemic environment is detected (Bouzakri et al.,

2011), as seen in sustained exercise sessions greater than 20 minutes in duration (Trefrs,

Williams, & Wasserman, 2015). The response of glucagon-like peptide-1 (GLP-1)

16 release was shown to be dependent on the concentration of exercise-induced levels of IL-

6. Secreted from intestinal cells, GLP-1 signals beta cells within the pancreas to increase insulin secretion and sensitivity (Ellingsgaard et al., 2015). The above information highlights the various ways the IL-6 produced acutely following exercise work both directly and indirectly to catalyze and sustain glucose metabolism.

Lipid metabolism. IL-6 also aides in lipid metabolism, the other vital metabolite needed for oxidative metabolism. Infusions of IL-6 have been shown to increase lipolysis and fatty acid circulation (Pedersen et al., 2004). In acute settings, it has been shown that IL-6 influences the production of lipoprotein lipase (LPL). By limiting LPL activity, triglyceride storage into adipocytes, lipogenesis, is thus limited as well. The effect of limiting lipogenesis increases circulating triglycerides. When exposed to IL-6, human adipose tissue shows a decrease in LPL activity, which would serve to limit the amount of energy stored within the exposed adipocytes (Greenberg et al., 1992). IL-6 has also been shown to increase adipocyte leptin production acutely (Trujillo et al.,

2004). IL-6 seems to share a similar inflammatory relationship in response to lipid metabolism, as with other cells with chronic exposure to high levels of IL-6 being linked to obesity and insulin resistance (Kern et al., 2001).

The measure of IL-6 production in response to exercise has largely been attributed to contracting muscle; in fact, it has been shown that during exercise, adipose tissue production of IL-6 is suppressed (Febbraio, 2003). Following the conclusion of exercise, production of IL-6 from adipose tissue is seen to rise, which suggests that it allows for increased lipolysis and fatty acid mobilization (Lyngso, Simonsen, & Bulow, 2002). For

17 those concerned with recovery following bouts of exercise, increased levels of IL-6 may lead to greater preservation of blood glucose and muscle glycogen levels and allow for a faster recovery.

Interleukin-6 Role in Hypertrophy

Although the muscle cell does not express an IL-6 receptor, the primary action of

IL-6 in response to exercise occurs primarily through satellite cell recruitment and proliferation. IL-6 plays a direct role in the activation of several metabolic pathways.

When mice were exposed to IL-6, a proliferation of murine (mouse) satellite cells, cyclin

D1 and c-myc, occurred (Serrano, Baeza-Raja, Perdiguero, Jardi, & Munoz-Canoves,

2008). However, some questions remain regarding the relationship between human and mouse satellite cell function (Boldrin, Muntoni, & Morgan, 2010). Through examination of mice missing the IL-6 gene, no differences were seen in the Phosphoinositide 3-kinase

(PI3K) and Protein Kinase B (PKB), which suggests that IL-6 does not influence protein synthesis (Serrano et al., 2008). Stimulated through the Insulin-like growth factor 1,

PIK3 is the primary regulator of acetyltransferases and MyoD; together, acetyltransferases and MyoD regulate myosin cell differentiation (Forcales & Purim,

2005; Diamond, Franklin, & Milfay, 1974). PKB serves to promote protein synthesis by activating the mTOR-p70 S6 kinase pathway, as well as to signal glucose uptake and glycogen synthesis in muscle cells (Sugita et al., 2004). The direct impact of IL-6 on both carbohydrate and lipid metabolism seems to be missing when the role of IL-6 on amino-acid metabolism is examined.

Cultured myoblast cells undergoing myogenesis have been shown to express IL-6.

During myogenesis, P38 mitogen-activated protein kinases (p38 MAPK) serves as an activator of myoblast differentiation. Nuclear factor-κB (NF-κB) also plays a key role during myogenesis proliferation and differentiation. NF-kB plays a dual role during myogenesis; it has been shown to inhibit differentiation through cyclin D1 expression

(Guttridge, Albanese, Reuther, Pestell, & Baldwin, 1999) and also triggers differentiation through activation of the insulin-like growth factor II (IGF-II) pathway (Canicio et al.,

2001). During myogenesis, NF-kB triggers IL-6 transcription, which in turn serves to interfere with the role of NF-kB while showing no interaction with p38 MAPK. This complex interaction suggests that longer exposure to IL-6 allows for longer periods of myoblast differentiation by inhibiting the negative role of NF-kB during differentiation.

These findings were supported with an inhibition of IL-6, which resulted in a reduced the amount of differentiation; overexpression showed greater levels of myogenesis (Baeza-

Raja & Munoz-Canoves, 2004). By aiding in myogenesis, the previous literature would suggest that greater production of IL-6 would create longer periods that favor adaptations to regular exercise training.

Exercise-derived Interleukin-6 as an Anti-Inflammatory Myokine

Traditionally, with regard to the established role of IL-6 within acute phase reaction of inflammatory response and its role in acute phase reaction to inflammation,

IL-6 appears to serve a very different role as a muscle-derived myokine. Traditional identifications of pro-inflammatory cytokines include TNF-α, Interleukin-1 (IL-1), and

IL-6 (Ostrowski et al., 1999). A dichotomy exists between TNF-α, IL-1, and IL-6 in

19 chronic inflammatory conditions with all cytokines being elevated in conditions linked to chronically elevated levels of inflammation. However, when produced following exercise, IL-6 appears to take on the role of an anti-inflammatory myokine (Febbraio et al., 2002).

When released from an exercising muscle, IL-6 has been shown to cause numerous reactions that help to regulate and diminish the inflammatory response. IL-6 production has been shown to inhibit the expression of both TNF-α and IL-1. IL-6 directly serves to suppress the actions of TNF-α through inhibition of lipopolysaccharide

(LPS)-induced TNF-α production (Petersen et al., 2006). When activated, LPS stimulates the release of pro-inflammatory cytokines from the monocytes (Bruggen, Nijenhuis, Van

Raaij, Verhoef, & Asbeck, 1999). Along with direct inhibition of TNF-α, IL-6 also produces soluble extracellular TNF receptors, sTNF-r1 and sTNF-r2 (Ostrowski et al.,

1999). IL-6 has also been shown to inhibit production of IL-1, as well as produce

Interleukin-1 Receptor Antagonist (IL-1ra), which serves to prevent the transcription of

IL-1 (Pedersen et al., 2000) and blocks the inflammatory responses to IL-1.

Enhanced plasma levels of the anti-inflammatory cytokine Interleukin-10 (IL-10) significantly increase following infusions of artificial IL-6 (Steensberg et al., 2003). IL-

10 has also been shown to serve in a similar function as IL-6, serving to inhibit production of IL-1 and TNF-α, mostly through prevention of transcription (Pedersen et al., 2004). IL-10 also serves to suppress the expression of Class II Major

Histocompatability Complex, which is responsible for production of pro-inflammatory cytokine production (Waal, Malefyt, Abrams, Bennett, Figdor, & Vries,1991). The

20 action of IL-6 as an anti-inflammatory cytokine would suggest that those who are able to produce greater levels of IL-6 in response to exercise would experience a diminished inflammatory response following exercise sessions.

The IL-6 Gene: The -174 G/C Polymorphism

A polymorphism in the gene in the promoter region of the IL-6 gene has been shown to alter circulating levels of plasma IL-6. The insertion of cytosine (C) in place of guanine (G) at the -174 position of the IL-6 promoter region (rs1800795) has been suggested to decrease circulating levels of IL-6 in healthy subjects (Fishman et al., 1998;

Bennermo et al., 2004), although these results are not universally accepted (Qi et al.,

2006). The -174 G/C polymorphism has been examined in multiple disease states including Alzheimer’s disease (Licastro et al., 2003), atherosclerosis, cardiovascular disease (Berrermo et al., 2004), diabetes (Illig et al., 2005), osteoporosis, juvenile arthritis

(Fishman et al., 1998), Hodgkin’s lymphoma (Hohaus et al., 2007), and stroke recovery

(Greisenegger et al., 2003). The insertion of C into the promoter region of the IL-6 gene allows for the binding of Nuclear Factor I (NF-I) proteins. The NFI-B3 protein, specifically, from the NF-I family of proteins has been shown to reduce transcriptional activity (Liu, Bernard, & Apt, 1997) of gene expression. Beyond resting values, individuals who are homozygous for the GG genotype showed greater increases in serum

IL-6 levels than individuals with the CC genotype when injected with an inflammatory stimulus (Bennermo et al., 2004).

The -174 G/C Polymorphism Role in Metabolism

Due to the role of IL-6 on metabolism and energy availability, multiple studies have examined how the G/C polymorphism influences metabolic activity in the healthy population (McKenzie et al., 2004; Underwood et al., 2012; Goyenechea, Patta, &

Martinez, 2007). When measuring energy expenditure, carriers of the genotype CC have been shown to have lower fasting insulin levels (Fernadez-Real et al., 2000; Kubaszek et al., 2003), lower fasting glucose levels (Huth et al., 2009; McKenzie et al., 2004), and lower energy expenditure rates (Kubaszek et al., 2003).

Using an oral glucose tolerance test (OGTT), the CC genotypes showed a decrease in serum glucose concentrations over time, lower levels of serum glycosylated hemoglobin (HbA1c), lower circulating white blood cell counts for Neutrophils,

Lymphocytes, and Monocytes (Fernadez-Real et al., 2000), and decreased levels of circulating free fatty acids (Fernadez-Real et al., 2000). Using the hyperinsulinemic- euglycemic clamp technique, the CC genotype carriers displayed lower metabolic rates, lower oxidative and non-oxidative glucose disposal, and decreased insulin sensitivity

(Kubaszek et al., 2003; Huth et al., 2009). CC carriers have also shown differences in fasting lipid levels. Subjects’ homozygotes for the C allele have displayed lower fasting triglycerides, circulating free fatty acids, VLDL (Very Low-Density Lipoprotein), and increased levels of HDL (High-Density Lipoprotein)-2-cholesterol (Fernadez-Real et al.,

2000; Halverstadt et al., 2005). These alterations to metabolic functions suggest that the -

174 G/C polymorphism may influence the availability of substrates such as glucose and free fatty acids, which are required for energy production during exercise.

The -174 G/C Polymorphism Response to Exercise Intervention

Relatively few studies have been performed to examine the influence of the -

174G/C polymorphism following an exercise session. Using an eccentric exercise protocol of one set of 50 repetitions on the subjects’ non-dominant arm, markers of muscle damage, creatine kinase levels, and response were measured through urinary collection prior to the exercise session as well as the following week after the exercise session. It was found that carriers of the CC genotype showed a steeper increase and reached a higher maximal level of CK concentration following eccentric arm exercise

(Yamin et al., 2008). This increase in CK in the CC genotype supports the notion that during exercise, IL-6 functions as a mytokine and serves to reduce muscular damage in response to exercise. This reduction in measures of muscle damage suggests that athletes who are able to produce more IL-6 during exercise may tolerate greater levels of imposed damage or may recover faster from a similar training levels due to reduced inflammatory response.

In a longitudinal study measuring response to a 6-month aerobic exercise program, 87 men and women between the ages 50-75 years old followed a 24-week supervised aerobic exercise program with progressive increases in intensity. All subjects were deemed healthy, free of any medical conditions, and possessed a BMI of 37kg/m2 or less. Once cleared for participation, subjects performed a graded treadmill test to assess maximal oxygen uptake, a dual-energy X-ray absorptiometry scan to assess body composition, and an OGTT to assess glucose and insulin sensitivity. Repeated testing of

57 finishers, individuals homozygous for the GG allele, showed a significantly lower

23 bodyweight, amounts of subcutaneous adipose tissue, and glucose exposure time following an OGTT after 6 months of aerobic training. Individuals who possessed at least one G allele, heterozygous GC or homozygous GG, displayed lower levels of intra- abdominal adipose tissue, body fat percentage, and improvements in insulin sensitivity scores following 6 months of aerobic training (McKenzie et al., 2004). Again, the following study suggests that carriers of the G allele may be able to increase glucose sensitivity, which allows for a larger amount of available energy within the cell to be used during exercise. Differences between levels of body fat were also seen between the groups following training, which suggests that G allele carriers may improve body composition in response better than CC carriers.

The -174G/C polymorphism has also been suggested to augment bone density and resorption rates. Using 434 postmenopausal women, significantly lower levels of C- telopeptide of Type I collagen (CTx), a marker of bone resorption rates, were seen when the women were grouped by genotype. CC carriers showed the lowest levels, followed by CG carriers, and finally, GG carriers showed the highest levels of CTx, which suggests that they would have higher amounts of bone resorption occurring. When separated by the median age of 71 years, the subjects who were at either GC or GG had significantly lower levels of Bone Mineral Density at the hip and forearm than CC carriers (Ferrari et al., 2001). Similar changes in bone density were seen in a group of

130 army recruits following 10 weeks of basic training. Significant differences were seen between soldiers when separated by genotype with differences in femoral cross-sectional area, measured as changes from baseline as CC +17.3%, GC +5.5%, and GG -6.8%

(Dhamrait et al., 2002). These findings suggest that the increased presence of IL-6 may hamper the bone remodeling typically associated with exercise training. The resulting lower bone density may result in an increased risk of bone fracture to the athlete trying to maintain intense training levels.

The -174 G/C Polymorphism Associations with Athletic Performance

It has been suggested through observational studies that athletes with the G allele are more likely to succeed in power-related events such as sprinting, throwing, and jumping. When the researchers looked at high-level athletes of Hispanic decent, the proposed role of IL-6 in helping in satellite cell recruitment was suggested to aid in training session recovery, which then allowed for greater training volumes and responses

(Ruiz et al., 2010). However, these differences between power performance athletes and endurance athletes have not been validated when expanding the ethnicities of the participants (Eynon et al., 2010). To date, no studies have been conducted to show how the single nucleotide polymorphism has an acute impact on exhaustive aerobic sporting performance.

CHAPTER III

METHODOLOGY

All procedures were approved by Integreview® Institutional Review Board (IRB) and Kent State University (IRB).

Inclusion/Exclusion Criteria

Initial contact was made with potential research subjects through word of mouth and social media posts. Subjects were pre-screened, and any potential participant with any known muscular skeletal injuries, inflammatory conditions, or cardiovascular issues were excluded from participation. Eligible participants were determined through a pre- screening message, which included a questionnaire and description of testing procedures

(Appendix 1). Upon determining eligibility and interest in participation, subjects then scheduled a date to report to the Center for Applied Health Sciences (CAHS; Stow, OH).

Protocol

After initial contact if the potential subject expresses interest a visit was then scheduled at the CAHS. If desired potential subjects were given the option to have a digital copy of the informed consent e-mailed to them prior to their first laboratory visit.

Laboratory Visit

All qualified subjects reported to the CAHS, subjects went through an orientation that covered the informed consent form, which they read and signed prior to any testing procedures. Following the informed consent administration, participants then answered a series of questions to determine if any subjects had a preexisting condition that would

25 26 have excluded them from participation. Female test subjects underwent a pregnancy test

(McKesson Consult® Rapid Diagnostic Pregnancy Test) prior to moving forward for any testing. These female subjects had access to a private bathroom for an unlimited time to complete the pregnancy test. A negative test allowed subjects to continue in the study, but a positive result lead to exclusion. Once the sample was collected testing results took roughly three minutes to determine.

Once the participants signed the informed consent, excluding conditions were eliminated, and the female subjects had taken the pregnancy test, the subjects’ height, weight, and date of birth were then be recorded. Once this data had been recorded, subjects received a DNA collection kit. Using an Oragene DNA collection kit (DNA

Genotek, Inc., Ottawa, ON, Canada), approximately 3 mL of saliva were collected in a tube labeled with the subjects’ identification code; subjects were given an unlimited amount of time to collect the required volume.

Following successful DNA collection, subjects will then undergo a Dual Energy

X-ray absorptiometry (DEXA) scan. For this scan, subjects were tested in one layer of clothing free of any metal material, typically a pair of spandex shorts and sports bra if necessary; if the subjects do not have the appropriate clothing, they will be provided a cotton hospital gown. A private bathroom was available if the subject chooses the option of changing in privacy. Female subjects were given the option to have a female research staff member complete the test if requested. Once the DEXA scan had been completed, subjects were be allowed to dress.

Finally, for Visit 1, subjects underwent a graded exercise test to determine their maximum volume of oxygen consumption. Prospective subjects followed the previously established testing procedures as used by Paavolainen (1999). Subjects begin running at

5.0 mph on a 1° incline. Every three minutes, the speed of the treadmill increased 0.6 mph until they reach the speed of 10.6 mph. At this point, the incline of the treadmill increased 1° every minute. At any point during the test, subjects were permitted to cease the testing session. A VO2 max score classification of “Good” or above was required for continued participation (Heyward, 1998).

Time Trial

The following visit was scheduled for a date within two weeks of the baseline testing visit. Upon arrival subjects confirmed they had consumed the provided meal bar and each subject confirmed their enrollment number. Once seated, a tourniquet was applied to the subject’s arm above the elbow. An alcohol swab was applied to the subject’s antecubital area of the preferred arm. A member of the CAHS medical team collected roughly 5mL venous blood sample into a chilled vacutainer EDTA collection tube. Upon collection completion, the medical team member then applied a sterile cotton gauze pad to the collection site and wrapped the gauze using coban wrap. Once the subjects confirmed their comfort, they will began the time trial.

After specimen collection, subjects were then instructed to complete each stage of the trial as quickly as they could, freely picking a pace that they see fit. Subjects then completed a simulated Duathlon on a stationary bike and motorized treadmill.

The time trial consisted of a 5km run, a 20km bike ride, and finished with a 2.5km run. These activities were closely matched to simulate the distances covered in a Sprint

Duathlon in the United States Triathlon Duathlon National Championships. The subjects only were given instructions about the distances they needed to cover per stage and told to try to finish the events as quickly as possible. No regulations or instructions were placed on the times or pacing of any events. During every phase of the time trial, subjects were permitted to consume water ad libitum.

Split times were recorded at even intervals over the course of every phase of the time trial. During the run, at every kilometer the subjects’ time, heart rate (HR), and rate of perceived exertion (RPE) score were recorded, as well as time and RPE score upon conclusion of both runs (5k & 2.5k). During the cycling stage of the event, time, HR, and

RPE score were recorded every two kilometers.

The time to complete each stage of the trial were recorded along with the combined time of all three events. Transitional time and time required for blood draws were not be factored into calculating the total time to completion but will be held to under

8 minutes. Following completion of the bike portion of the time trial, subjects then went to the phlebotomist’s chair for another 5mL blood sample. Again, upon giving verbal confirmation confirming their comfort subjects then proceeded to the last phase of the time trial for the final 2.5km run. Finally, following the completion of the run, subjects then again returned to the phlebotomist’s chair for a final blood draw. With the conclusion of the final blood draw, the subjects were given the choice to eat a Clif®

Builder’s Protein bar if they desire before leaving the facility.

Specimen Handling

The coded Oragene R500 kits (DNA, Genotek, Kanata, Ontario, CA) were sent to

GenoFIND Services (GenoFIND, Salt Lake, Utah) in order to be processed for the -

174G/C single nucleotide polymorphism. Subjects who did not complete the time trial had their samples discarded through biohazard disposal.

Once the blood samples were collected, a single drop was placed on a Contour®

Next EZ Meter to determine blood glucose levels. The EDTA tubes were then be spun using a centrifuge. Following a 10-minute cycle in the centrifuge, the separated plasma was then aliquoted from the collection tube and placed into two separate transfer tubes

(Primary and Duplicate). A minimum of 2 mL of plasma were stored into each tube from every sample. All samples were stored in a ScienTemp -80°F freezer. Once all subjects had finished the study, the Primary sample tubes were then transported to the Kent State

Exercise Physiology lab.

Specimen Analysis

SNP of the -174 G/C polymorphism analysis was determined through

Pyrosequencing single tube assays at the EpigenDx® Laboratory. Upon the final subjects’ specimen collection, plasma samples were removed from the -80 °F freezer and transported to the Kent State Exercise Physiology lab. The plasma samples were then analyzed via an Elisa plate reader to determine plasma IL-6 content per sample.

Primary Outcome Measures

Production of IL-6 following the conclusion of the duathlon was examined based on the genotype of the athlete at the -174G/C polymorphism. IL-6 levels were measured

30 through plasma samples obtained from the subjects three separate samples. IL-6 plasma content was measured through an Enzyme-linked immunosorbent assay (ELISA) performed at the Kent State Exercise Physiology Biochemical Laboratory. The -174G/C polymorphism was determined through genotype sequencing. Using EpigenDx, Inc

Services (EpigenDx, Hopkinton, Massachusetts), determination of subjects’ -174G/C single nucleotide polymorphism (SNP) was determined and reported into one of three outcomes (G/G, G/C, and C/C).

Secondary Outcome Measures

Secondary measures were recorded to examine the relationship with IL-6 production. These measures were recorded prior to determination of IL-6 content or individual genotype.

Lean Mass

The amount of muscle involved in an exercise session has been shown to be related to reported levels of circulating IL-6. Previous studies have shown that the larger the amount of muscle mass involved in an exercise routine, the larger the amount of circulating IL-6 that will be produced (Pedersen, 2010). Therefore, the subjects’ recorded lean mass measured via the DEXA scans was accounted for to examine differences in circulating IL-6 that result from differences in lean mass between subjects.

Event Times

Both total event time and individual event times (run, bike, and run) were recorded to establish an evaluation of performance. Evaluation of performance was

31 determined to be better with faster times of completion of both the total event times and each individual stage of the simulated duathlon. Correlations between performance times of total and individual events was examined between circulating IL-6 levels, as well, to see what possible influence each genotype group will have on athletic performance as determined by event times; lower event times will be judged as more desirable.

Blood Glucose

Circulating blood glucose levels was recorded from each blood sample obtained.

Previous work has shown that levels of blood glucose have a negative association with circulating IL-6 levels (Keller et al., 2003; Steensberg et al., 2001; Cox, Pyne, Callister,

& Gleeson, 2008). Blood glucose levels were accounted for to control any differences that may be noted in IL-6 production due to individual nutritional differences.

Data Analysis

Data from all subjects is presented as mean ± standard deviation. All data will be analyzed using Statistical Package for Social Sciences software (IBM SPSS, Version

24.0, Armonk, NY). Sample size was determined using G*Power 3.1.9.2 with an alpha set at 0.05 and power level set to 0.80. All results are reported with an a priori alpha of p≤0.05 determined to be a significant finding. For significant findings effect size is reported using partial eta squared (η2).

A person’s correlation test was run on the association of muscle mass and glucose levels, as both have been estimated to have an influence on circulating levels of IL-6. If either variable had a reported coefficient r of 0.5 or higher, it will be deemed to have a strong association with IL-6 levels. If baseline measures or selected athletic traits are

32 found to be significantly different between groups, these variables will be used as a covariate for future analysis of variance tests (ANOVA).

The statistical analysis for hypothesis 1 involved separate repeated measures

ANOVA that compare the group (subject’s genotype [G/G, G/C, and C/C]) and amount of measured IL-6 at the varying time frames (Baseline to Mid to Post, Baseline to Mid, and Mid to Post). Additionally, a one-way ANOVA was used to examine difference between groups using the change (delta) in IL-6 levels from the Baseline sample to the

Post Sample (Post-Baseline).

The testing of hypotheses 2 consisted of four separate one-way ANOVAs composed of group (phenotype) by event times (5 KM run, 20KM bike, 2.5 KM run, and total time). If a significance was found (p=0.05 or less) in any of the conditions, a repeated ANOVA measures was used to determine the difference between groups and recorded split times. Associated measures of difficulty (HR and RPE) were compared between groups using a repeated measures ANOVA for each individual event (5 KM run,

20 KM bike, and 2.5 KM run).

Hypothesis 3 used an one-way ANOVAs to examine differences between groups in athletic traits which may impact athletic performance including maximal oxygen uptake (VO2 Max) and body composition (body fat percentage, lean mass, and fat mass).

Finally using a Pearson correlation (r) associations between performance times, body composition, and blood glucose were all be measured against circulating IL-6.

CHAPTER IV

MANUSCRIPT 1

Influence of the -174G/C Single Nucleotide Polymorphism on the Interleukin-6 Gene on Athletic Traits and Aerobic Performance.

Scott M. Habowski,1 J. Derek Kingsley1, Betsy Raub,2 and William Kedia2

Kent State University, Kent, OH; 1 and The Center for Applied Health Sciences, Stow,

OH 2

Introduction

Interleukin-6 (IL-6) has traditionally been identified as one of the initial cytokines in response to injury or infection, having roles in both the acute phase response (Van

Snick, 1990) and conditions of chronic inflammation (Petersen, 2005). Furthermore, researchers have linked elevated levels of IL-6 to multiple conditions including arthritis, asthma, Crohn’s Disease, various types of cancer, and numerous others (Neurath, 2011).

Despite previous research that has demonstrated IL-6 as an inflammatory cytokine, IL-6 becomes a myokine when generated by a muscle cell and acts functionally as an anti- inflammatory (Pedersen, 2007; Trefts, 2016). This contradictory role largely depends on the conditions in which the molecule, IL-6, is produced and the organelle of origin.

When produced independently of the standard acute phase inflammatory response, IL-6 acts to inhibit other traditionally identifiable inflammatory cytokines such as Tumor

Necrosis Factor-alpha (TNF-α) and Interleukin-1 (IL-1) (Pedersen, 2006); it thereby

33 34 effectively serves as an anti-inflammatory myokine. Along with helping to manage inflammation, IL-6 also helps to activate metabolic activity via lipolysis (Pedersen, 2004) and increased glucose production within the liver (Pedersen, 2007). This finding suggests that the presence of IL-6 would benefit aerobic endurance during exercise by managing inflammation and aiding in metabolism.

IL-6 has been shown to increase despite vastly different types of exercise interventions, which include eccentric isokinetic arm extensions (Croisier, 1999), 6 minutes of high-intensity rowing (Neilsen, 1996), and steady-state running (Ostrowski,

1998). The most dramatic increases have been attributed to aerobic exercise, showing up to a 100-fold increase beyond resting levels (Steensberg, 2000). This marked increase in circulating IL-6 levels has been associated with the intensity, duration, and mode of exercise; increased demands have shown increased IL-6 production (Ostrowski, 1998).

Duration of the exercise bout has been suggested to account for 51% of the exercise- associated increases of IL-6 (Fisher, 2006). In addition, acute exercise that employs larger amounts of muscle mass, such as running compared to arm ergometry, produces a larger response in circulating IL-6 levels (Nieman, 1998). Furthermore, during fasted exercise, IL-6 levels may show a 70-fold greater increase when compared to the fed state

(Keller, 2001). Collectively, these data suggest that during aerobic exercise, when stores of energy within the muscles are depleted, IL-6 helps to stimulate hepatic glucose production (Keller, 2001) and fatty acid circulation (Lyngso, 2002) as a means to compensate for the reduction in muscle glycogen content (MacDonald, 2005).

The IL-6 gene accounts for the transcription of IL-6, which is a part of the initial inflammatory response (Nieman, 2005). Penkowa (2003), using muscle biopsies following a 3-hour cycling session at 60% of their VO2 max, demonstrated through histochemistry analysis the mRNA IL-6 content in the obtained muscle samples reflected the rise in circulating IL-6. A single nucleotide polymorphism (SNP) has been found when the amino acid cytosine (C) is found in place of the amino acid guanine (G) at the -

174 position (-174G/C SNP) of the promoter region of the IL-6 gene. Data have demonstrated that this SNP results in significant increases in circulating IL-6 for both healthy (Fishman, 1998; Bennermo, 2004) and unhealthy subjects (Illig, 2005; Liu 1997).

The -174G/C SNP also affects the metabolic response in those individuals with C present at either allele, showing lower resting energy expenditures (Kubaszek, 2003), lower fasting insulin levels, diminished response to feeding, lower levels of circulating glucose

(Fernadez-Real, 2000), and lower levels of fasting triglycerides (Halverstadtm, 2005).

Despite the suggested influence on metabolic regulation of IL-6, relatively little research has been completed on the -174G/C SNP and what, if any, impact it may have in either aiding in, or response to, aerobic exercise. It has been suggested that Spanish athletes with the G allele present may have an advantage in power events, but these results have yet to been duplicated with other populations. In young smokers, those with the C allele displayed a lower level of power output (watts/kg of bodyweight) in a graded cycling test

(Ortlepp, 2003).

Therefore, the purpose of the present study was to determine the impact of the -

174G/C SNP during a simulated athletic event. Secondary measures included

36 determining if differences in athletic traits and feelings of difficulty are manifested differently between genotypes. Athletic traits included measuring body composition via a Dual-energy X-ray absorptiometry (DEXA) scan, and aerobic fitness as determined through maximal oxygen uptake (VO2max). Assessment of difficulty was determined using the subjects’ heart rate (HR) and rate of perceived exertion (RPE). We hypothesized that the subjects with the G/G allele would display the benefits of additional circulating IL-6 on exercise performance: They would have lower event times for the duathlon, experience lower levels of difficulty, possess favorable body composition, and achieve higher levels of aerobic fitness. Furthermore, we expected the performance results to be present during the final stage of the duathlon (2.5 km run) due the release of

IL-6 during the later stages of aerobic exercise.

Methods

After providing consent, subjects attended two separate visits to the research facility for data collection. After the subjects had cleared the initial screen questions, data collection began with recording of basic anthropometric measurements including height, weight, resting blood pressure, and heart rate. Body composition was determined using a DEXA Scan. Finally, subjects performed a maximal exercise test to voluntary exhaustion to determine VO2 max. Prior to leaving the laboratory, all subjects were provided with a standardized meal bar (PowerBar®, Performance Energy Bar) to consume 60 minutes prior to the next laboratory visit. These bars included 4 grams of fat, 44 grams of carbohydrates, and 10 grams of protein, the entire bar contained 230 calories. Subjects then reported back to the research facility within 2 weeks of the initial

37 baseline measurements to perform a simulated sprint duathlon. Before the beginning of the events, subjects confirmed that they had consumed the standardized bar 60 minutes before the scheduled visit time and had refrained from strenuous exercise or activities for

48 hours prior. Next, the medical staff obtained saliva samples and stored them for determination of the -174G/C genotype. The subjects performed the simulated sprint duathlon with only the instructions to finish the totality of the events as quickly as possible. All subjects completed a 5-kilometer run on a treadmill, a 20-kilometer bike ride on a mounted bicycle, and finally, a 2.5-kilometer run on the same treadmill. Each individual event time was recorded, and upon completing the final run, all individual events were added together to determine a complete event time. The subjects received up to 5 minutes between events, and all were permitted to consume water ad libitum. At standard intervals over the course of all events, total time, HR, and RPE were recorded.

Subjects

Nineteen apparently healthy men aged between 18 and 40 volunteered to participate in the study (Table 1). Upon providing written consent to participate, potential volunteers were then cleared of any known medical conditions including musculoskeletal injuries, cardiovascular conditions, autoimmune disorders, chronic inflammatory conditions, bleeding, or platelet disorders. Finally, all subjects self- reported participating in a minimum of two aerobic workouts a week and verbally confirmed that they felt they could sustain a continuous bout of exercise for approximately 2 hours. IntegReview® IRB (Austin, TX), an independent review board, approved all research-related procedures, activities, and documents. All research was

38 conducted in accordance with the Declaration of Helsinki and FDA outlines for Good

Clinical Practice.

Procedures

All testing measures recorder were collected over two separate days following the administration of the informed consent form.

Anthropometric measurements. Height was determined using a mounted stadiometer to the nearest cm, and weight was determined using a calibrated scale (Seca

Medical Scale, Hanover, MD). Resting Blood Pressure and Heart Rate were measured using an automated blood pressure monitor (Omron HEM-907XL, Bannockburn, IL).

Lean mass, fat mass, and body fat percentage were determined via a DEXA scan (GE

Electrical Lunar DPX Pro, Boston, MA) and analyzed by the manufacturer’s software

(enCORE Version 15).

Maximal oxygen consumption. Maximal oxygen uptake was tested using methods that Paavolainen (1999) outlined. In short, initial speed was at 5 miles per hour at a 1 incline on a treadmill (Sole TT8, Taipei, Taiwan). Then speed was increased by

0.6 miles per hour at 3-minute intervals. Once subjects reached the speed of 10.6 mph, the incline was increased 1 in 1-minute intervals. Subjects were instructed to run until voluntary exhaustion, at which point they were free to discontinue the test by stepping off the treadmill. Oxygen consumption was determined using a Parvo Medics TrueOne

Metabolic System (OUSW 4.3.4, Sandy, UT).

Genetic collection. Subjects provided roughly 3 mL of saliva in an Oragene OG-

500 (DNA Genotek, Ottawa, Ontario) collection kit. Each kit was labeled with a unique

5-digit number, and then on separate sheet, a subject identifier was recorded with the 5- digit number. All samples were stored in a locked cabinet until the conclusion of the data collection phase. Once every subject had completed all study visits, DNA samples were sent to EpigenDx (Hopkinton, MA), and using pyrosequencing, the -174G/C was determined for every subject.

Simulated Sprint duathlon. All simulated sprint duathlon events occurred at a single research location in a consistent ambient environment (23-25˚C). The duathlon constituted of a 5km run, a 20km bike ride, and a final 2.5km run (Figure 1). All subjects were fitted with a Polar® H1 Heart Rate Monitor (Bethpage, NY) prior to the beginning of the events. Subjects performed all running events on a Sole® TT8 Treadmill (Salt

Lake City, UT), and they performed the biking event on a FluidPro® Bike Trainer

(Cascade Health and Fitness, Woodinville, WA). During each stage, subjects could monitor their progress and were free to adjust treadmill speed and bike gears as they wished.

Sprint Duathlon

5 KM Run Baseline 2 KM 4 KM 6 KM 8 KM 10 KM 12 KM 14 KM 16 KM 18 KM 20 KM 2.5 KM Run

Baseline 1 KM 2 KM 3 KM 4 KM 5 KM 20 KM Bike Ride Baseline 1 KM 2 KM 2.5 KM

Figure 1, The order of events during the simulated sprint duathlon. HR and RPE were recorded at the noted distances.

Statistical Analyses

Subjects were separated into groups based on the presence of C allele at the -174 positions on the IL-6 gene (G/G, or the C/C or G/C alleles). Significance was set a priori at p≤0.05. Group baseline athletic traits (VO2 max, Lean Mass, Fat Mass, and Body Fat

Percentage) were compared with an independent samples t-test with effect size reported for significant findings using Cohen’s d (d). Duathlon times (individual events and total) and resulting measures of difficulty (HR and RPE) were compared among groups using a two-way repeated measure analysis of variance (ANOVA) with a Sidak post hoc test to control for multiple comparisons. A Greehouse-Geisser epsilon correction was used if

Mauchley’s test of Sphericity was violated. Partial eta squared (ŋ2) was used to assess the effect size for each variable found to be significantly different between groups for each respective ANOVA. Values are presented using as mean ± SD. All statistical analyses were completed using IBM SPSS version 24 (Armonk, NY).

Results

Frequency of the C allele matched the reported frequency reported by others

(Fishman, 1998) occurring in 40% of alleles. The groups were not statistically significantly different in anthropometric measures (Table 1) or when examining athletic traits (Table 2), which include VO2 max (F(1,17) =1.18; p=0.29), Body Fat Percentage

(F(1,17) =0.07; p=0.79), Lean Mass (F(1,17) =0.41 p=0.53), or Fat Mass (F(1,17) =0.01; p=0.96) during the simulated duathlon heart rate over the individual events: the 5km Run

2 2 (F(1,17) =0.79; p=0.56; ŋ =0.04), the 20km Bike Ride (F(1,17) =1.46; p=0.25; ŋ =0.79), or

2 during the 2.5km Run (F(1,17) =0.70; p=0.46; ŋ =0.04).

Table 1

Anthropometric Measures

Total (n= 19) G/G (n=8) G/C, C/C (n=11)

Age (yrs) 30 (±5) 28. (±4) 32 (±5)

Height (cm) 180.6 (±7.3) 179.8 (±8.7) 181.2 (±6.5)

Weight (kg) 85.0 (±9.8) 83.3 (±11.8) 86.3 (±8.6)

BMI (kg/m2) 26.0 (±2.2) 25.7 (±2.0) 26.3 (±2.4)

Data expressed as mean ± standard deviation.

Table 2

Athletic Traits

Total (n= 19) G/G (n=8) G/C, C/C (n=11)

VO2 Max (ml/kg/min) 50.8 (±5.5) 49.3 (±5.3) 52.0 (±5.5)

Body fat (%) 20.8 (±6.6) 21.2 (±5.4) 20.4 (±7.6)

Lean Mass (kg) 65.7 (±8.9) 64.2 (±9.1) 66.9 (±9.1)

Fat Mass (kg) 17.3 (±6.2) 17.4 (±5.6) 17.2 (±6.9)

Data expressed as mean ± standard deviation.

Reported RPE scores resulted in statistically significant differences between the groups over the course of the entire event (p<0.01; ŋ2=0.279). When measured by individual events, neither the first 5km run (F(1,17)=1.13; p=0.34) nor the 20km bike ride

(F(1,17) =1.17; p=0.35) resulted in a significant difference between groups. Significant differences (p=0.03; d=1.0) prior to the start of the final 2.5km run (Figure 2) were found with those homozygous for G/G (8.5 ± 2.2) reporting a higher RPE score than those with Cytosine (6.7 ± 1.1) present. As a result, baseline RPE scores were used as a covariate. Accounting for reported 2.5 km baseline RPE scores, significant differences

2 existed (F(1,16) =4.55; p=0.02; ŋ =0.22) in RPE scores during the final event of the duathlon. The final RPE score recorded at the conclusion of the 2.5 km run resulted in

2 significant differences (F(1,16) =14.37; p=<0.01; ŋ =0.47) between groups; those within the C group reported significantly higher RPE (17.6 ±1.4) scores than those in the G/G group (16.3±1.8) at the conclusion of the 2.5km run.

Figure 2, RPE scores base on group during the final event of the simulated sprint duathlon. Data expressed as mean ± standard deviation. # indicates p=0.03, * indicates p= <0.01 for ACOVA result.

Performance times between groups were not found to be different in 5km

2 performance times (F(1,17) =0.41; p=0.57; ŋ =0.23), 20 km biking times (F(1,17) =1.10;

2 2 p=0.31; ŋ =0.61), or 2.5km run times (F(1,17) =2.20; p=0.16; ŋ =0.16). For the entirety

2 of the duathlon, the groups did not show a difference (F(1,17) =1.53; p=0.26; ŋ =0.08) in performance times. The GG group had an average duathlon time of 1:38:12 (± 16:56 min), while those in the C group had an average time of 1:28:44 (±14:51 min).

Discussion

The primary finding of this study is that the subjects with Cytosine at the -174 position on the promoter region of the Interleukin-6 gene reported higher average RPE scores at the finish of the simulated duathlon. The difference in RPE scores observed at the final and at the most intense portion of the simulated duathlon in the present study, supports the hypothesis that subjects with the G/G allele have felt a lower level of difficulty, which may be attributed to differences in the amount of IL-6 in circulation.

Robson-Ansley (2004) demonstrated that prior to an aerobic exercise session, infusions at a volume of 0.05µg/kg of bodyweight of recombinant IL-6 (rhIL-6), chemically similarly structured as IL-6, impair performance times and increase sensations of fatigue and higher levels mood disturbance. The limitation to this finding is that the source of IL-6 was the infusion of rhIL-6, not the exercising muscle. Over the course of a 6-day mountain bike race, only the soluble IL-6 receptor was found to be related to levels of fatigue, not IL-6 specifically. However, these samples were obtained the morning after, not post-exercise, when IL-6 levels are expected to be highest (Robson-

Ansley, 2009). IL-6 has been shown to increase sympathetic nervous activity by

44 increasing circulating levels of norepinephrine (Torpy, 2000) and by increasing metabolism via adrenocorticotropic (ATCH), cortisol, and thyroid-stimulating hormone

(Tsigos, 1997). The mechanism by which IL-6 may alter interpretations of exercise difficulty should be further examined to determine if these findings are the result of neural or metabolic interactions.

Previous work has linked subjects with C at the -174 position on the IL-6 gene to decreased insulin sensitivity, lower circulating levels of glucose, (Kubaszek, 2003;

Fernandez-Real, 2000a), and lower levels of free fatty acids (Fernandez-Real, 2000b,

Halverstadt, 2005). Collectively, these previous studies may explain the differences in

RPE scores that could be attributed to limitations in metabolism. However, studies to explain the neural impact that the -174G/C may have on psychological measures in athletes are lacking.

All baseline athletic characteristics that may be advantageous in endurance exercise performance seem to support previous work; neither maximal oxygen uptake

(McKenzie 2004) nor differences in body composition (McKenzie, 2004; Yamin, 2008) were found to be related to the -174G/C SNP on the IL-6 gene. Over the course of the duathlon, the groups did not display significant differences in performance times at any point. To create conditions as close to the nature of an actual duathlon as possible, all subjects were permitted to complete the events at a pace which they chose; they were only directed to complete the entire duathlon as quickly as they could. This condition was unique because this was the first study, to our knowledge, to examine the effect that -

174G/C SNP may have on athletic performance by using event performance time as a

45 primary variable. Controlling for the mode of exercise that the subjects routinely perform would perhaps allow more precise control of the differences for which the -174G/C variant may account because adaptations to routine training would be closer among subjects.

In conclusion, the impact of the -174G/C SNP on the IL-6 gene appears to play a role in feelings of difficulty during intense aerobic exercise. These findings align with much of the previous work that has shown that exercise-driven increases in IL-6 are found only during the later stages of aerobic exercise. It would stand to reason that differences in RPE scores would be more likely present at the later stages of our simulated duathlon. These findings present many exciting avenues for future research to determine the impact of IL-6 on exercise performance and any potential links to athletic genotypes. Future studies may wish to employ more precise measures of fatigue and markers of metabolism to differentiate between genetic impact of -174G/C SNP on the central nervous system and potential metabolic limitations. Work should also be done in order to draw these applications to difference athletic populations and expand to female athletes. Further, as the relationship of exercise performance and human genetics continues to grow, population-specific studies will be needed to further refine the field, which is still in its relative infancy. Using a standard athletic competition, which would include external factors such as external feedback of fellow competitors and being allowed to link success to order of finish, would allow more precise conclusions to be drawn for those who seek to maximize athletic performance.

Practical Applications

The resulting data from this study suggest that athletes’ genotype rates of perceived exertion may be expected to appear different depending on their genotype.

Those who seek to monitor perceptions of training sessions may see altered scores due to a biological reason as opposed to measuring the true difficulty of the training. Our study also suggests that assessments based on a person’s genetic sequence at the -174SNP for the IL-6 gene would be done in error, as our study and those of others have not found supporting evidence, which may suggest an inherited advantage.

Acknowledgments

The authors have no relevant conflicts of interest.

CHAPTER V

MANUSCRIPT 2

Differences in Interleukin-6 Production and Circulating Glucose following a

Simulated Sprint-Duathlon; The impact of the -174G/C Single Nucleotide

Polymorphism on Expression.

Scott M. Habowski,1 J. Derek Kingsley,1 Adam Jajtner,1 Betsy Raub,2 and William

Kedia2

Kent State University, Kent, OH; 1 and The Center for Applied Health Sciences, Stow,

OH 2

Introduction

The cytokine Interleukin-6 (IL-6) functions within the human body as an inflammatory cytokine during conditions of acute or chronic injury and inflammation.

IL-6, along with other cytokines, has traditionally been recognized as one of the primary diagnostic markers of systemic inflammation (Tanka, 2013). The IL-6 molecule, when produced in response to aerobic exercise, serves an inverse role in preventing the actions of other inflammatory molecules (Febbraio, 2002); in this case, it functions as an anti- inflammatory myokine. Beyond acting as an anti-inflammatory myokine, the release of

IL-6 during and after aerobic exercise has been shown to signal gluconeogenesis and lipolysis (Pedersen, 2007), which are metabolic necessities for sustained energy production during high-intensity exercise sessions of long duration. Ostrowski (1998)

47 48 recorded up to a 100-fold increase from resting values following aerobic exercise sessions.

The dramatic increases in circulating IL-6 in response to aerobic exercise have been linked to the intensity, duration, and the amount of muscle mass involved in the session (Fischer, 2006). With the larger increases linked to higher intensities, exercise- derived IL-6 functions to support metabolism by increasing the availability of the metabolites needed for aerobic metabolism (Febbraio, 2002). IL-6 has an established inverse relationship with muscle glycogen content, showing muscles that have been depleted of glycogen to produce greater amounts of IL-6 during and following exercise

(Keller, 2001; Steensberg, 2001). Infusions of recombinant IL-6 have resulted in increases in gluconeogenesis (Tsigos, 1997) and lipolysis (Pedersen, 2004), providing the needed metabolic substrates necessary to maintain aerobic metabolism.

Genetic differences in the Interleukin-6 gene have been established to display significantly different circulating levels of IL-6 in patients from a multitude of medical conditions (Bennermo, 2004). This work has identified a single nucleotide polymorphism (SNP) at the -174 position on the promoter region of the Interleukin-6 gene (-174G/C SNP; rs1800795). At this site, the insertion of the amino acid cytosine (C) in place of the amino acid guanine (G) within the genetic sequence has been recognized to alter transcription rates of IL-6 (Fishman, 1998; Terry, 2000). The insertion of the amino acid cystine within the genome functionally suppresses transcription rates by allowing for methyltransferase-1 (DMNT-1) to attach a methyl group to cytosine, thereby compressing histone structure (Hodge, 2001; Horsburgh, 2005). It also allows for the

49 attachment of Nuclear factor I (NF-I) proteins, which also reduce transcriptional rates

(Liu, 1997). Interestingly, the associated increase of IL-6 following exercise has been shown to alter the methylation status of 11 other genes significantly (Robson-Ansley,

2014), many of those which play a role in metabolism or immune function.

The impact of the -174G/C SNP genetically based difference has gone beyond people diagnosed with medical conditions, which may fall under the traditional role of

IL-6 as a marker of inflammation and has worked to support the role of IL-6 in metabolism. Those with the C allele have shown lower fasting glucose levels (Huth,

2009), lower rates of resting energy expenditure (Kubaszek, 2003), lower circulating triglycerides (Halverstadt, 2005), and lower levels of blood glucose following intervention (Fernadez-Real, 2000). Collectively, these results would suggest that those with the G at both positions (G/G) at the promoter region would have the genetic predisposition for an advantageous phenotype for those engaged in training sessions of the appropriate intensity to deplete muscle energy stores.

With the established impact on metabolic function, and in varying states of disease, relatively little research has been conducted on the impact the -174G/C SNP may have in relation to exercise and IL-6 response, despite the well-established relationship to exercise. Previous work has suggested the G/G genotype may favor athletes in events that demand high power output such as the shotput and high jump (Ruiz, 2010), although these results have failed to be duplicated in other populations (Eynon, 2010). In response to various exercise intervention programs, subjects with the GG allele showed significantly lower levels of bodyweight and subcutaneous adipose tissue, and higher

50 rates of glucose clearance than those with the C allele (McKenzie, 2004). Previous work in army recruits has shown that those with the CG genotype produced significantly larger amounts of IL-6 following an acute training session. Following an 8-week training program, the same group demonstrated the greatest improvements in VO2 max

(Huuskonen, 2009). Because of the relatively limited work that has examined the impact of what the -174G/C SNP may have on exercise athletes, practitioners and researchers would benefit from understanding what impact genetic differences may have on IL-6 production and the concomitant alterations in metabolism and inflammation management.

Therefore, the purpose of the present study was to determine the influence of -

174G/C SNP on IL-6 production following a simulated sprint duathlon and performance times. A secondary aim was to determine if circulating levels of IL-6 had an influence on event performance times, circulating levels of blood glucose, and rates of perceived exertion. Tertiary objectives were to measure the relationship between IL-6 and event times against measures of body composition and aerobic capacity. We tested the hypothesis that subjects with the GG allele will display inherent advantages for several measures during the study, which ultimately may aid athletic performance. It may be expected that those with the GG allele will display faster total duathlon times and a faster performance in the final stage (2.5km run) of the duathlon due to an increase in circulating IL-6. This advantage may also manifest itself through IL-6’s ability to increase gluconeogenesis and lipolysis, which may lead to improved metabolic performance.

Methods

All potential subjects were provided a written informed consent form and given a chance to ask questions. Upon their giving both verbal and written consent, they were enrolled in the study. Initial anthropometric measurements were recorded, including height, weight, and seated blood pressure and heart rate. To determine the -174G/C SNP genotype, all subjects provided a saliva sample, which was then stored until the conclusion of the study. All body composition measurements (Lean Mass, Fat Mass,

Visceral Adipose Tissue [VAT], and body fat percentage) were assessed using a dual- energy x-ray absorptiometry (DEXA) scan. To assess aerobic capacity, subjects then performed a maximal exercise test to determine aerobic fitness levels (VO2 max). After concluding the aerobic assessment, all subjects were scheduled for their simulated duathlon visit. They were given a standardized meal bar (Performance Energy Bar,

PowerBar®) and instructed to avoid heavy exercise sessions for 48 hours prior to their scheduled visit. All bars had approximately 4 grams of fat, 44 grams of carbohydrates, and 10 grams of protein, the entire bar contained 230 calories

Subjects then reported back to the research center within two weeks of their initial assessment visit to perform the simulated sprint duathlon. Prior to initiation of the duathlon, all subjects confirmed that they had consumed the provided meal bar. The simulated sprint duathlon consisted of a 5-kilometer run, a 20-kilometer bike ride, and a final 2.5-kilometer run. All events took place within the research center on the same treadmill and mounted bike. To determine IL-6 and glucose, blood samples were obtained prior to the start of the event (Baseline), following the 20km bike ride (Middle),

52 and upon conclusion of the 2.5km run (Post). The second blood draw was referred to as

Middle to simplify explanations, not in reference to the time within the duathlon (Figure

1). At even intervals along every stage of the duathlon, Rate of Perceived Exertion

(RPE) and event time were recorded.

Subjects

Eighteen males between the ages of 23-40 years volunteered for the study. After reading the informed consent and agreeing to the procedures outlined, subjects were then enrolled. Prior to collection of any study information, all subjects reported being free of any preexisting cardiovascular, autoimmune, metabolic, or bleeding disorders as well as showing no signs of illness, infection, or injury within the previous 30 days. Finally, all subjects reported to engaging in aerobic exercise at least twice a week and confirmed that they felt they could sustain a bout of aerobic exercise for approximately two hours. To qualify for the study, all subjects had to meet a minimal level of “Good” or above for their age group as identified by Haywood’s (1998) standards for aerobic fitness based on

VO2 max. An independent review board, IntegReview® IRB (Austin, TX), reviewed and approved of all study procedures, interventions, and materials. Research was conducted within the outlines set by the FDA for good clinical practice and the Declaration of

Helsinki.

Procedures

The collection of data measures first assessed athletic traits including subject anthropometric measurements, genetic collection, and maximal oxygen consumption.

The following visit included testing procedures collected circulating IL-6 and performance times.

Anthropometric measurements. Weight was measured in kilograms (kg) on a calibrated digital scale (Seca® Medical Scale, Hanover, MD), and height was measured in centimeters using a mounted stadiometer. This information was then used to determine Body Mass Index (BMI) scores. To determine body composition, subjects underwent a DEXA scan (GE® Electrical Lunar DPX Pro, Boston, MA) and were analyzed by the manufacturer’s software (enCORE® Version 15) to determine each subject’s lean mass, fat mass, VAT, and body fat percentage.

Maximal oxygen consumption. Adopted from Paavolainen (1999), aerobic fitness was determined from a progressive exercise testing protocol. Beginning at a 1˚ incline going at a speed of 5.0 miles per hour (mph), speed was increased 0.6 mph in 3- minute intervals. Following the incremental increases, once the speed of 10.6 mph was achieved, the incline was raised in 1-minute intervals until exhaustion. A Parvo Medics

TrueOne® Metabolic System (OUSW 4.3.4, Sandy, UT) was used to determine VO2 max. Averaging measures in 30-second intervals, oxygen consumption scores were recorded in milliliters of oxygen/per bodyweight in kg/ per minute (ml/kg/min) with the highest sustained average of oxygen consumption used to determine aerobic fitness levels

(VO2 max).

Genetic collection and processing. Approximately 3mL of saliva was collected in an Oragene OG-500 (DNA Genotek, Ottawa, Ontario) collection kit following a

54 minimum of a 30-minute window without eating, chewing, or drinking. Once a sufficient sample had been obtained, each tube was labeled with a unique five-digit code. This code was then recorded on a separate sheet with the corresponding subject number. All samples and code sheets were stored in a locked cabinet in a temperature-controlled room. Upon the conclusion of the last subject’s visit, all samples were shipped to

EpigenDx® (Hopkinton, MA), where each sample was tested to determine the genotype of the -174G/C SNP on the IL-6 gene using pyrosequencing.

Simulated Sprint duathlon. The simulated sprint duathlon consisted of a 5km run, a 20km bike ride, and a 2.5km run (Figure 3). Subjects were permitted to determine their own pacing through the entirety of the event, adjusting bike gears and treadmill speed as they saw fit. Prior to beginning the duathlon, each subject was fitted with a

Polar® H1 Heart Rate Monitor (Bethpage, NY) and told to attempt to finish the entirety of the distance as quickly as possible. Both running events were performed on a Sole®

TT8 Treadmill (Salt Lake City, UT), and the 20km bike ride was performed on a stationary FluidPro® Bike Trainer (Cascade Health and Fitness, Woodinville, WA). A break between events was permitted, not to exceed 5 minutes, to obtain a blood sample.

Finally, subjects were permitted to consume water ad libitum but were restricted from consuming any sources of carbohydrate, protein, electrolyte, or fats until the conclusion of all events.

Sprint Duathlon 5 KM Run 20 KM Bike Ride 2.5 KM Run

Baseline Middle Post IL-6 IL-6 IL-6 Glucose Glucose Glucose Figure 3, Timeline of events and samples during the simulated sprint duathlon. Blood collection and storage. A blood sample was obtained using a disposable

21-gauge needle from the subjects’ antecubital vein in a refrigerated 10mL EDTA tube

(BD Vacutainer®, Franklin Lakes, NJ). Circulating blood glucose was measured using a

Contour® Next EZ Blood Glucose Meter (Bayer® HealthCare, Mishawanka, IN). The tube was spun in a centrifuge (LabCorp®, Model 642E) at 3380 revolutions per minute for 15 minutes. The sample was then aliquoted into separate storage vials, which were then stored in a ScienTemp® (Adrian, MI) -80˚ C freezer until processing.

IL-6 Analysis. Interleukin-6 was determined via ELISA assay analysis at the Kent State

Exercise Physiology Biochemical Laboratory. Plasma concentrations were determined using Quantikine® High Sensitivity Enzyme Linked Immunosorbant Assay (ELISA):

Human IL-6 Immunoassay (R & D Systems, Minneapolis, MN). The mean coefficient of variation for IL-6 testing was 4.2%.

Statistical Analyses

All tests were run with an established a priori of p ≤0.05 to determine statistically significant findings. Analysis was performed by separating subjects by genotype (G/G,

C/G, C/C) at the -174 positions on the IL-6 gene. Initial analysis employed a one-way analysis of variance (ANOVA) for baseline anthropometric measures, which include:

VO2 max, body composition (lean mass, fat mass, visceral fat, and body fat percentage), final RPE scores, and changes (Delta) in circulating IL-6 and glucose from baseline to middle, baseline to post, and middle to post. A repeated measures ANOVA was used to test the effects the genotype had over time (Baseline, Middle, Post) on circulating IL-6 and glucose. If the sample data points were found to violate the assumption of sphericity, the α was adjusted in accordance with the Greehouse-Geisser epsilon correction. For variables found to be significantly different, effect size will be determined using partial eta squared (η2).

For all tertiary measures, a Pearson correlation (r) was used to assess relations between changes in IL-6 changes for all subjects over the course of the simulated duathlon to establish what contributions exercise-induced IL-6 production may have in glucose, performance times, and RPE. Finally, the relation between body composition measures will be used to examine the relation to IL-6 levels. Values are presented using as mean ± SD. All statistical analyses were completed using IBM SPSS version 24

(Armonk, NY).

Results

Genetic testing produced results expected well within the Hardy-Weinberg equation. Anthropometric measurements (Table 3) at baseline resulted in a significant

2 difference (F(2,15) =4.67, p=0.03; η = 0.37) between the groups in body fat percentage

(Figure 2). Those with the C/C (13.6% ±6.7) allele displayed significantly lower body fat percentage than those with the C/G (24.3% ±5.1) allele (p=0.02; r=-0.67) and those with the G/G (21.2% ±5.4) allele (p=0.05; r=-0.53).

Table 3

Anthropometric Measurements

Total (n= 18) G/G (n=8) C/G (n=6) C/C (n=4) Age (yrs) 30 (±4) 28 (±4) 30 (±5) 32 (±3) Height (cm) 180.7 (±7.5) 179.8 (±8.7) 182.0 (±8.0) 181.0 (±5.6) Weight (kg) 85.4 (±10.0) 83.3 (±11.8) 88.0 (±8.8) 85.7 (±9.5) BMI (kg.m2) 26.1 (±2.3) 25.7 (±2.0) 26.5 (±2.1) 26.2 (±3.4) Body fat (%)* 20.7 (±6.8) 21.2 (±5.4) 24.8 (±5.4) 13.6 (±6.7) Lean Mass (kg) 66.0 (±9.1) 64.2 (±9.1) 64.2 (±8.8) 72.6 (±8.8) Fat Mass (kg) 17.3 (±6.4) 17.4 (±5.6) 21.0 (±5.0) 11.7 (±7.0)

VO2max (ml/kg/min) 50.7 (±5.5) 49.3 (±5.3) 52.4 (±3.8) 50.8 (±8.6) Data expressed as mean ± standard deviation. * indicates p=0.03.

Over the course of the duathlon, IL-6 production did not result in a difference among the groups (p=0.30) (Figure 4). The difference in IL-6 from post 20km bike to

2 the finish (Delta) resulted in significantly different (F(2,15) =3.76, p=0.05; η = 0.37) changes between genotypes (Figure 5). The C/G genotype (+2.7 pg/mL; ±2.2) resulted in significantly larger (p=0.03; r=0.49) increases in IL-6 during the final event than those with the G/G genotype (+0.66 pg/mL; ±1.4), as well as those with the C/C genotype

(+0.25 pg/mL; ±0.7) (p=0.03; r=0.60) (Figure 4). No differences between genotypes were observed in circulating glucose (p=0.595) over the course of the duathlon or at any point between events.

Figure 4, IL-6 production over the course of the simulated duathlon. Data expressed as mean ± standard deviation

Figure 5, The difference (delta) between IL-6 following the 20km bike ride and following the 2.5km run. Data expressed as mean ± standard deviation. * indicates p=0.05.

Tertiary measures among all subjects revealed various correlations between several variables over the course of the duathlon. Baseline measures of IL-6 had a medium correlation (p=0.03; r= 0.49) to post IL-6 values and a large correlation

(p=<0.01; r= 0.60) to body fat percentage. Body fat percentage was also positively associated (p=<0.01; r=0.60) to post IL-6 levels. Lean Mass was negatively associated with IL-6 at the middle (p=<0.01; r=-0.72) and post (p=<0.01; r=-0.70) time points

(Figure 6). Post IL-6 levels were also strongly correlated to blood glucose at the middle draw (p=0.01; r=0.55) and the post draw (p=0.02; r=0.54). Blood glucose was correlated to lower event times (p=0.02; r=--0.53) at the middle time point and total event time (p=<0.01; r=-0.67).

Figure 6, The correlation (r=-0.70) between muscle mass (g) and circulating IL-6 (pg/mL) at the end of the simulated sprint duathlon.

Discussion

Over the entire course of the simulated duathlon, the separate genotypes failed to display differences in IL-6 production. This information contradicts our stated hypothesis that those with the G/G phenotype would produce the largest amount of IL-6 following aerobic exercise. The presence of the C/G allele allows the attachment of a methyl group that functionally suppresses gene expression. This fact suggests that the

C/G phenotype displays lower IL-6 levels due to increased methylation at the promoter region. Hodge (2001) demonstrated that exposure to IL-6 increases methylation activity in erythroleukemia cells and thereby suggested that overexposure to inflammatory cytokines may inhibit the expression of genes important in tumor suppression. This mechanism supports the traditional view of association between IL-6 and levels of inflammation.

However, the dual-role of IL-6 as a pro-inflammatory cytokine in the inflammatory response, and as an anti-inflammatory myokine when produced following exercise, may also hold true when it comes to traditional mechanisms of methylation at the C/G alleles across the genome. Barres (2012) found that acute gene activation following aerobic exercise increases due to hydroxylation at the amino acid cytosine, resulting in demethylation at the promoter sites of several genes associated with metabolism (PGC-1α, PPAR-δ, & PDK4). Comparing aerobic exercise sessions of intensities at 40% and 80% of VO2 max resulted in increases in transcription rates and greater demethylation of the several genes following the higher intensity session. This relationship with intensity is similar to the previously established link between exercise

61 intensity and myocyte-derived IL-6. Our findings showed that those with the C/G genotype had a significantly larger increase in IL-6 production during the final stage of the duathlon, and this increase may have occurred through a similar process of hydroxylation. This proposed mechanism rejects the previously stated hypothesis that the

G/G allele would produce a larger response in IL-6 production during the final stage of the duathlon.

Our findings suggest that the C/G allele may lead to increased transcription of IL-

6 during and following aerobic exercise. Our findings support the previous work of

Huuskonen (2009), who found army recruits with the C/G allele on the IL-6 gene being the only phenotype to display significantly higher levels of IL-6 following acute bouts of aerobic exercise. Previous work that has attributed the interaction of exercise and impact of gene expressions (epigenetics) may suggest that acute exercise sessions increase global methylation across the genome, but this effect is far from being universal at each individual gene (Horsburgh, 2015). Aerobic exercise alters genetic expression through two major mechanisms: First, exercise causes hypomethylation through alterations in

DMNT-1 activity, which allows for demethylation at promoter sites. Second, it allows histone modifications through the actions of Histone Deacetylase 5 (HDAC5) and

Histone Acetyltransferase (HAT), which functionally serve to open the chromatic structure, permitting transcription (Rasmussen, 2014). It stands to reason that these alterations may serve as the mechanisms for exercise-related increases in IL-6, although no study to date has sought to determine this relationship.

A possible explanation of the contradictory role of the 174-G/C SNP on IL-6 levels in disease and following exercise appears to be the initiation of production. Absent of the epigenetic changes associated with exercise, those with C/G genotype can be expected to have a decreased rate of IL-6 expression through methylation of at cytosine by DMNT-1 at the C/G base pair within promoter regions (Hodge, 2001; Terry, 2000).

The epigenetic alterations found following acute exercise sessions result in demethylation and histone modification (acetylation), which allow for increased transcriptional activity

(Barres, 2012; Vital de Silva, 2017). Changes in methylation status following acute aerobic exercise may then more dramatically impact those with the C/G genotype and allow for increased IL-6 transcription.

Another unique finding of the present study is the strong negative correlation between IL-6 production and lean mass among between all subjects. This finding contradicts much of the published research, which has suggested that by employing larger amounts of muscle mass during aerobic exercise sessions, IL-6 production will increase

(Ostrowski, 2000; Nieman, 1998; Fischer, 2006). Our data does match other trends by presenting a correlation to body fat percentage to IL-6 levels observed at baseline.

Conclusion

Though our findings have contradicted the original hypothesis, we have added to the limited amounts of evidence suggesting that individuals with the C/G genotype may have different rates of expression of the IL-6 gene following acute endurance exercise.

Future work should be aimed to distinguish the transcription-signaling mechanisms

63 behind inflammatory cytokine and aerobic exercise-induced myokine release. This need is highlighted by the rejection of the stated hypothesis: that those subjects with the GG allele would produce more IL-6 during aerobic exercise and, therefore, possess an athletic advantage. This hypothesis was formed based on much of the current literature, which has indicated that those with GG allele have higher circulating levels of IL-6 in various disease states and following interventions than those with either C/G or C/C genotypes.

Our findings highlight that the transcription of IL-6 appears to differ based on environmental stimulus. By understanding the initiation of exercise-induced IL-6 transcription, future practitioners may be able to take advantage of the previously highlighted benefits of exercise-induced IL-6 for both athletic and diseased populations.

Another unique finding of the current study is the suggestion of an inverse relationship between lean mass and IL-6 production; this contradiction to previously established work warrants further exploration. Much of the related research, which has established the relationship between increases in IL-6 production resulting from exercise, has employed standardized exercise sessions, controlling exercise intensity through standardized heart rate intensities. Our study allowed participants to determine their own pacing for all events, which was done in order to draw closer comparisons to athletic competitions and allow for total event time to be used as measured outcome. Future work should be conducted following various athletic events within actual competitions to further understand the contributions of IL-6 to athletic performance.

APPENDIXES

APPENDIX A

TELEPHONE SCREENING

Name:

Contact (Social Media/Phone/Email):

Hello, Thank you for expressing your interest in participating in our research study. The purpose of this study is to examine the effects of a gene variant on the production the molecule Interleukin-6 during and following exercise. The Interleukin-6 molecule is thought to have various effects on metabolism, which may influence exercise performance.

The study involves a visit to our laboratory in Stow. During this visit you will be asked to complete a simulated sprint duathlon. This will require you to run 5k on a treadmill, ride 20k on a stationary bike, and finally, finish with 2.5k on the treadmill. You will be free to pick any pace you choose; we just ask that you complete the events as quickly as possible.

Before the events we will be measuring body fat and a gene variant. Your results will be shared with you if you choose. We will also be doing three blood draws over the course of the race, which measure the Interleukin-6 molecule. The total volume of blood taken is roughly 15mL; this is about 4% of the volume given during a typical donation. Did you have any further questions?

Schedule appointment time:

Thank You,

(Staff Name)

APPENDIX B

IRB APPROVAL

IMPORTANT

PLEASE READ YOUR ENTIRE APPROVAL PACKET THOROUGHLY, PAYING SPECIAL ATTENTION TO THE APPROVAL LETTER. THE LETTER CONTAINS INTEGREVIEW’S REPORTING REQUIREMENTS WITH WHICH YOU ARE OBLIGATED TO COMPLY.

IN ADDITION, IT IS YOUR RESPONSIBILITY TO ENSURE THAT PERIODIC CHECKS ARE DONE IN IRBMANAGER TO CONFIRM YOU ARE UTILIZING THE MOST CURRENT INFORMED CONSENT.

To access request forms and/or obtain your IRB documents, you will always start from your Home page: • Click on the specific protocol number you wish to submit for; which can be found under ‘My Protocols’. • Once you click on the protocol, it will re-direct you to the Study Summary Page (SSP).

Obtaining documents from IRBManager: To obtain your IRB action letter, including new/updated informed consents when applicable, once on the SSP you can: • Scroll down to the ‘Events’ tab • You can click on the Event itself or on the number listed in the column labeled “Att” (i.e. attachments) • Approval documents will be listed under the sub-heading “IntegReview Approval Documents”

Submitting on IRBManager: All request forms (other than New Study Submission Forms) are protocol driven. You can obtain by: • Clicking on ‘Start xForm’ - Under ‘Actions’ (left side of screen) • Select the appropriate xForm (listed alphabetically)

IRB Statement of Compliance

IntegReview IRB, an independent IRB located in Austin, Texas, was established in 1999 and offers five weekly meetings for review of U.S., Latin American and Japan research sites, quality review by experienced individuals, competitive fees, and quality assurance/control. Designed to accelerate the IRB process without compromising accuracy or the protection of human subjects, IntegReview IRB delivers study documents to the investigator within two business days of board review.

IntegReview IRB is committed to meeting rigorous standards for quality and maintaining sound policies and procedures involved in the protection of human research participants. IntegReview IRB was initially awarded full accreditation of its human research protection program (HRPP) by the Accreditation of Human Research Protection Programs, Inc.® (AAHRPP) in June 2007. AAHRPP accreditation will expire in September of 2020.

Written standard operating procedures govern IntegReview IRB for initial, continuing, full board, expedited and exempt review of clinical research studies. IntegReview IRB complies with the regulations as defined in the United States Food and Drug Administration (FDA), Code of Federal Regulations, Title 21, Parts 50, 54, 56, 312 and 812, International Conference on Harmonisation (ICH) Guidelines for Good Clinical Practices, E6, the Department of Health and Human Services (DHHS) regulations as identified in the Code of Federal Regulations, Title 45, Part 46, other regulations as applicable, as well as local and state laws.

Lynn A. Meyer, CCRP President

Version 1/1/2013 Version 9/12/2014 Version 2/16/2015 Version 9/18/2015 Version 9/9/2016

December 8, 2016

Principal Investigator: Scott Habowski Sponsor: The Center for Applied Health Sciences (Kent State University) Protocol Number: KSU-001 Study Title: “EFFECTS OF IL-6 GENE POLYMORPHISM -174G/C ON INTERLEUKIN-6 PRODUCTION AND ENDURANCE EXERCISE PERFORMANCE”

Dear Mr. Habowski:

A convened IRB meeting of IntegReview was held on the above-referenced date. The following full board action was taken on initial review of the above-referenced study:

Approved: Principal Investigator Investigative site(s) as submitted with initial submission documents Protocol Version 1 dated 16 November 2016 Informed Consent, English language, dated December 8, 2016 (refer to IntegReview modifications as reflected on the following document containing revision marks)

IntegReview does not review stand alone HIPAA documents. Federal regulations do not require IRBs to review and approve stand-alone HIPAA authorizations. Following is a website presenting the IRBs role under the Privacy Rule from the National Institutes of Health: http://privacyruleandresearch.nih.gov/irbandprivacyrule.asp. Please refer to paragraph 12, which identifies IRB obligations regarding these stand-alone documents.

IMPORTANT • The following changes in approved research may not be implemented until you have received approval from IntegReview except where necessary to eliminate apparent immediate hazards to the human subjects: Protocol Amendments Change in the Principal Investigator and/or Sub-investigators (only if the Sub- investigators will be performing study-related procedures that the PI is not qualified through expertise to perform) Change in the address at the study site or the addition of a study site(s) • Only Informed Consent documents containing IntegReview’s approval stamp may be utilized: There must be procedures in place to guarantee that consent has been voluntarily obtained and properly documented. For participants that do not speak English, the informed consent document must be in a language understandable to them. Only IntegReview staff may initiate modifications to Informed Consent documents. The Informed Consent document for your site will be maintained in our computer files, and IntegReview will make all revisions following board approval.

3815 S. Capital of Texas Hwy, Suite 320, Austin, TX 78704 Tel. 512.326.3001 Local Fax. 512.697.0085 http://www.integreview.com

IRB Registration Numbers: IRB00008463, IRB00003657, IRB00004920, IRB00001035, IRB00006075

MEMBERSHIP ROSTER As of November 15, 2016

Sponsor Name: The Center for Applied Health Sciences (Kent State University) Protocol Number: KSU-001 Principal Investigator Name: Scott Habowski Meeting Date: December 8, 2016

C Denotes consultants (non-voting) assisting in the review of the study when the knowledge and expertise in a particular therapeutic area is not available among the voting members A Denotes members abstaining from the vote Denotes (voting) members who were in attendance at the meeting and reviewed the study information

NAME DEGREES/ BOARD EXPERIENCE AFFILIATION ALTERNATE CERTS. POSITION WITH IRB FOR M Susan Parker Ginnings, R.Ph. Scientific Previously employed as Hospital Pharmacy Non-affiliated Scientific members Chairman Supervisor Paid Consultant Th, Susan Parker Ginnings M Levi Machado, Co-chair CCRP Non- IRB Coordinator, administrative support for Full time Employee Non-scientific scientific IntegReview members M Olga Obrda B.S. (Chemistry) Scientific BD Representative; previously employed as Non-affiliated Scientific members Project Management and Business Development Paid Consultant for CRO M Jennifer Christensen Pharm. D., BCPS Scientific Internal Medicine, Investigational Drugs, Non-affiliated Scientific members Neuropharmacology research scientist Paid Consultant M Susan Krivacic B.A. (German), Scientific Consultant and patient advocate for oncology Non-affiliated Scientific members M.P.Aff (Public trials; serves as a Board member for NCI’s Adult Paid Consultant Affairs) Early Stage Central IRB (CIRB); previously employed as Senior Executive and Executive Director for CRO; Global Project Assoc. Director for a CRO (Orphan Diseases Division)

M Denotes regular Monday board members T Denotes regular Tuesday board members W Denotes regular Wednesday board members Th Denotes regular Thursday board members F Denotes regular Friday board members Other members are alternates All regular Board members can serve as alternates on other Boards as specified by their positions (e.g. Scientific for Scientific) Non-scientific members represent the general perspective of study participants Note: In addition to our regular members, we have access to specialists in therapeutic areas not represented on this roster. Page 1 of 6

Sponsor Name: The Center for Applied Health Sciences (Kent State University) Protocol Number: KSU-001 Principal Investigator Name: Scott Habowski Meeting Date: December 8, 2016

NAME DEGREES/ BOARD EXPERIENCE AFFILIATION ALTERNATE CERTS. POSITION WITH IRB FOR M Karen Haslund M.D. Scientific Licensed physician, Pediatrics Non-affiliated Scientific members Paid Consultant T Charles F. Ryan, Chairman Ph.D., M.S. Scientific Representative for first time in human studies; Non-affiliated Scientific members W Charles F. Ryan (Pharmacology & Pharmacology, Toxicology, Radiation Safety, Paid Consultant Toxicology), Radioisotope, Nutritional/Food supplements and R.Ph. Medical Foods T Tonya Reed, Co-chair Non- IRB Coordinator, administrative support for Full time Employee Non-scientific scientific IntegReview; previously employed as a Project members Assistant for CRO T Sara Bartos M.D. Scientific Licensed physician, Internal Medicine Non-affiliated Scientific members Paid Consultant T M. Alexander Kenaston Ph.D., M.S. Scientific Toxicology Research Scientist, representative for Non-affiliated Scientific members (Toxicology), first time in human studies, Pharmacology/ Paid Consultant R.N., CIP Toxicology, Licensed Registered Nurse; previously employed as Project Manager, CRA and CRC for CRO T Marcy Goodfleisch B.S., M.A. (Liberal Non- Adjunct University Professor; Ethicist; Management Non-affiliated Non-scientific Studies); Mediator scientific & Communication Consultant. Former Clinic Paid Consultant members (Civil & Family Administrator for nationally recognized HIV Clinic Dispute Resolution); and large FQHC community health center. Graduate work in Communications and English

Sponsor Name: The Center for Applied Health Sciences (Kent State University) Protocol Number: KSU-001 Principal Investigator Name: Scott Habowski Meeting Date: December 8, 2016

NAME DEGREES/ BOARD EXPERIENCE AFFILIATION ALTERNATE CERTS. POSITION WITH IRB FOR T Michael D. Aldridge Ph.D. (Nursing Scientific Assistant Professor of Nursing; previously Non-affiliated Scientific members Education), R.N., employed in various Nursing roles for pediatric Paid Consultant CNE intensive care unit, including Specialty Education Coordinator for pediatric ICU, previous experience as an IRB member T Christine du Castel M.D. Scientific Medical Advisor; previously licensed to practice Non-affiliated Scientific members General Medicine in France Paid Consultant W Carolyn Hensler, B.S. (Physical Non- Quality Assurance and Quality Control Non-affiliated Non-scientific Chairman Education) scientific Administrator for CRO; previously employed as a Paid Consultant members Clinical Research Monitor; and Project Manager W Angelica Martinez, Co- CCRP Non- IRB Coordinator, administrative support for Full time Employee Non-scientific Chair scientific IntegReview members W Raymond Carr R.Ph. Scientific Staff Pharmacist Non-affiliated Scientific members Paid Consultant W Christopher P. Martin Pharm.D., M.S., Scientific Clinical Assistant Professor, Division of Non-affiliated Scientific members BCPS Pharmacotherapy UT; Clinical Pharmacy Paid Consultant Coordinator; Assistant Professor University of Oklahoma Health Sciences Center, College of Pharmacy W Robert A. Blum Pharm.D. Scientific Various experience as a principal investigator for Non-affiliated Scientific members research studies Paid Consultant Th Frederick Kopec, J.D., B.A. Non- Licensed, Practice of Law, Ethicist Non-affiliated Non-scientific Chairman (Philosophy) scientific Paid Consultant members Th Christina Willis, Co- B.S. (Clinical Scientific IRB Coordinator, various administrative support for Full time Employee Scientific members chair Research), CCRP IntegReview, including Quality Control; previously employed as Activate Specialist for CRO

Sponsor Name: The Center for Applied Health Sciences (Kent State University) Protocol Number: KSU-001 Principal Investigator Name: Scott Habowski Meeting Date: December 8, 2016

NAME DEGREES/ BOARD EXPERIENCE AFFILIATION ALTERNATE CERTS. POSITION WITH IRB FOR Th Michael Romain M.D. Scientific Licensed physician, Internal Medicine Non-affiliated Scientific members Paid Consultant Th Mary O’Connell Scientific Quality & Regulatory Affairs Manager; previously Non-affiliated Scientific members employed as Clinical Research Recruiter, Data Paid Consultant Associate, Coordinator and QC Auditor for CRO, IRB Administrator, Emergency Medical Technician Paramedic Th Matthew Pfeiffer Ph.D. Scientific Project Manager and CRA for CRO; Representative Non-affiliated Scientific members (Pharmacology & for first in human studies; clinical and research Paid Consultant Toxicology) experience; Pharmacology, Toxicology, CNS, Infectious disease, Metabolic/Endocrine Disorders, Oncology F Mary Ruwart, Chairman Ph.D. Scientific Research Scientist, Drug Delivery Systems, Non-affiliated Scientific members (Biophysics); Diabetes, GI Diseases, Drug Metabolism Paid Consultant B.S. (Biochemistry) F Tamara Britt, Co-chair B.S., M.A., CIP, Scientific IRB Coordinator, administrative support for Full time Employee Scientific members CCRP IntegReview; previously employed as CRA for CRO, Research/Regulatory Coordinator (Oncology, Psychiatry, Neurology, emergency medicine, military research) F Laurajo Ryan Pharm.D., MSc Scientific Clinical Associate Professor of Pharmacotherapy Non-affiliated Scientific members (Clinical UT Austin, Department of Medicine UTHSCSA, Paid Consultant Investigations), Clinical Pharmacist Specialist South Texas Veterans BCPS, CDE Administration

Sponsor Name: The Center for Applied Health Sciences (Kent State University) Protocol Number: KSU-001 Principal Investigator Name: Scott Habowski Meeting Date: December 8, 2016

NAME DEGREES/ BOARD EXPERIENCE AFFILIATION ALTERNATE CERTS. POSITION WITH IRB FOR F Dennis Brannon R.Ph., B.S. Scientific Clinical Research Consultant, Director of Pharmacy; Non-affiliated Scientific members (Animal Science) Executive Director Clinical Development; Paid Consultant previously employed as Senior Project Manager and CRA for CRO F Ashley Hutson Non- Previously volunteered as research subject in Non-affiliated Non-scientific scientific clinical trials; homemaker Paid Consultant members F Eduardo E. Sandoval M.D., MBA Scientific Clinical Research Consultant; previously employed Non-affiliated Scientific members (Healthcare as Medical Affairs/Medical Officer, Regional Senior Paid Consultant Administration) Clinical Research Associate, Quality Assurance Auditor, Clinical Research Manager Victoria Govea CCRP Non- Management support for IntegReview; former IRB Full time Employee Non-scientific scientific Coordinator, Co-chair for IntegReview members Lynn Goldman B.S. (Nutrition), Scientific Management support for IntegReview; former IRB Full time Employee Scientific members MSHP Coordinator, Co-chair; previously employed as (Healthcare Research Coordinator, Health Care Administration Administration), and Education, Clinical Nutrition, Registered RD, LD, CCRP Dietician/Certified Pediatric Nutrition Specialist Kimberly S. Cowley Ed.D., E.S., Non- Educational researcher/evaluator; Managing Non-affiliated Non-scientific M.A., B.A. scientific Evaluator; previously employed as Director of Paid Consultant members Research, Evaluation & Expertise, Executive Director, served as IRB member and Chair Christina H. Walker M.D., B.S. Scientific Licensed physician Non-affiliated Scientific members Paid Consultant Bennie C. Lopez MBA Non- Representative for adult and juvenile prisoner Non-affiliated Non-Scientific Scientific population; Teacher with Austin ISD Alternative Paid Consultant Learning Center; previously employed as Corrections Officer and retired military M Denotes regular Monday board members T Denotes regular Tuesday board members W Denotes regular Wednesday board members Th Denotes regular Thursday board members F Denotes regular Friday board members Other members are alternates All regular Board members can serve as alternates on other Boards as specified by their positions (e.g. Scientific for Scientific) Non-scientific members represent the general perspective of study participants Note: In addition to our regular members, we have access to specialists in therapeutic areas not represented on this roster. Page 5 of 6

Sponsor Name: The Center for Applied Health Sciences (Kent State University) Protocol Number: KSU-001 Principal Investigator Name: Scott Habowski Meeting Date: December 8, 2016

NAME DEGREES/ BOARD EXPERIENCE AFFILIATION ALTERNATE CERTS. POSITION WITH IRB FOR Jami Brackeen CST, CCRP Scientific Former IRB Coordinator, Co-chair, administrative Full time Employee Scientific members support for IntegReview; former Quality Control Associate for IRB Regulatory Compliance; Certified Surgical Technologist Rosa S. Sandoval B.S. (Chemistry), Scientific Former Senior IRB Coordinator, administrative Full time Employee Scientific members CCRP support for IntegReview; previously employed as Clinical Research Coordinator and Medical Research Assistant for University

APPENDIX C

IL-6 GENE VARIANT STUDY

SUBJECT #

Anthropometrics and Vital signs Weight: ______lbs ______kg Height: ______in. ______cm BMI: ______

BP: ______HR: ______Pregnancy test result: + - N/A (if + excluded from study)

Comments from staff: ______

Testing Day

Date: ______

Any changes in medical conditions? ______

Refrained from exercise for 48 hours prior to treatment? ______

Did subject consume minimal 48 oz of water 18hrs prior to visit?______

Did subject consume standardized breakfast and water prior to visit?______

SUBJECT #

ADVERSE EVENTS REPORT FOR PROTOCOL (PN-1051-001-2016)

Subject Number: ______Subject Initials: / / / None (check if no adverse events were experienced)

Document any non-serious or serious adverse events which occur since Screening Visit. Any other complaints or adverse events reported to CAHS within 72 hours of completing the study will also be captured on this form. SEVERITY GRADE: 1 = mild 2 = moderate 3 = severe but not life threatening 4 = life threatening 5 = death RELATIONSHIP TO STUDY PROCEDURE: 1 = Unlikely 2 = Possible 3 = Probable

TREATMENT ACTION TAKEN: 1 = None 2 = Medication 3= Procedure 4 = Medication & Procedure 9 = None of Above RELATIONSHIP TO STUDY PRODUCT: 1 = Unlikely 2 = Possible 3 = Probable 4 = N/A SERIOUS: 0 = No 1 = Inpatient Hospitalization 2 = Persistent or Significant Disability 3= Life Threatening 4 = Death 5 = Important Medical Event

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APPENDIX D

EXERCISE RESEARCH STUDY

Approved August 21, 2017 IntegReview IRB

Seeking endurance trained volunteers for research study measuring relationship between Interleukin-6 gene difference and endurance exercise.

Study Involves Exercise Testing free of charge includes… -Includes Body fat test, VO2 Max Test, and Genetic Test of IL-6 Gene. Body Fat Scan - Perform a simulated Duathlon VO2 Max Testing - 5km run, 20km Bike ride, & 2.5km run Genetic Testing - 3 blood samples taken over the course of duathlon

Contact Scott: 330-842-2472 –or- [email protected]

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APPENDIX E

INFORMED CONSENT

APPROVED BY INTEGREVIEW IRB AUGUST 21, 2017

THIS IS AN IMPORTANT DOCUMENT - KEEP FOR FUTURE REFERENCE VERSION CONTROL

INFORMED CONSENT DOCUMENT AGREEMENT TO BE IN A RESEARCH STUDY NAME OF SPONSOR: The Center for Applied Health Sciences PROTOCOL NUMBER AND TITLE OF STUDY: KSU-001; “EFFECTS OF THE IL-6 GENE POLYMORPHISM -174G/C ON INTERLEUKIN-6 PRODUCTION AND ENDURANCE EXERCISE PERFORMANCE” NAME OF PERSON IN CHARGE OF THE RESEARCH STUDY (INVESTIGATOR): Scott Habowski, Ph.D. (candidate) TELEPHONE NUMBER(S), DAYTIME: 330-926-6927 AFTER HOURS: 330-328-1870 INTRODUCTION You are being invited to volunteer for a research study. You must read and sign this form before you agree to take part in this study. This form will give you more information about this study. Please ask as many questions as you need to before you decide if you want to be in the study. You should not sign this form if you have any questions that have not been answered. The investigator is completing this research study in partial fulfillment of the requirements for his degree as a doctor of physiology. The investigator is not being paid to conduct this research study. You must be honest with the investigator about your health history or you may harm yourself by participating in this study. PURPOSE OF THE STUDY The purpose of this study is to assess the influence of your genetics on exercise performance. Specifically, the study will examine a single nucleotide polymorphism and how this affects production of the Interleukin-6 molecule and how this may possibly influence exercise performance. A “polymorphism” is a variation in the DNA that is present in at least 1% of the population. HOW LONG THE STUDY WILL LAST AND WHO MAY PARTICIPATE The study will last approximately 12 weeks and include 3 visits to the testing facility. Approximately 20 healthy men and women, ages 18-40, will participate in this study. TO BE IN THIS STUDY You cannot be in this study if you are in another research study or if you have been in any other research study in the last 30 days. Protocol number KSU-001 Scott Habowski, Ph.D. (candidate) Informed Consent Page 2 of 8 APPROVED BY INTEGREVIEW IRB AUGUST 21, 2017 THIS IS AN IMPORTANT DOCUMENT - KEEP FOR FUTURE REFERENCE VERSION CONTROL

Subject Responsibilities: While participating in this research study, you will need to: • • Be willing and able to follow the study directions and procedures • • Tell the study staff about any side effects or problems • • Ask questions as you think of them • • Tell the investigator or the study staff if you change your mind about staying in the study

You cannot be in this study if you: • • Have a history of cardiovascular disease or conditions. • • Have a history of malignancy in the past 5 years except for non-melanoma skin cancer (basal cell or squamous cell cancer of the skin). • • Have had prior gastrointestinal bypass surgery (Lapband, etc.) • • Have an autoimmune disorder (e.g., rheumatoid arthritis, Crohn’s disease, ulcerative colitis, Lupus, HIV/AIDS, etc.). • • Have a musculoskeletal injury or issues, which you feel, would prevent you from completing the event requirements. • • History of bleeding diathesis, platelet or coagulation disorders, or antiplatelet/anticoagulation therapy. • • Have a history of diabetes. • • Are a woman who is currently pregnant, planning to become pregnant, or breastfeeding.

WHAT WILL HAPPEN DURING THE STUDY Visit 1: Before the study starts, you will come to the testing facility for a screening visit. During this visit, you will be asked to read and sign this consent form. Study staff will provide you with an additional overview of the study requirements and testing procedures. You will answer questions about your health history. Study staff will review the inclusion and exclusion criteria for the study with you to ensure that you qualify for participation. Additionally, women who are able to have children will have a urine pregnancy test performed. If you qualify for participation in the study, you will be asked to return for Visit 2 approximately 7 days after Visit 1. Please refrain from exercise for 48 hours prior to Visit 2. Visit 2: At this visit, you will recall everything you ate in the last 24 hours and have your height, weight, heart rate and blood pressure measured. Then, a dual energy x-ray absorptiometry (DEXA) will be used to determine lean mass. This scan will use X-ray technology to assess the amount of fat, muscle, and bone that your body contains. Then, a 3 mL saliva sample will be collected to study your DNA. This is for the genetic testing portion of the study. Finally, you will have a test to measure the amount of air you can breathe into your lungs while exercising. For this test, you will breathe into a mask while exercising. After completing these tests, you will be given a standardized diet plan and scheduled to return for Visit 3 approximately 7 days later. Please refrain from exercise for 48 hours prior to Visit 3. Protocol number KSU-001 Scott Habowski, Ph.D. (candidate) Informed Consent Page 3 of 8 APPROVED BY INTEGREVIEW IRB AUGUST 21, 2017

Visit 3: The morning of Visit 3, you will consume a standardized breakfast according to your diet plan, and you will also be asked to consume a PowerBar Performance Energy Bar and a 20-ounce bottle of water. You must consume both the PowerBar and bottle of water 60 minutes prior to your scheduled time for Visit 3. At Visit 3, you will have your heart rate and blood pressure measured. Then, you will complete a duathalon. This includes a 5 kilometer run, 20 kilometer bike ride, followed by another 2.5 kilometer run. You will complete the duathalon as quickly as possible. Blood samples will be collected prior to the duathalon, following the bike phase, and upon finishing the duathalon. Heart Rate and Rate of Perceived Exertion will also be recorded at these times. Blood Samples: Blood samples will be collected at three specified time points mentioned in Visit 3. Blood samples will be taken by single needle-sticks. A total of about 15 mL (1 tablespoon) of blood will be drawn during the entire study. For comparison, the standard blood donation is about 480 mL (two cups). Genetic Sample: The collection of a saliva sample will be used to collect roughly 3 mL of volume. This will be required to test the single nucleotide polymorphism (SNP) in the IL-6 gene. The SNP is an alteration in one line of roughly 3 billion total base pairs in Human DNA. We will only be testing this SNP, no other tests will be done using your sample. All tests will be coded and the laboratory providing the genetic results will only receive your subject number with no other means of personal identification. However, if your test results got into the wrong hands, the confidentiality of your health information may be lost. The investigators will make every effort to make sure no one gets your test results except those you read about in this form. You will be given the option of finding your personal gene variant for the gene being tested if you desire. There are also current limited protections afforded to you by a US Federal law, the Genetic Information Nondiscrimination Act. (GINA), which generally makes it illegal for health insurance companies, group health plans, and most employers to discriminate against you based on your genetic information. All health insurance companies and group health plans must follow this law and all employers with 15 or more employees must follow this law. RISKS OR DISCOMFORTS If you do not understand what any of these side effects mean, please ask the investigator or study staff to explain these terms to you. You must tell the investigator or study staff about all side effects that you have. If you are not honest about your side effects, you may harm yourself by staying in this study. Protocol number KSU-001 Scott Habowski, Ph.D. (candidate) Informed Consent Page 4 of 8 APPROVED BY INTEGREVIEW IRB AUGUST 21, 2017 THIS IS AN IMPORTANT DOCUMENT - KEEP FOR FUTURE REFERENCE VERSION CONTROL

Blood Samples There may be side effects of having blood drawn such as: • • Fainting • • Redness • • Pain • • Bruising • • Bleeding • • Infection • • Blood clots, which may cause inflammation, swelling and pain

If you feel faint tell the study staff right away. Exercise: You may have some of following symptoms after exhaustive exercise: • • Difficulty breathing • • Faintness • • Fatigue

DEXA Scan: The DEXA scan (dual-energy x-ray absorptiometry) lasts about 10 minutes, and exposes you to less radiation than a standard chest x-ray (about the same amount of radiation exposure as taking a trans-continental flight). BIRTH CONTROL, DANGERS OF PREGNANCY AND BREASTFEEDING If you are a female, you must not get pregnant, plan to become pregnant, or breastfeed while in this study. The only certain way to not get pregnant is to not have sex. If you are a female and choose to have sex, you must use a type of birth control. Methods of birth control for this study include: abstinence, or a barrier type of birth control (e.g., condom with spermicide, diaphragm with spermicide). Please ask your study doctor if you have any questions regarding the birth control that should be used while in this study. Even if you use birth control during the study, there is a chance you could become pregnant. If you are pregnant or become pregnant during the study, the study procedures may involve unforeseeable risks to the unborn baby. POSSIBLE BENEFITS OF THE STUDY You will get no medical benefit from this study. Protocol number KSU-001 Scott Habowski, Ph.D. (candidate) Informed Consent Page 5 of 8 APPROVED BY INTEGREVIEW IRB AUGUST 21, 2017 THIS IS AN IMPORTANT DOCUMENT - KEEP FOR FUTURE REFERENCE VERSION

ALTERNATIVES TO PARTICIPATING IN THE STUDY Since this study is for research only, the only other choice would be not to be in the study. CONFIDENTIALITY Your records of being in this study will be kept private except when ordered by law. The following people will have access to your study records: • • The investigator • • Sponsor company or research institution [including monitor(s) and auditor(s)] • • Other country, state or federal regulatory agencies • • IntegReview IRB

The Institutional Review Board (IRB), IntegReview, and accrediting agencies may inspect and copy your records, which may have your name on them. Therefore, total confidentiality cannot be guaranteed. If the study results are presented at meetings or printed in publications, your name will not be used. IN CASE OF STUDY RELATED INJURY There are no plans to offer to treat or compensate you if you are injured or become ill while participating in this study. If you think you have a study related injury, please contact the investigator immediately. Please be aware that some insurance plans may not pay for research-related injuries. You should contact your insurance company for more information. LEGAL RIGHTS You will not lose any of your legal rights by signing this consent form. CONTACT INFORMATION If you have questions, concerns, or complaints about this study or to report a study related injury, contact the investigator: Scott Habowski, Ph.D. (candidate) Daytime: 330-926-6927 After hours: 330-328-1870 If you do not want to talk to the investigator or study staff, if you have concerns or complaints about the research, or to ask questions about your rights as a study subject you may contact IntegReview. IntegReview’s policy indicates that all concerns/complaints are to be submitted in writing for review at a convened IRB meeting to: Mailing Address: OR Email Address: Chairperson

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