THE EFFECTS OF AEROBIC AND RESISTANCE TRAINING ON INSULIN SENSITIVITY IN PRIMARY HUMAN SKELETAL MUSCLE CELLS by Christian Jones May 2023 Director of Thesis: Joseph Houmard, PhD Major Department: Kinesiology Insulin is a hormone that plays a role in the regulation of glucose homeostasis, through the regulation of blood glucose levels³⁶. Insulin sensitivity is defined as how well the body responds to insulin, and when this is impaired, it results in insulin resistance. Insulin resistance refers to a decrease in insulin-mediated glucose disposal in insulin-sensitive tissues². Impaired insulin action can result in insulin resistance, which can lead to illnesses such as cardiovascular disease, type 2 diabetes, obesity, and metabolic syndrome. PURPOSE: The purpose is to explore various training modalities and their effects on insulin action, with the intent to answer the question which mode of training, aerobics or resistance has a greater impact on insulin sensitivity. I speculate that the resistance exercisers will show greater improvements in insulin action, compared to the endurance and control groups. METHODS: To examine insulin action, glycogen synthesis was performed on primary human skeletal muscle cells that were derived from 3 groups: high active endurance, highly active resistance, and sedentary control. The samples underwent proliferation and were grown in a cell culture media incubator. After reaching 70-80% confluency, myogenic cells were isolated and differentiated. After cells went through five days of differentiation, they were ready for glycogen synthesis, which was determined using liquid scintillation counting. RESULTS: There were 11 participants in the highly active endurance group, 10 participants in the highly active resistance group, and 4 participants in the sedentary control group. The sedentary group had a greater BMI than the endurance and resistance groups. There was an increase in glycogen synthesis in all groups when stimulated with insulin (p < 0.0001). There was a significant difference between the highly active resistance and sedentary control groups, when fold change was calculated (p = 0.0060), with the change in the sedentary controls being greater than highly active resistance group. There were no significant correlations between the results and the group characteristics (age, BMI, and oxygen consumption). CONCLUSION: The present study showed that there was an increase in glycogen synthesis in all subjects, regardless of group, when comparing basal to insulin stimulated values. The results of this current study line up with results found in several others that have looked at aerobic trained individuals, however; they have failed to investigate resistance training. It is evident that resistance training has the same effects as aerobic training, but the topic needs to be further explored to confirm this finding. The Effects of Aerobic and Resistance Training on Insulin Sensitivity in Primary Human Skeletal Muscle Cells A Thesis Presented to The Faculty of the Department of Kinesiology East Carolina University In Partial Fulfillment of the Requirements for The Master of Science in Kinesiology Exercise Physiology Concentration Christian Jones East Carolina University May 2023 © Christian Jones, 2023 THE EFFECTS OF AEROBIC AND RESISTANCE TRAINING ON INSULIN SENSITIVITY IN PRIMARY HUMAN SKELETAL MUSCLE CELLS By Christian Jones APPROVED BY: DIRECTOR OF THESIS: Joseph Houmard, PhD COMMITTEE MEMBER: Nicholas Broskey, PhD COMMITTEE MEMBER: Chuck Tanner, MA COMMITTEE MEMBER: Donghai Zheng, PhD CHAIR OF THE DEPARTMENT OF KINESIOLOGY: J.K. Yun, PhD DEAN OF THE GRADUATE SCHOOL: Kathleen Cox, PhD Table of Contents List of Tables .......................................................................................................................... vi List of Figures........................................................................................................................ vii Chapter I: Introduction ............................................................................................................ 1 Background ......................................................................................................................... 1 Purpose and Question ........................................................................................................... 1 Hypothesis ........................................................................................................................... 2 Delimitations ........................................................................................................................ 2 Limitations........................................................................................................................... 2 Operational Definitions ......................................................................................................... 3 Chapter II: Review of Literature ............................................................................................... 4 Background: Chronic Health Conditions................................................................................ 4 Background: The Problem .................................................................................................... 5 Insulin Actions ..................................................................................................................... 6 Aerobic Training .................................................................................................................11 Resistance Training .............................................................................................................13 Muscle Cell Cultures............................................................................................................15 Conclusion ..........................................................................................................................18 Chapter III: Methods ..............................................................................................................19 Experimental Design ............................................................................................................19 Participants.........................................................................................................................19 Screening Assessments .........................................................................................................22 Analysis ..............................................................................................................................22 Statistical Analysis ...............................................................................................................24 Chapter IV: Results.................................................................................................................25 Subject Characteristics ........................................................................................................25 Insulin-Stimulated Glycogen Synthesis ..................................................................................25 Chapter V: Discussion .............................................................................................................27 Works Cited ...........................................................................................................................37 Appendix: Approval of Submission Letter ................................................................................43 List of Tables 1. Participant Characteristics……………………………………………………………….32 List of Figures 1. Glycogen Synthesis……………………………………………………………………....33 2. Fold Change……………………………………………………………………………...34 3. Glycogen Synthesis (Females Only)……………………………………………………..35 4. Fold Change (Females Only)…………………………………………………………….36 Chapter I: Introduction Background Insulin is a hormone that plays a large role in the regulation of glucose homeostasis. This is accomplished through the stimulation of glucose uptake in insulin -responsive tissues, mainly skeletal muscle³⁶. Insulin sensitivity is how well the cells respond to insulin¹. The more insulin sensitive someone is, the more effectively the cells take up glucose in response to insulin and the less amount of insulin is needed to lower blood glucose levels. Essentially, insulin resistance is decreased insulin-stimulated glucose uptake¹. This results in greater amounts of insulin needed to get glucose into the cells. There are multiple causes that could lead to this impairment including defects in glucose phosphorylation ¹. The primary location for glucose disposal is skeletal muscle, which was the source of samples used in this research. Insulin resistance can lead to chronic illnesses such as type 2 diabetes, obesity, and metabolic syndrome¹. Exercise is a tool that is often recommended for those who lack insulin sensitivity. Exploring and understanding how exercise affects the mechanisms of insulin sensitivity can provide exercise professionals with information needed to create prescriptions tailored to combat insulin resistance. Purpose and Question The purpose of this study was to explore various training modalities and their effects on insulin action, specifically the mechanisms of improving insulin sensitivity. The goal was to answer the question: which mode of training, aerobic or resistance, has a greater impact on insulin sensitivity? To directly determine insulin action, specifically glycogen synthesis, in skeletal 2 muscle, we examined glycogen synthesis in primary human skeletal muscle cells derived from muscle biopsies of the research subjects. Hypothesis The speculation is that the resistance exercisers will show greater improvements in insulin action, compared to the endurance and control groups due to the cells from those donors being larger. Delimitations Some delimitations for this study were: • Age range ❖ The age range for this study was 18 years and older. This is beneficial because it allowed for the study of samples from a larger population. • Health status ❖ To participate in this study, participants were required to be free of any serious health conditions. This assured that there are no underlying issues or mutations of the sample. Limitations Possible limitations for this study were: • Sample size ❖ The sample was made up of 25 tissue samples. This was not very representative of the population. • Cell model ❖ It cannot be guaranteed that the cell models represent everything in the donor. 3 Operational Definitions The main terms that needed to be defined were: • Insulin ❖ A peptide hormone secreted by the beta cells in the pancreatic islets of Langerhans. Insulin’s primary job is to regulate and maintain blood glucose levels. • Insulin resistance ❖ A decrease in insulin-mediated glucose disposal in insulin-sensitive tissues and increased hepatic glucose production. • Human skeletal muscle culture cells ❖ A model developed from satellite cells that have been isolated from a donor, proliferated into myoblasts, sorted, and differentiated into mature myotubes. • Sedentary ❖ Participants not engaging in any regular, structured endurance or resistance exercise more than one day per week that lasts on average more than 60 minutes in the past year. • Highly active endurance exercise ❖ Participants who participate in ≥ 240 minutes/week of endurance training for more than one year. • Highly active resistance exercise ❖ Participants who participate in resistance training of 3 or more upper and lower body groups at least 2 times per week for at least one year, utilizing a prescription sufficient to increase strength and muscle mass. Chapter II: Review of Literature Background: Chronic Health Conditions Obesity Obesity is a chronic disease that is increasing in epidemic proportions¹⁸. It is a medical condition due to a chronic imbalance between energy intake and energy expenditure¹⁴. Obesity- linked insulin resistance is primarily due to fatty acid overload in non -adipose tissues, specifically the liver and skeletal muscle¹². Severe obesity is increasing at a disproportionate rate compared to a milder grade of obesity¹⁸. Severe obesity is categorized by a body mass index (BMI) ≥ 40 kg/m2 and is associated with a decrease in insulin action and signaling, which in turn causes whole body insulin resistance. In the muscles of severely obese patients, with or without type 2 diabetes, there is little or no stimulation of glucose transport. In those with a BMI of about 30 kg/m2, there was a significant decrease in insulin responsiveness, and after which there was virtually no insulin-induced stimulation. Data has shown that in those with a BMI ≤ 20 kg/m2, glucose transport was stimulated by approximately 3-4-fold. Insulin stimulation of glycogen formation, glucose oxidation, and non-oxidized glycolysis are also depressed in muscle of those who suffer from obesity. Diabetes Type 2 diabetes is characterized by high plasma glucose levels and the tissues failure to respond to insulin¹⁴. The hyperglycemic effect of diabetes is associated with impaired carbohydrates, lipids, and protein metabolism with lack of insulin secretion or decreased sensitivity to the metabolic effects of insulin²². Insulin resistance is a hallmark of type 2 diabetes³. Insulin resistance impairs the ability of muscle cells to take up and store glucose, which leads to high levels of glucose in the blood²². In an insulin resistant state, target cells show 5 an attenuated response to insulin, which is compensated for by increased insulin secretion ³. Compared to lean individuals, the dose-response curve of insulin was gradually shifted to the right and flattened in patients with type 2 diabetes. A study showed that subjects with type 2 diabetes displayed a great degree of derangements in insulin sensitivity in skeletal muscle, along with a great degree of intramyocellular lipid accumulation ³⁴. Background: The Problem What is the problem? Insulin is a peptide hormone that is secreted by the beta cells in the pancreatic islets of Langerhans. The primary role of insulin is to regulate and maintain normal blood glucose levels, and this is achieved by facilitating cellular glucose uptake, regulating carbohydrates, lipids, and protein metabolism, and promoting cell division and growth³⁶. Insulin also promotes glycogen and lipid synthesis, while suppressing lipolysis and gluconeogenesis. Insulin sensitivity refers to how well the body responds to insulin. When this sensitivity is impaired, it leads to an insulin resistant state. Insulin resistance is defined as a decrease in insulin-mediated glucose disposal in insulin-sensitive tissues and increased hepatic glucose production². Peripheral insulin resistance refers to a lower capacity to utilize glucose in skeletal muscle, which negatively affects glucose transport and glycogen synthesis. This impairment leads to hyperglycemia and hyperlipidemia, and the lack of insulin leads to protein-wasting, ketoacidosis, and ultimately, death. Why is insulin sensitivity important? Insulin sensitivity is important because when the body does not respond to insulin, it can be the underlying cause of multiple chronic illnesses, such as Type 2 diabetes and obesity. According to The Centers for Disease Control and Prevention, approximately 30.3 million 6 American have Type 2 diabetes and 84.1 million are prediabetic²⁶. Obesity is a global epidemic that is continuing to rise relentlessly, affecting over 2 billion people, which is approximately 30% of the world population¹º. The Global Burden of Disease Group reported in 2017 that “since 1980, the prevalence of obesity has doubled in more than 70 countries and has continuously increased in most other countries.” Insulin resistance is an underlying cause of both issues; therefore, it is imperative to scientifically explore this topic in detail. Finding effective ways to combat and prevent insulin resistance could prove beneficial to help reduce these chronic issues. How does the literature relate to the problem? The literature being reviewed relates to the problem because it not only discusses the problem, but ways to combat it. The role of physical activity has been shown to increase insulin effectiveness in skeletal muscle²⁵. There seems to be an effect of exercise on insulin sensitivity that depends on various factors such as mode, intensity, and frequency of training. The literature being reviewed discusses how exercise, specifically aerobic and resistance, can affect insulin sensitivity, along with ways to evaluate these changes. Insulin Actions Insulin’s role Insulin acts to regulate glucose homeostasis by stimulating glucose uptake in insulin - responsive target tissues such as adipocytes, and skeletal and cardiac muscle, as well as by suppressing hepatic glucose production¹⁵. Insulin stimulates the uptake of glucose by increasing the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Insulin also suppresses lipolysis, resulting in a decrease in circulating free fatty acid levels, which may mediate the action of insulin on hepatic glucose production. 7 This hormone has other metabolic effects to increase storage of lipids and proteins as well as to promote cell growth and differentiation. The stimulation of cell growth and differentiation using insulin is done through the MAPK-ERK pathway, which is activated by insulin receptor- mediated phosphorylation of Shc or IRS¹⁵. The non-metabolic effects of insulin have been described in non-classical insulin-responsive organs as having increased pancreatic ꞵ-cell survival, endothelium-dependent vasodilation, and renal sodium transport³. Insulin has also been shown to influence brain function to regulate glucose metabolism and food -seeking behavior³. The journey though the body The journey of insulin throughout the body can be broken down into five stages, starting with biogenesis in the pancreas and ending with degradation in the kidneys³³. To begin, insulin is transcribed and expressed in the ꞵ-cells of the pancreas. Humans have a single insulin gene, INS, that is located on chromosome 11 and is controlled by numerous enhancer elements ³³. These enhancer elements are required for the expression of the insulin gene and contribute to the regulation of INS transcription in response to glucose and autocrine insulin signaling. After leaving the pancreas, the portal vein delivers insulin to the liver³³. Because it is the first stop, the liver is exposed to higher concentrations of insulin than other insulin -responsive tissues. The delivery of insulin to the liver occurs in discrete pulses roughly every 5 minutes. This pulsatile insulin delivery system is an important physiological signal that regulates both hepatic insulin action and insulin clearance. Insulin clearance is a process that regulates th e amount of insulin reaching peripheral tissues. The concentration of insulin arriving to the liver via the portal vein can be up to 10-fold higher than the concentration in the peripheral circulation, and the maintenance of this portal-systemic gradient is mediated by the substantial 8 insulin degradation by the liver. Approximately 50-80% of insulin arriving to the liver via the portal vein is degraded during the first-pass hepatic clearance, and ~25% of the circulating insulin is degraded upon the second pass through the liver, so that the circulating concentration of insulin is one third of that in the portal circulation. Portal circulation delivers insulin to the capillaries of the sinusoids, which are not supported by the basement membrane and their endothelial cells contain fenestrations, together permitting the exchange of contents between the blood and the surrounding liver cells. This allows the insulin to easily diffuse out of the circulation and into the perisinusoidal space, where it encounters the hepatocytes. Insulin that is not degraded in the liver exits through the hepatic vein, reaching the heart, which pumps insulin into the arterial circulation to be delivered to its target tissues³³. The peripheral actions of insulin begin inside the vessels of the systemic circulation. Here insulin exerts its hemodynamic effects on endothelial cells to promote blood flow and ensure its delivery to the peripheral tissues³³. Insulin crosses the blood-brain barrier through a receptor-mediated process and begins its action on the central and peripheral tissues. The hormone regulates appetite, exerts trophic and developmental actions on neurons and glial cells, modulates cognition, memory, and mood. Insulin acting centrally also evokes efferent inputs into peripheral tissue metabolism, which contributes to gluconeogenesis suppression in the liver. It also contributes to thermoregulation by activating heat-liberating mechanisms in adipose tissue. Stage four of insulin’s journey occurs in the muscle and adipose tissues. Here, the major role of insulin is to increase the uptake of carbon sources and store them for energetic needs of tissue³³. The main job of insulin is to regulate glucose uptake, which is done through the process of GLUT4 translocation, a series of signals that cooperate in bringing glucose transporters to the cell surface. GLUT4 translocation takes place rapidly after the binding of insulin to its receptors 9 of the myocytes and adipocytes and does not involve internalization of the hormone. Insulin binds to its receptor on the surface of muscle or fat cells and activates the canonical insulin - signaling cascade to PI3K and Akt. Downstream of Akt, phosphorylation of AS160 allows for the full activation of Rab8A and Rab13, located in the muscle cells, and Rab10, located in the adipocytes. Simultaneously, downstream of PI3K, insulin leads to activation of Rac1 that promotes a dynamic cycle of cortical actin remodeling. Together, these actions tether GLUT4 vesicles to the actin cytoskeleton near the plasma membrane. After about 30 minutes of its initial release from the pancreas, insulin is no longer detectable in circulation and its half -life while in circulation is approximately 6 minutes³³. In addition to clearance in the liver, insulin is slowly internalized by most cells, including myoblasts and adipocytes, where it is routed to lysosomes for degradation; this only accounts for a fraction of insulin destruction. Most of the degradation of the circulating hormone remaining after the second pass through the liver occurs when it enters the kidney. In the kidneys, there are three ways to dispose of insulin. First, it enters the luminal space and reaches the proximal tubule, where it is rapidly reabsorbed by the renal epithelial cells³³. Second, an equal amount also enters the renal tubular cells from the contralumial side facing the renal peritubular capillaries. Here, the insulin receptors on the epithelial cells bind insulin and transport it intracellularly for degradation. Lastly, a small fraction is reabsorbed back to the renal circulation through retroendocytosis. Insulin Sensitivity: what does it look like in the body? Insulin sensitivity is a term that refers to how well cells respond to insulin. The higher the levels of insulin sensitivity, the more effectively the cells transport and use glucose. When 10 insulin sensitivity levels are normal, the body requires small amounts of insulin to lower blood glucose levels. When insulin sensitivity levels are low, this means the body isn’t responding to insulin production, which results in an insulin resistant state. Insulin resistance: what does it look like in the body? When insulin sensitivity becomes impaired, it is referred to as insulin resistance. Poor insulin sensitivity is characterized by impaired insulin action on whole-body glucose uptake⁷. This results in elevated blood glucose, impaired glycemic control, and a risk of pancreatic ꞵ cell failure. Those who suffer from insulin resistance require greater amounts of insulin to get glucose into the cells²⁶. Skeletal muscle is the major site for glucose digestion, but in an insulin resistant state, insulin stimulated glucose disposal is impaired¹. This impairment is due to impaired insulin signaling and multiple postreceptor intracellular defects including impaired glucose transport and phosphorylation, and glucose oxidation and glycogen synthesis. At th e cellular level, a common feature of insulin resistance is the presence of increased lipid accumulation in insulin-responsive tissues³⁴. When adipose tissue capacity to store lipids is sub- optimal, lipids are ectopically stored in the skeletal muscle and liver and this ectopic fat disrupts substrate utilization in these tissues, leading to a decrease in insulin sensitivity ³⁴. In an insulin resistant state, the cellular response to insulin is reduced and the activation of insulin signaling pathways requires increased concentration of insulin³. Insulin resistance can lead to health conditions such as: Type 2 diabetes, metabolic syndrome, dyslipidemia, hypertension, PCOS, non-alcoholic fatty liver disease, cancer, and obstructive sleep apnea³⁶. Insulin resistance has also been discovered in insulin-responsive organs including the liver, skeletal muscle, and white adipose tissue³. 11 Gender and age differences Falkner et al. have established that lower insulin sensitivity is more prevalent in young women than in young men; however, there was no difference when insulin-mediated glucose utilization was corrected for body fat. It has also been established that there is significantly higher peripheral insulin sensitivity and significantly lower insulin-receptor binding in females as compared with males⁹. There have been studies that show significantly higher insulin sensitivity in females in the follicular phase of the menstrual cycle and lower in the luteal phase. A study conducted by JM Escalante Pulido and M. Alpizar Salazar resulted in the opposite: fewer insulin-receptors binding and decreased receptor numbers in females during the follicular phase⁹. Certain sex hormones; estriol, estradiol, and estrone; have been shown to induce a state of insulin resistance⁹. There is a slight decrease in peripheral insulin sensitivity with ageing in both males and females, with the reduction in females being more prominent. Despite the decrease in insulin sensitivity, females have an increase in insulin-receptor binding with age, while males have a decrease. It is thought that this increase is most likely counterbalanced by the unfavorable effect of the increasing androgen levels on post-receptor processes in insulin action. Aerobic Training Definition Aerobic training is characterized by many contractions performed with a development of a relatively low force¹⁴. Aerobic training increases the capacity of muscle for aerobic metabolism, promoting its adaptation toward a more oxidative phenotype. Aerobic training also leads to fiber-type transformation, mitochondrial biogenesis, angiogenesis, and other adaptive 12 changes in the skeletal muscle. These changes are what allow previously untrained people to increase their ability for prolonged periods of exercise. Effects of aerobic training on insulin sensitivity/resistance There have been many studies done that evaluate the effects of aerobic training on insulin sensitivity and resistance. One study that looked at the effects of moderate to intense levels of aerobic activity discovered greater differences in insulin sensitivity in the exercise group compared to the control group²⁵. Another study was conducted that involved a 6-month aerobic protocol involving various pieces of cardio equipment³º. The results indicated an increase in insulin sensitivity in overweight and obese sedentary women with a history of gestational diabetes and type 2 diabetes. For this reason, it is thought that endurance training greatly improves insulin sensitivity. Aerobic training and obesity A study examined the relationship between aerobic exercise and insulin action in severely obese individuals¹⁸. It was found that exercise training improved insulin action through reduced fasting and two-hour insulin concentrations as well as the insulin area under the curve during the oral glucose tolerance test. A study conducted by D. De Strijcker et al. (2018) showed that a 10 - week high intensity training (HIT) intervention was effective in improving insulin sensitivity in overweight and obese males. This study also revealed a significant negative association between changes in basal respiratory exchange ratio and changes in oral glucose tolerance test ¹³. This shows that when basal respiratory exchange ratio is decreasing, lipid utilization and oxidation is more effective, which increases insulin sensitivity. 13 Aerobic training and aging A single bout of aerobic exercise significantly improved endothelial function and insulin - induced vasodilation in older subjects¹⁶. Aerobic exercise also improved the anabolic response of skeletal muscle protein synthesis to insulin in healthy older subjects. It was found that aerobic training improved insulin signaling; this was related to higher regional and whole-body insulin- stimulated glucose uptake. These findings are consistent with previous reports indicating that a single bout of aerobic exercise improves insulin sensitivity ¹⁶. Another study reported that acute aerobic exercise can restore anabolic sensitivity to insulin in older adults ²¹. This study showed that aerobic exercise training increases skeletal muscle mass; therefore, it is effective for counteracting muscle loss in aging adults. Two studies showed that the change in insulin - stimulated glucose disposal was similar between elderly endurance trained and younger endurance trained groups, as well as between the elderly sedentary and younger sedentary controls²⁸. Resistance Training Definition Resistance training consists of a small number of contractions (typically 10 -20) with development of a relatively high force¹⁴. This type of training typically results in hypertrophy of muscle cells, accompanied by an increase in strength without major changes in the biochemical makeup. The muscle gain is mainly due to an increase in the number of myofibrils, where the fast fiber types are mostly responsible for the net increase in muscle size. Effects of resistance training on insulin sensitivity/resistance 14 While there is a substantive amount of research regarding the effects of aerobic training on insulin sensitivity, there is not much on the effects of resistance training on insulin sensitivity. A few investigations have shown an inverse, linear relationship between strength training and insulin resistance²⁶. It is thought that this relationship is a result of the mass effect, which suggests that the muscle hypertrophy gained from strength training increases insulin sensitivity. Some research also indicates that differences in waist circumferences have a significant influence on the relationship between strength training and insulin sensitivity. Molsted et al. conducted a 16-week resistance intervention that resulted in significant improvement in glucose tolerance in patients with type 2 diabetes⁷. The findings of Black et al. supported resistance exercise as a modality for those with impaired fasting glucose. It was also suggested that high -intensity, multiple set resistance exercise programs are beneficial for insulin sensitivity ⁸. This muscle hypertrophy leads to an increase in the amount of skeletal muscle tissue and GLUT 4 receptors, which in turn increases insulin sensitivity²⁶. Resistance training and obesity Obesity-linked insulin resistance is due to fatty acid overload in non-adipose tissues, particularly skeletal muscle¹⁴. While resistance training doesn’t necessarily result in weight loss the way aerobic training does, it does result in significant changes in body composition. Resistance training induces significant gains in strength and lean body mass, which leads to changes in percent body fat³⁷. It has also been shown that moderate to high intensity strength training can lead to marked gains in muscle hypertrophy¹⁴. Another study done involving circuit resistance training concluded that resistance training improved body composition as well as insulin sensitivity among inactive obese men²º. Overall, there is little research that examines the 15 effects of resistance training and even less looking at resistance training and its relationship with obesity. Resistance training and aging The effects of resistance training and aging have received noticeably less attention than the effects of aerobic training and aging. Despite this, the American College of Sports Medicine stated that resistance training is a safe and effective intervention for older adults. In the past, resistance training has been linked to improvements in whole-body insulin sensitivity in older adults¹². A study that involved elderly individuals performing moderate-intensity resistance training for 3-6 months, found an increase in whole-body insulin sensitivity by ~10-30%¹². This is a direct result from an increase in skeletal muscle glucose uptake. A separate study showed that a 4-month resistance training program normalized insulin-stimulated glucose uptake in the vastus lateralis in older adult women. A study conducted by Miller et al. concluded that a 16 - week resistance training program led to an increased insulin-stimulated nonoxidative glucose disposal by 40%; increasing whole-body sensitivity by 22%¹². This suggests that resistance training could improve skeletal muscle glycogen metabolism¹². Muscle Cell Cultures Biopsies – collection process The percutaneous or ‘semi-open’ biopsy technique is one that is used by both researchers and clinicians because it is safe and highly effective. The percutaneous biopsy technique is done using the Bergstrӧm needle to obtain skeletal muscle tissue samples f rom the vastus lateralis, as well as other muscle groups. The needle consists of an outer cannula with a small opening at the side of the tip and an inner trocar with a cutting blade at the distal end. After numbing the area 16 with local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the outer trocar, the inner trocar is pulled back, skeletal muscle is drawn into the opening of the outer cannula by the suction, and the inner trocar closes rapidly, which cuts and clips the skeletal muscle tissue sample. When locating the incision site, it is important to note that the vastus lateralis is more anterior and proximal when contracted than when it is relaxed. Gauging skinfold thickness helps the operator estimate the amount of tissue superficial to the fascia; information that is crucial in the biopsy process³². The percutaneous biopsy method is a strong method to use because it is stra ightforward and there are minimal risks³². The primary site for the muscle biopsy is the skeletal muscle from the vastus lateralis⁶. This site is ideal for creating human skeletal muscle cells (HSMC) because skeletal muscle is the major site for disposal of ingested glucose¹. Skeletal muscle comprises approximately 45% of the body mass and is responsible for approximately 75% of the glucose disposal after a meal¹⁸. Based on these findings, insulin resistance could possibly remain expressed in HSMC⁶. It was first seen in the 1990s that HSkMCs from individuals with type 2 diabetes exhibited impaired insulin-stimulated glucose transport and decreased glycogen synthase activity³⁸. One advantage of using human skeletal muscle cell cultures in experiments is that cell cultures can be developed from the small amount of human skeletal muscle. Another advantage of using HSkMC is that it retains some metabolic characteristics of the donor. Cultures that are derived from human skeletal muscle may thus offer the possibility of studying innate characteristics of human metabolism. The resemblance between HSkMC and the in vivo condition in relation to the actions of insulin shows that HSkMC offers a model to study the 17 mechanisms of insulin resistance with type 2 diabetes. Human skeletal muscle cell cultures have been used to help discern the characteristics of insulin mediated glucose uptake in skeletal muscle and study potential defects that may be present with type 2 diabetes. Decreased glycogen synthesis has also been reported in HSkMC obtained from patients with diabetes⁵. It has been discovered that lipid metabolism is reduced in the skeletal muscle of those who suffer from diabetes and obesity. When human skeletal muscle cell cultures are incubated with pharmacological agents, they can provide insight into the mechanisms by which these treatments can improve and/or insulin action⁵. Insulin action in HSkMC and exercise A recent article was published on a study that looked at the effects of aerobic training on mitochondrial respiratory capacity in human skeletal muscle stem cells³⁸. They previously found that HSkMCs from individuals with type 2 diabetes had significant improvements in carbohydrate-supported respiratory capacity after 10 weeks of aerobic training. In the current study, the team compared lean active individuals and sedentary obese individuals at baseline with lean/overweight sedentary pre-training individuals³⁸. They then compared the HSkMCs of lean/overweight sedentary individuals before and after 18 days of aerobic training. The researchers found that aerobic training consistently improved skeletal muscle metabolic flexibility, mitochondrial capacity, and insulin action ³⁸. Bourlier et al. found that 12 weeks of aerobic and strength training resulted in significant increases in fatty acid oxidation in HSkMCs from overweight individuals³⁸. There have been no studies examining the effects of resistance training on insulin action in HSkMC. 18 Conclusion Studies summary The literature reviewed discusses the effects of exercise on insulin action. While it is unclear which is more beneficial, aerobic or resistance training, it is known that multiple bouts of exercise yield more benefits than single bouts¹⁷. A single bout of exercise increases skeletal muscle glucose uptake, but this is short-lived and disappears after 48 hours¹⁷. In contrast, repeated physical activity results in a consistent increase in insulin action in skeletal muscle ¹⁷. Studies have also shown that exercise training may be the key factor necessary for stimulating mitochondrial biogenesis in insulin resistance³⁴. While some research has been done regarding insulin action and exercise, most of it focuses on aerobic exercisers. There is still little known about insulin action in life long exercisers, particularly resistance exercisers. Research Possibilities The MoTrMyo study focuses on insulin activity in healthy individuals who have previously gone through aerobic or resistance training. This study also partners with the MoTrPac study to look at changes in insulin activity in healthy sedentary individuals. The goal of both studies is to determine how beneficial exercise is to improve insulin sensitivity. Chapter III: Methods Experimental Design Subjects for this study were a part of the “Investigating the effects of aerobic and resistance training in vivo on skeletal muscle metabolism in vitro in primary human muscle cells” study (MoTrMyo), which is a sub study of the Molecular Transducers of Physical Activity Consortium (MoTrPAC). MoTrPAC is a nationwide study that recruits healthy individuals with the intent to explore how exercise improves health. Samples for this study were collected from multiple clinical sites including: the AdventHealth Translational Research Institute, Duke University, the Pennington Biomedical Research Center, the University of Colorado, and the University of Texas Health Science Center. Human skeletal muscle cell cultures were derived from a total of 25 healthy subjects, split into three groups: 11 highly active endurance, 10 highly active resistance, 4 sedentary controls. Samples were matched across groups for age, race, and gender. Samples were also selected for various oxygen consumption values (VO2max) and BMI. Using radioisotopes, insulin sensitivity was determined in HSkMC. This was done under two conditions: with and without insulin stimulation. Participants Basic Criteria Gender, race, and ethnicity Men and women are eligible. All races and ethnic groups are eligible for the study. For the overall study of sedentary participants, randomization is stratified by sex with the goal of recruiting >30% minority and 25-40% in each of three age bins: 18-39, 40-59 and 60+. 20 Age Participants must be 18 years of age or older to be eligible. There is no upper age limit for eligibility. If a participant meets all eligibility and health screening criteria, they are invited to participate. If potential participants are under the age of 18, they can be rescreened at age 18. Weight Participants must have a BMI between 19 and 34.9 kg/m², calculated from their height and weight measurements. Participants over 34.9 kg/m² pose possible concerns with weight limits on equipment as well as concerns with being able to obtain adequate muscle and adipose tissues during biopsies. Inclusion Criteria Sedentary participants To be considered sedentary, participants should not be engaging in any regular, structured, endurance or resistance exercise that lasts on average more than 60 minutes more than one day per week in the past year, which could result in increased heart rate or rapid breathing, muscle fatigue, and/or sweating. Highly active endurance exerciser (HAEE) To be considered for the HAEE group, participants must participate in ≥240 minutes/week of endurance training for more than a year. This can include running, brisk/power walking, cycling, elliptical etc., which results in increased heart rate, rapid breathing, and sweating. • Must include cycling at least 2 days per week for at least 120 minutes. 21 • Resistance training in the past year must be no more than 2 days per week and involve no more than 2 groups of upper body resistance exercise, and no more than 2 days per week of lower body resistance exercise. Highly active resistance exerciser (HARE) To be considered for the HARE group, participants must participate in resistance training of 3 or more upper and lower body groups at least 2 times a week for at least a year, utilizing a prescription sufficient to increase strength and muscle mass. • Endurance training in the past year must be less than 90 minutes per week of moderate- to-vigorous endurance exercise. Exclusion Criteria Participants were excluded from the study if they had any serious medical conditions such as: diabetes, elevated blood pressure, or cardiovascular conditions. These cardiovascular conditions include congestive heart failure, coronary artery disease, significant valvular disease, congenital heart failure, or symptomatic peripheral artery disease. They were also excluded if they consumed excess amounts of alcohol or used tobacco products. Participants were also excluded from the study if they were suffering from any mental impairments or are taking any serious medications. Groups Muscle samples were collected from three groups: HAEE, HARE, and sedentary controls. 22 Screening Assessments Anthropometric Measures Height, weight, and waist circumference were used to assess the body composition of participants. Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer. Weight was measured to the nearest 0.1 kg using a digital scale. Waist circumference was measured to the nearest 0.1 cm using a Gulick tape measure. Height and weight were used to determine body mass index (BMI). Dual energy x-ray absorptiometry (DXA) The DXA scan was also used to assess body composition, but in more detail. This scan assessed fat, lean and bone mass, from which percent fat is derived. This was done using a Hologic Horizon A DXA machine (Hologic Horizon A, Marlborough, MA). Cardiopulmonary exercise test (CPET) The CPET was performed using a Lode Excailbur Sport electrically braked cycle ergometer. Oxygen consumption (VO2max) was measured using Provomedics True One metabolic measuring system (TrueOne® 2400 MMS, Parvomedics, Salt Lake City, UT). A 12 - lead ECG was used to monitor heart rhythms and blood pressure was monitored by a lab assistant. Analysis Skeletal muscle biopsies Muscle tissue samples were obtained with the needle biopsy technique from the quadriceps (vastus lateralis). A tissue sample of 100 mg was consistently obtained with a double 23 pass from a single insertion. Muscle was obtained in the fasting state for biochemical analyses and for deposition into the MoTrPAC repository. All biopsies were performed by IRB-approved personnel at each of the participating sites. All tissue/samples were coded according to the MoTrPAC protocol (i.e., subject numbers will be identical). For this study, control subjects will undergo one pre-intervention muscle biopsy, while HAEE and HARE subjects will also undergo one muscle biopsy. Human skeletal muscle cell culture Samples used for this study were Passage 2 cells, obtained from different clinical MoTrPac sites. They have gone through the culture process and been cryopreserved. Once we received these samples, they were seeded and underwent the proliferation process, in which they were raised up in culture media and incubated in a cell culture incubator (37℃, 5% CO2). During the proliferation process cells started growing in a collagen coated T25 flask and treated with 16% FBS growth media (500 mL low glucose DMEM supplemented with 16% HI-FBS, 0.1% 50 mg/mL gentamicin, 0.1% 1 uM Dexamethasone, 0.1% human EGF, and 0.02% 250 ug/mL Amphotericin B). Upon reaching 70-80% confluency, cells were split and transferred into two collagen coated T75 flasks and treated with 10% FBS growth media (500 mL low glucose DMEM supplemented with 10% HI-FBS, 0.1% 50 mg/mL gentamicin, 0.1% 1 uM Dexamethasone, 0.1% human EGF, 0.02% 250 ug/mL Amphotericin B). Once cells reach ~70- 80% confluency, they underwent sorting, which ensures that experimenters only worked with muscle cells. During the sorting process, the CD56- cells were separated from the CD56+ cells, which was the focus. Next cells went through differentiation, which is when myoblasts mature into myotubes. During this process, cells were incubated for 5 -6 days in differentiation media 24 (500 mL DMEM low glucose pyruvate, 0.2% BSA, 2% penicillin/streptomycin). After 5-6 days of differentiation, the myotubes are ready to start the experiment process. Insulin-Stimulated Glycogen Synthesis Insulin-stimulated glycogen synthesis was measured in cells as an index of insulin sensitivity. Myotubes underwent 3 hours of serum starvation, 2-hour incubation with 14C- glucose, two washes with DPBS, and then were lysed with 0.5% SDS. An aliquot of the lysate was combined with carrier glycogen (1mg) and denatured at 100°C for an hour. Ice-cold ethanol was then added to the denatured lysates, and samples were spun overnight at 4°C for glycogen precipitation. On day 2 of the experiment, glycogen pellets were centrifuged at 11,100g for 15 minutes at 4°C, washed with 70% ethanol, and centrifuged again. The glycogen pellets were then resuspended in dH2O, and the incorporation of 14C-glucose into glycogen was determined with liquid scintillation counting. Statistical Analysis A repeated measures two-way ANOVA analysis was used to determine statistical significance. Factors used were groups (sedentary control, HAEE, and HARE) and condition (basal and insulin stimulated). Statistical significance was set at 𝑝 ≤ 0.05. When significance was found, a post-hoc analysis was done to see where the differences occurred. To normalize individual differences to basal conditions, a one by three-fold change was calculated by computing the ration of the change between the insulin condition and the basal condition. Chapter IV: Results Subject Characteristics Highly active endurance and resistance exercisers were matched with sedentary controls by age and gender. As shown in Table 1, the sedentary controls had higher BMIs and lower relative oxygen consumption values than both the highly active groups. Insulin-Stimulated Glycogen Synthesis Statistical significance was set at 𝑝 ≤ 0.05. Glycogen synthesis increased in all groups with insulin stimulation (p < 0.0001) compared to basal conditions (Fig. 1). There was a significant increase in glycogen synthesis within the HAEE (p = 0.0028) with insulin stimulation compared to basal conditions (Fig. 1). This significant increase was also seen with the sedentary controls (p = 0.0215; Fig. 1). The increase in glycogen synthesis in the HARE group had a greater significance than the other two groups (p < 0.0001; Fig. 1). There was no significance between basal glycogen synthesis levels with the groups. Glycogen synthesis data was also reported as fold changes (Fig 2). There was an overall significance in the treatment effect (p = 0.0545; Fig 2), in this case insulin stimulation. When taking a deeper look, there was no significant difference between the HAEE and HARE groups; however, there was a significant difference between the HARE and sedentary control groups (p = 0.0060; Fig 2). Data was also analyzed looking only at female participants. While there is an overall treatment effect with insulin stimulation (p < 0.0001), there is no significant increase between basal and insulin conditions within the groups (Fig. 3). Fold change was also looked at only in female subjects, which showed a significance in the treatment effect (p = 0.0112; Fig. 4). There was no significance between the HAEE and HARE groups (Fig. 4); however, there was a 26 significant difference between the HAEE and SED groups (p = 0.0377), as well as the HARE and SED groups (p = 0.0101). When looking at the correlations between the rate of glycogen production and the variables listed in Table 1, there were no significant relationships. Chapter V: Discussion Samples for this study were a part of the MoTrMyo study and received from multiple sites from across the country. Subjects were matched for age and gender. Table 1 shows that the subjects in the sedentary control group had higher BMIs and lower oxygen VO2max values compared to the other groups. They all underwent muscle biopsies, which were used to establish primary human skeletal muscle cultures. These cultures retain the same characteristics as their donors, which allowed researchers to study the innate characteristics of human metabolism. The current study showed that there was no significant difference in insulin stimulated glycogen synthesis between the exercise trained groups. The subjects had similar basal level glycogen synthesis and when stimulated with insulin, all the groups saw a significant increase in glycogen synthesis (Fig 1). This shows that insulin stimulation increases glycogen stores, regardless of training mode. With insulin stimulation, there was about a 1.5 -fold increase in glycogen synthesis. With fold change, there was a greater increase in the sedentary control group when compared to the HAEE and HARE groups (Fig 2). The basal value for glycogen synthesis tended to be lower in the sedentary group when compared to the HAEE and HARE groups, which may explain the fold-change differences. These results could also be related to the fact that there are only four subjects in the sedentary group, compared to the 11 subjects and 10 subjects in the endurance and resistance groups, respectively. Because the sedentary group is only made up of female subjects, glycogen synthesis was also analyzed for all female subjects. There was an overall significant increase in insulin stimulated glycogen synthesis when compared to basal conditions, but not within the groups (Fig. 3). Like the original results (Fig. 2), there was also a 1-5-fold increase in glycogen synthesis among the female subjects (Fig. 4). Also like the original data there was no significance between the HAEE and HARE. There was, 28 however, a significant difference between the HAEE and sedentary groups and between the HARE and sedentary groups (Fig. 4). These results can possibly be attributed to the difference in sample sizes, as the sedentary group has less participants. There was a study done looking at the effects of resistance and endurance training on whole body insulin sensitivity in young nonobese women, that found an increase with both groups²⁹. The increase was greater in the endurance group, but this was seen when insulin sensitivity was expressed on an absolute basis or indexed per kg fat-free mass²⁹. A study comparing the metabolic effects, including insulin sensitivity, of aerobic and resistance training in type 2 diabetic subjects, found that there was similar efficacy between both training modalities. The researchers found that insulin sensitivity significantly increased by ~30% in the aerobic group and ~15% in the resistance group, with no statistical significance between the two groups⁴. These results were compared to two other studies done by Sigal et al. and Church et al. that looked at HbA1c as a measure of whole-body insulin sensitivity, and both had similar outcomes. Church et al. conducted a study looking at effects of aerobic and resistance training on hemoglobin A1c levels in patients with type 2 diabetes. The researchers found that neither the resistance training nor the aerobic training groups saw significant changes in HbA1c levels, when compared to the control group. The study also had a combination group, wh ich combined both aerobic and resistance training, and when compared to the control group there was a significant decrease in HbA1c levels¹¹. The findings of these two studies seemingly suggest that maybe training volume has a greater impact on insulin sensitivity, rather than mode. Park et al. conducted a study using electrical pulse stimulation in myotubes to simulate muscle contraction, with the aim of investigating insulin action in lean and obese individuals. The research team found that electrical pulse stimulation of primary human skeletal muscle 29 myotubes improved insulin action in both lean and severely obese subjects ²⁷. These findings show that exercise influences insulin sensitivity in skeletal muscles due to the contraction of the muscle. With that being said, the study fails to identify which type of exercise has a greater impact. Bergstӧrm and Hultman used one legged cycling (using the non -exercised leg as a control) performed to exhaustion to examine glycogen synthesis in the quadriceps. They took biopsies in both legs immediately after exercise and each day after for 3 days. It was reported that by day 3, the glycogen synthesis of the exercised leg was twice that in the control leg. Because the glycogen synthesis in the control leg was seemingly unchanged, the results were attributed to some effect of exercise in the exercises muscle ³⁵. The results show that there is some effect of exercise training, at least with aerobic exercise. Since this study, there have been other studies done to replicate these findings. Richter et al. also did a one-legged exercise study and found a 2-fold increase in glucose uptake in response to low physiological concentrations of insulin in the exercised leg when compared to the rested leg³⁵. These findings line up with the findings of the current study. A study conducted by Dela et al. using one -legged cycling demonstrated similar results, with the trained leg having 30% higher glucose uptake stimulated by a physiological concentration of insulin compared to the untrained leg³⁵. These studies, and the one conducted by Bergstӧrm and Hultman, line up with the current study finding that aerobic exercise is an effective tool for increasing insulin sensitivity in vitro; however, they solely focus on aerobic training and fail to explore the relationship between resistance training and insulin sensitivity. A more recent study was done that looked at the effects of in vivo exercise on in vitro metabolic adaptations. Researchers compared biopsies taken before and after 12 weeks of combined aerobic and resistance training. They reported no changes in the basal level of 30 glycogen synthesis were observed in the myotubes, and insulin significantly increased glycogen synthesis by about 1.5-fold both before and after exercise²⁴. The findings of this study also line up with the findings of the current study, suggesting that exercise, regardless of modality, is beneficial for the increase of insulin sensitivity in vitro. Berggren et al. conducted a study comparing endurance trained athletes and sedentary individuals to examine noninsulin and insulin-mediated glucose uptake in human skeletal muscle cells. Like the experimental process of the current study, the researchers took biopsies and grew them up in vitro and looked at insulin stimulated glucose uptake. They found that there was a dose response for glucose uptake with an increase in insulin concentrations, with the glucose uptake being ≈1.5-fold over basal⁶. The absolute uptake of glucose in the cells of the endurance- trained was significantly higher both in the absence of insulin and at insulin concentration. The fold stimulation over basal between the sedentary and trained subjects at each insulin concentration did not significantly differ⁶. Again, the results of this study line up with the current study; there is a significant increase in glucose uptake when stimulated with insulin, but there is not a significant difference in fold change between exercise and control groups. Another research team looked at the impact that aerobic training has on mitochondrial metabolism in HSkMC³⁸. They found that while training robustly increases in vivo skeletal muscle mitochondrial capacity in lean and obese sedentary subjects, there was no effect of aerobic training on insulin or homeostatic model assessment for insulin resistance (HOMA-IR). While the team found no effect on insulin or HOMA-IR, they did find that changes induced by aerobic training in skeletal muscle in vivo are retained in HSkMC in vitro. This finding supports the idea that training can improve the intrinsic skeletal muscle mitochondrial capacity ³⁸. This study supports the underlying idea of the current one, that HSkMCs retain the characteristics of their donors. 31 It is known that a single session of aerobic exercise can increase glucose uptake by muscle during exercise, increases the ability of insulin to promote glucose uptake, and increases glycogen accumulation after exercise³⁵. There has also been some indication that resistance training may be effective in preventing insulin resistance³⁵. These findings are mainly the result of whole-body insulin sensitivity studies. Despite this, there hasn’t been a lot of research done comparing aerobic and resistance and their relationship to insulin sensitivity specifically in skeletal muscle. Most of the studies done with in vivo exercise effects have focused on aerobic training alone. The results of this current study line up with the results of whole-body studies and in vivo studies that look at aerobic training. While there have been studies proving the effects of aerobic training on insulin sensitivity in skeletal muscle, there needs to be more done to prove the effect of resistance training. At this moment it seems that the two modalities provide similar outcomes. It also seems that a combination of the two types of training would provide greater outcomes with insulin sensitivity. There needs to be further investigation to confirm either of these ideas. 32 Tables and Figures Table 1. Participant Characteristics HIGHLY ACTIVE ENDURANCE EXERCISERS HIGHLY ACTIVE RESISTANCE EXERCISERS SEDENTARY CONTROLS N 11 10 4 SEX (MALE/FEMALE) 4/7 4/6 0/4 AGE (YEARS) 40.2 ± 8.7 36.5 ± 11.5 40.8 ± 9.2 BMI (𝐤𝐠/𝐦𝟐) 23.5 ± 3.0 26.2 ± 1.8 29.3 ± 4.0* RELATIVE 𝐕𝐎𝟐𝐦𝐚𝐱 (ML/KG/MIN) 47.0 ± 10.4 34.0 ± 2.0 24.5 ± 5.3* ISOMETRIC KNEE PEAK TORQUE 185.6 ± 64.1 310.5 ± 80.7 195.0 ± 65.4 Data are means ± SE. 33 Figure 1. Glycogen Synthesis HAEE HARE SED 0.00 0.02 0.04 0.06 0.08 Groups n m o l/ m in /m g Basal Insulin Stimulated ✱✱✱✱ ✱✱✱✱ Figure 1. Displays the glycogen synthesis under basal and insulin stimulated conditions. HAEE, highly active endurance exercisers; HARE, highly active resistance exercisers; SED, sedentary controls. There is an overall treatment effect of insulin stimulation. The significant increase in the HAEE and SED groups is marked with two asterisks (**) and the more significant increase in the HARE group is marked with four asterisks (****). There was no significant difference between the basal levels of the groups. 34 Figure 2. Fold Change H A EE H A R E S E D 0.0 0.5 1.0 1.5 2.0 Groups In s u li n S ti m u la te d /B a s a l HAEE HARE SED ✱✱ Figure 2. Displays fold change. HAEE, highly active endurance exercisers; HARE, highly active resistance exercisers; SED, sedentary controls. There is an overall treatment effect, with a 1.5- fold increase in glycogen synthesis with insulin stimulation. The significant difference between the HARE and SED groups is noted with two asterisks (**). 35 Figure 3. Glycogen Synthesis (Females Only) HAEE HARE SED 0.00 0.02 0.04 0.06 0.08 Groups n m o l/ m in /m g Basal Insulin Stimulated Figure 3. Displays the glycogen synthesis under basal and insulin stimulated conditions in female subjects only. HAEE, highly active endurance exercisers; HARE, highly active resistance exercisers; SED, sedentary controls. There is an overall treatment effect of insulin stimulation, however; there is no significant effect within the groups. 36 Figure 4. Fold Change (Females Only) H A EE H A R E S E D 0.0 0.5 1.0 1.5 2.0 Groups In s u li n S ti m u la te d /B a s a l HAEE HARE SED ns ✱ ✱ Figure 4. Displays fold change of female subjects only. HAEE, highly active endurance exercisers; HARE, highly active resistance exercisers; SED, sedentary controls. 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American Journal of Cell Physiology, 323(2), C606– C616. https://doi.org/10.1152/ajpcell.00146.2022 https://doi.org/10.3791/51812 https://doi.org/10.1083/jcb.201802095 https://www.sciencedirect.com/science/article/pii/S0303720713002566 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1204764/ https://doi.org/10.1152/japplphysiol.01370.2011 Appendix Office of Human Subjects Research Institutional Review Boards 1620 McElderry Street, Reed Hall, Suite B-130 Baltimore, Maryland 21205-1911 410-955-3008 410-955-4367 Fax e-mail: jhmeirb@jhmi.edu APPROVAL OF SUBMISSION February 20, 2023 Dear Dr. Lauren Sparks: On 2/20/2023, the IRB reviewed the following submission: Type of Review: Continuing Review Title of Study: Investigating the effects of aerobic and resistance training in vivo on skeletal muscle metabolism in vitro in primary human muscle cells (MoTrMyo) Investigator: Lauren Sparks IRB ID: CR00000021 Review Type: Convened Committee: IRB-5 Committee Chair: Dr. Joseph Carrese The IRB approved the study from 2/20/2023 to 02/19/2024. JHM IRB is serving as the Single IRB for this study. Continuing Review approval applies to the following participating sites: • Adventist Health System, Sunbelt, Inc. d/b/a AdventHealth Orlando • Duke University Health System, Inc. • East Carolina University • LSU's Pennington Biomedical Research Center • University of Colorado Denver • University of Texas Health Science Center at San Antonio • Wake Forest University Health Sciences The above-referenced participating sites should be provided a copy of the continuing review approval letter. mailto:jhmeirb@jhmi.edu 44 Participants have been enrolled and enrollment is ongoing. Documents approved as part of this Continuing Review include: • SIRB CR PSite Summary Sheet_CR 2023_edit 2.13.23.xlsx • Seven Site Specific Annual Review Logs • One Site Specific Deviation Log (PBRC) Date of Approval and Expiration Date: The approval and expiration date for this research are listed above. If the approval lapses, the research must stop and you must submit a request to the IRB to determine whether it is in the best interests of individual participants to continue with protocol-related procedures. Modifications: All proposed changes to the research must be submitted using a Modification. The changes must be approved by the JHM IRB prior to implementation, with the following exception: changes made to eliminate apparent immediate hazards to participants may be made immediately and promptly reported to the IRB. Continuing Review: Continuing Review Applications should be submitted at least 6 weeks prior to the study expiration date. Failure to allow sufficient time for review may result in a lapse of approval. If the Continuing Review Application is not submitted prior to the expiration date, your study will be terminated and a New Application must be submitted to continue the research. Unanticipated Problems: All unanticipated problems must be submitted using a RNI. https://sirb.jhu.edu/sirb/sd/Doc/0/9U0V6U5BPC8URT402I280LIG00/SIRB%20CR%20PSite%20Summary%20Sheet_CR%202023_edit%202.13.23.xlsx