Anti-Catabolic Effects of Conjugated Linoleic Acid and Omega-3

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2012 Anti-Catabolic Effects of Conjugated Linoleic Acid and Omega-3 Polyunsaturated Fatty Acid Administration in Resting or Loaded Skeletal Muscles of Middle Aged Mice during 20 Weeks of High Fat Diet Sang-Rok Lee

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COLLEGE OF HUMAN SCIENCES

ANTI-CATABOLIC EFFECTS OF CONJUGATED LINOLEIC ACID AND OMEGA-3

POLYUNSATURATED FATTY ACID ADMINISTRATION IN RESTING OR

LOADED SKELETAL MUSCLES OF MIDDLE AGED MICE

DURING 20 WEEKS OF HIGH FAT DIET

SANG-ROK LEE

A Dissertation submitted to the Department of Nutrition, Food and Exercise Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Spring Semester, 2012 Sang-Rok Lee defended this dissertation on March 23, 2012.

The members of the supervisory committee were:

Jeong-Su Kim Professor Directing Dissertation

Samuel C Grant University Representative

Lynn B Panton Committee Member

The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements.

I dedicate this dissertation to my mom and dad Bok-ae Kim and Kwan-hae Lee, my wife, Eun-sook Yoo, and my daughters, Erin and Annie. Mom and dad, you have been a great influence in my life. You have always supported my aspirations and prayed for my future success. From the beginning, you have taught me to live within God. My wife, Eun-sook, you have always stood by me through thick and thin. Without a doubt, nothing could have been completed without your love and dedication. I thank God you are my wife. Erin and Annie, you have become the reason for my life. I always thank GOD for sending you to me. Like my parents did, I will do all that I can for your future.

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ACKNOWLEDGEMENTS

The author wishes to thank the following people for their involvement in this dissertation:

My major professor and advisor, Dr. Jeong-Su Kim. I cannot find the words to express my gratitude for your incredible dedication. Since I joined your lab, you have always been invested in my educational growth and development as a scientist throughout my PhD program. Not only as an academic mentor, but you have provided guidance on the right way for living within Jesus. It has been an honor to be your doctoral student and I will never forget your commitment and priceless efforts. My committee member Dr. Panton. I sincerely thank you for everything you have done for me throughout my time in the PhD program. You always have treated me like family and it was a great opportunity to have you on my committee. Your outstanding insight and advice enhanced the quality of my dissertation. I will never forget your generosity and dedication. My committee member Dr. Samuel Grant. Thank you for being one of my committee members and for providing the opportunity to work in one of the greatest research institutes in the world. You have provided great insight and direction for my dissertation. It was my honor to collaborate with your laboratory and I will remember all of your advice and dedication. To my colleagues in the Kim muscle lab. It has been a pleasure to work with this lab team. Eddie and Andy, I know that I could not complete my dissertation without your dedication. You have always helped to ensure that my research was completed well. I will never forget your great contribution and time spending with you. Mike and Dr. Park, I thoroughly enjoyed working with both of you, I really appreciated the support you provided for my dissertation. To my colleague Ihssann. I truly appreciate your hard work and dedication for my dissertation. It would have been hard to complete this project without your incredible efforts. I will remember your great help. Dr. Michael Ormsbee, I sincerely appreciate your help on this research project, especially for providing the supplements. Your help allowed my dissertation to go smoothly. Thank you. To my colleagues Neema, Shrin, and Marcus. Thanks for everything you have done for my dissertation. I sincerely appreciate your dedication.

Finally and most importantly, I would like to thank my savior and Lord Jesus Christ. I believe you always have been with me. You are the only way to solve the things given to me and get through the tribulations I have encountered.

TABLE OF CONTENTS

LIST OF TABLES ...... viii LIST OF FIGURES ...... ix ABSTRACT ...... xi 1. CHAPTER ONE: INTRODUCTION ...... 1 1.1 SIGNIFICANCE OF STUDY ...... 3 1.2 SPECIFIC AIMS ...... 3 1.3 RESEARCH HYPOTHESES ...... 4 1.4 ASSUMPTIONS ...... 4 1.5 DELIMITATIONS ...... 4 1.6 LIMITATIONS ...... 5 1.5 DEFINITIONS OF TERMS ...... 5 2. CHAPTER TWO: REVIEW OF RELATED LITERATURE ...... 7 2.1 AGE-MEDIATED IMPAIRMENTS AND SARCOPENIA ...... 7 2.1.1 Hormonal changes and sarcopenia...... 7 2.1.2 Alterations of motor unit and sarcopenia ...... 7 2.1.3 Oxidative stress-mediated mitochondria impairments and sarcopenia ...... 8 2.1.4 Oxidative stress-mediated apoptosis and sarcopenia ...... 9 2.2 INFLAMMATION-MEDIATED IMPAIRMENTS AND MUSCLE WASTING .....10 2.2.1 Pro-and anti-inflammatory cytokines ...... 10 2.2.2 Chronic low-grade inflammation during aging...... 12 2.2.3 Chronic inflammation-related diseases and muscular abnormalities in aged populations ...... 13 2.3 SARCOPENIC OBESITY ...... 14 2.3.1 High fat diet-induced obesity ...... 14 2.3.2 Inflammation-induced changes in body composition and disability During aging ...... 15 2.3.3 Sarcopenic obesity and inflammatory cytokines-mediated insulin resistance .15 2.3.4 Sarcopenic obesity and adipokine-induced insulin resistance ...... 19 2.4 INFLAMMATION-MEDIATED SARCOPENIA ...... 21 2.4.1 Inflammatory cytokine-induced impairments in anabolic systems and sarcopenia ...... 21 2.4.2 TNF-α-mediated apoptosis and sarcopenia...... 24 2.5 EXERCISE-INDUCED CHANGES IN INFLAMMATION LEVEL AND SARCOPENIA ...... 24 2.5.1 Aerobic-exercise training and inflammation in aged muscle ...... 24 2.5.2 Resistance-exercise training and inflammation in aged muscle ...... 25 2.6 ANTI-INFLAMMATORY SUPPLEMENTS AND SARCOPENIA ...... 28 2.6.1 Conjugated linoleic acid and inflammation in aged muscle ...... 28 2.6.2 Omega-3 poly unsaturated fatty acid and inflammation in aged muscle ...... 29 2.7 MAGNETIC RESONANCE IMAGING TRACKING CHANGES IN SKELETAL MUSCLE ...... 31 2.7.1 Diffusion-weighted MR imaging ...... 31

2.8 SUMMARY AND FUTURE RESEARCH IMPLICATIONS...... 32 3. CHAPTER THREE: RESEARCH DESIGN AND METHODS...... 33 3.1 OVERVIEW OF EXPERIMENTAL DESIGN ...... 33 3.2 RESISTANCE EXERCISE TRAINING ...... 33 3.3 CLA/N-3 MIX ADMINISTRATION ...... 34 3.4 SPECIFIC AIM 1 (BODY COMPOSITION AND MYOFIBER DIMENSIONS) ....35 3.5 SPECIFIC AIM 2 (NEUROMUSCULAR FUNCTIONALITY) ...... 36 3.6 SPECIFIC AIM 3 (MUSCLE MRNA EXPRESSION)...... 37 3.7 STATISTICAL ANALYSIS AND SAMPLE SIZE DETERMINATION ...... 39 4. CHAPTER FOUR: RESULTS ...... 40 4.1 FOOD AND CLA/N-3 CONSUMPTION ...... 40 4.2 MORBIDITY AND MORTALITY ...... 40 4.3 DUAL X-RAY ABSORPTIOMETRY DETERMINED BODY COMPOSITION ....42 4.4 NEUROMUSCULAR FUNCTIONALITY ...... 46 4.5 MYOFIBER DIMENSIONS ...... 49 4.6 MUSCLE MASS ...... 51 4.7 CHANGES IN TRANSCRIPT FACTORS ...... 55 5. CHAPTER FIVE: DISCUSSION ...... 77 5.1 MORBIDITY AND MORTALITY ...... 77 5.2 BODY COMPOSITION ...... 78 5.3 MUSCLE MASS AND MYOFIBER DIMENSIONS ...... 81 5.4 MUSCLE STRENGTH AND SENSORIMOTOR FUNCTION ...... 82 5.5 REGULATOR OF INFLAMMATION ...... 83 5.6 REGULATORS OF PROTEIN TURNOVER ...... 85 5.7 REGULATORS OF MITOGENESIS ...... 86 5.8 REGULATORS OF MYOGENESIS ...... 87 5.9 CONCLUSIONS...... 87

APPENDICES ...... 89

ANIMAL CARE AND USE COMMITTEE APPROVAL OF STUDY PROTOCOL ...... 89

REFERENCES ...... 90

BIOGRAPHICAL SKETCH ...... 113

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LIST OF TABLES

1. Profile of diets ...... 41

2. Daily food and energy consumption ...... 42

3. DXA determined body composition ...... 42

4. Neuromuscular functionality measurements ...... 46

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LIST OF FIGURES

1. Comparison of Total Body Mass ...... 43

2. Comparison of Lean Body Mass ...... 44

3. Comparison of Fat Mass ...... 45

4. Grip Strength Test ...... 47

5. Incline Plane Test ...... 48

6. Fractional anisotropies ...... 49

7. Eigenvalues (λ) 1, β, and γ ...... 50

8. Gastrocnemius muscle wet weight ...... 51

9. Soleus muscle wet weight ...... 52

10. Quadriceps muscle wet weight ...... 53

11. Hamstring muscle wet weight ...... 54

12. Relative mRNA expression of TNF-α in the gastrocnemius ...... 55

13. Relative mRNA expression of TNF-α in the quadriceps ...... 56

14. Relative mRNA expression of IL-1 in the gastrocnemius ...... 57

15. Relative mRNA expression of IL-1 in the soleus ...... 58

16. Relative mRNA expression of IL-1 in the quadriceps ...... 59

17. Relative mRNA expression of IL-6 in the gastrocnemius ...... 60

18. Relative mRNA expression of IL-6 in the soleus ...... 61

19. Relative mRNA expression of IL-6 in the quadriceps ...... 62

20. Relative mRNA expression of IL-15 in the gastrocnemius ...... 63

21. Relative mRNA expression of IL-15 in the soleus ...... 64

22. Relative mRNA expression of Akt in the quadriceps ...... 65

23. Relative mRNA expression of mTOR in the gastrocnemius ...... 66

24. Relative mRNA expression of atrogin-1 in the gastrocnemius ...... 67

25. Relative mRNA expression of atrogin-1 in the soleus ...... 68

26. Relative mRNA expression of MURF1 in the gastrocnemius ...... 69

27. Relative mRNA expression of MURF1 in the soleus ...... 70

28. Relative mRNA expression of MURF1 in the quadriceps ...... 71

29. Relative mRNA expression of IGF-IEa in the soleus ...... 72

30. Relative mRNA expression of MyoD in the soleus ...... 73

31. Relative mRNA expression of MyoD in the quadriceps ...... 74

32. Relative mRNA expression of myogenin in the gastrocnemius ...... 75

33. Relative mRNA expression of myogenin in the quadriceps ...... 76

ABSTRACT

Sarcopenic obesity, a recent medical term, refers to a new trend in aged individuals who simultaneously demonstrate reductions and increases in lean mass and fat mass, respectively, leading to poor quality of life. It is currently thought that increases in fat mass with age start the actual sarcopenic process by increasing inflammatory factors. Chronic resistance exercise training (RET) is considered the most cost effective intervention to combat sarcopenia, improving muscle strength. Conjugated linoleic acid (CLA) and omega-3 polyunsaturated fatty acid (n-3) attract great attention for their anti-inflammatory properties, possibly reducing risks of muscle wasting. However, the efficacy of CLA/n-3 on resting or loaded muscles during chronic high fat diet (HFD)-induced inflammation has not been fully elucidated. Therefore, the overarching aim of the present study was to investigate the impact of HFD-induced inflammation on sarcopenia and the effects of 20-wk CLA/n-3 administration on muscle at rest or with RET in the middle aged mice. Nine-month old C57BL/6 male mice were randomly assigned to five groups (n=10/group): 1) Normal diet (C), 2) High fat diet (H), 3) HFD+RET (HE), 4) HFD+CLA/n-3 (HCN), and 5) HFD+RET+CLA/n-3 (HECN). Progressive RET (4 sets of 3 repetitions with 1-min inter-set rest) was conducted using a ladder climbing device 3x/wk for 20 wks. The combined supplement was comprised of 1% CLA (0.5% of c9, t11 and 0.5% of t10, c12) and 1% n-3. Body composition (dual X-ray absorptiometry, DXA), myofiber dimensions (magnetic resonance diffusion tensor imaging, MR DTI), grip strength, and sensorimotor function (incline plane test) were assessed pre- and post-experiment. Muscle isolation (i.e. gastrocnemius, soleus, quadriceps, and hamstrings) was performed to determine muscle wet weight and RT-PCR was used to analyze transcript levels of target factors involved in muscle inflammation and muscle fiber growth and regeneration: pro-inflammatory regulators [tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1)], anti-inflammatory regulators (IL-6 and IL-15), protein synthesis [protein kinase B (Akt), mammalian target of rapamycin (mTOR)], protein degradation [atrogin-1, muscle ring finger 1 (MURF1)], mitogenic factor [insulin-like growth factor-I Ea (IGF-IEa)], and muscle regeneration (MyoD, and myogenin). ANOVAs were utilized, and significance was set at p ≤ 0.05. There were significant group x time interactions for lean body mass (LBM), fat mass (FM), grip strength, and sensorimotor function. FM increased in H (+74%), HE (+142%), and HECN (+43%) but not in C and HCN. LBM decreased in C (- 27%), H (-31%), and HE (-55%) while no change was found in HECN. Interestingly, TBM in

HCN was significantly lower than both H and HE. Strength significantly declined in H (-15%) and HCN (-17%) but was maintained in C. Sensorimotor function markedly declined in H (- 11%) with no change in C, HCN, and HE. Interestingly, CLA/n-3 administration appeared to facilitate greater RET-mediated improvements in strength (+22%) and sensorimotor coordination (+17%). There was a significant group effect in muscle wet weight. Gastrocnemius wet weight significantly decreased in C (-27%), H (-39%), and HCN (-35%) from baseline but was maintained in HE and HECN. Soleus wet weight significantly decreased in H (-24%) while maintained in C, HE, and HCN. In contrast, soleus wet weight was greater in HECN compared to C, H, and HCN. Fractional anisotropy (FA) was significantly decreased in HECN (-22%). While eigenvalue (λ) β decreased in HECN (-8%), λγ significantly increased in HCN (+16%) from baseline. A 20-wk CLA/n-3 administration improved the inflammatory state in HFD-fed middle aged animals. TNF-α mRNA expression was greater in H compared to C, HE, HCN, and HECN in the gastrocnemius. Moreover, HE, HCN, and HECN demonstrated greater IL-6 mRNA expression compared to baseline in the gastrocnemius. HCN showed elevated IL-15 mRNA expression compared to baseline and H in the gastrocnemius. Both H and HE had significantly lower IL-15 expression than C and HECN in the soleus. Based on our findings, a long-term HFD negatively altered body composition, muscle wet weight, and functional capacity in middle aged mice. Daily CLA/n-3 administration attenuated these impairments while facilitating RET- induced improvement in functional capacity, possibly by improving the inflammatory state. Finally, we are the first group to employ DTI to detect HFD-induced and intervention-mediated alterations in myofiber dimensions with in vivo model utilizing the most powerful MR technology. Future research may need to be conducted in middle aged obese populations to verify our findings.

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CHAPTER ONE INTRODUCTION

Sarcopenia, the progressive loss of muscle mass and strength [1], is considered one of the primary factors that reduce the quality of life in the aging population [2]. Between the ages of 20 and 80 years, humans inevitably undergo muscle wasting by approximately 40%, resulting in the loss of strength, metabolic rate, respiratory function, and morbidity [3]. The consequences of these age-related decrements increase risk factors in the elderly such as incidence of falls [4-6], physical disability [7, 8], and mortality [9, 10]. Failure of compensatory efforts that diminish degenerative processes in skeletal muscle might progress sarcopenia [11]. This impairment includes the attrition of myogenic mechanisms responsible for maintaining muscle mass and protein turnover [12-15]. Although the principal cellular and molecular mechanisms of sarcopenia and its intervention are not well established, recent studies have proposed that inflammatory mediators are a key associated with potential etiological factors of sarcopenia [16, 17]. Inflammation is closely related to various diseases afflicting the elderly, such as osteoarthritis, cardiovascular disease, and chronic heart failure (CHF) [18]. In addition, it has been suggested that elevated concentrations of pro-inflammatory cytokines with decreased levels of growth factor correlate with sarcopenia [19]. Sarcopenia is linked not only to a decrement in muscle mass, but also with an increase in body fat. The coexistence of these body composition alterations have been described as ‘sarcopenic obesity’ [20]. It is generally accepted that the primary factors, which induce obesity, include high energy-dense foods such as in a high fat diet (HFD), resulting in greater passive energy consumption, and environments, which limit physical activity, reducing energy expenditure. Obesity provokes an increased level of inflammatory biomarkers [21] via adipose tissue, a primary source of pro-inflammatory cytokines [22] and adipokines [23]. Elevated cytokine levels coincide with decreased lean body mass and increased fat mass in the aged population [21]. Recent evidence suggests that chronic low-grade systemic inflammation during aging is closely associated with sarcopenic progression [24, 25]. Moreover, abnormally elevated levels of pro-inflammatory cytokines possibly promote sarcopenia through induction of the cysteine-aspartic proteases (caspase) cascade [26], activation of nuclear factor kappa B (NF-kB) [27], and impairment of cellular anabolism such as reduced insulin like growth factor-I (IGF-I)

1 concentration [28]. Inflammation can deteriorate skeletal muscle through direct catabolic effects or indirect mechanisms (e.g., reduction in growth hormone (GH) and IGF-I concentrations and/or anorexia) [29]. It was demonstrated that IGF-I, a stimulant of muscle growth, has an inverse correlation with inflammation [30]. The development of sarcopenia, frailty, and mortality appears connected to reduced levels of IGF-I [31]. In fact, aged women with low IGF-I levels and augmented interleukin-6 (IL-6) concentrations exhibit impaired functional performance and a greater mortality rate [32]. Therefore, since obesity promotes muscle wasting, it is convincible that HFD-induced obesity during middle age possibly triggers an earlier onset and acceleration of the sarcopenic process. Long-term resistance exercise training (RET) is recognized as an effective intervention to combat sarcopenia, facilitating the preservation and improvement of muscle mass in older individuals. However, aged muscles display blunted hypertrophic responses to RET compared to young individuals [33]. These impaired responses might stem from greater ultrastructural damage [34], slower rates of recovery [35] and a blunted acute inflammatory response [36], causing defective repair and regeneration of damaged muscle [37]. While chronic inflammation is associated with sarcopenia [31], the acute inflammatory response to a bout of exercise promotes repair [38] and regeneration of damaged myofibers through provision of growth factors [39]. The blunted inflammatory response to the mechanical stimuli might interfere with the adaptive response necessary for remodeling in damaged muscle in the elderly. Therefore, a different intervention strategy using anti-inflammatory supplements may attenuate resting inflammation while simultaneously sensitizing the acute inflammatory response to exercise necessary for regeneration of skeletal muscle. Conjugated linoleic acid (CLA) and omega-3 polyunsaturated fatty acids (n-3) have attracted attention for their anti-inflammatory and anti-oxidant properties. However, no investigations exist either examining the effects of HFD during middle age or demonstrating the benefits of CLA/n-3 administration in resting or exercising muscles during HFD-induced inflammation. Therefore, the objectives of the present study were 1) to investigate the impact of HFD-induced chronic inflammation on sarcopenia and 2) to examine the effects of 20-wk CLA/n-3 administration at rest or with programmed RET on improvements in muscle wasting during the aging progress through the middle ages of animals.

Significance of Study

Sarcopenia generally progresses beyond the 5th decade of life and leads to a host of functional limitations and comorbidities markedly impairing quality of life [40]. The most severe consequence of sarcopenia occurs when an individual loses his or her functional independence [41]. The sarcopenic process is characterized by decreased muscle mass and strength, resulting in disability and frailty. In addition to physical impairment, these outcomes lead to substantial financial burden for effective treatment. A previous report indicated that approximately one-third of women and two-thirds of men aged 60 years or older experience muscle atrophy, and in the year 2000, sarcopenia attributed to $18.5 billion in direct health care cost in the US [42]. This economic burden related to sarcopenia is expected to increase resulting from a progressive escalation in the elderly populations [42]. Therefore, understanding the factors that delay or possibly reverse sarcopenia exists as a public health priority, improving quality of life of the elderly and blunting related economic costs.

Specific Aims

The specific aims of the present study were to determine the degree by which a 20-wk administration of CLA/n-3 administration with and without RET will modulate: 1) body composition using dual energy X-ray absorptiometry (DXA), myofiber dimensions using the Magnetic Resonance Diffusion Tensor Imaging (MR DTI) technology in middle aged mice fed a HFD; 2) neuromuscular functionality in middle aged mice fed a HFD by measuring muscle strength (grip strength test) and sensorimotor capacity (inclined plane test); and 3) myogenic capacity, protein turnover, and inflammation status by in vitro analyses using reverse transcription polymerase chain reaction (RT-PCR) technique to compare molecular biomarkers associated with related regulatory factors on collected muscle samples of the middle aged mice fed a HFD. Specific aim #3 will be pursued by examining changes in muscle mRNA expression of target biomarkers involved in muscle regeneration [mitogenic (IGF-IEa) and myogenic (MyoD and myogenin)], protein synthesis [protein kinase B (Akt) and mammalian target of rapamycin (mTOR)] and protein degradation [atrogin-1 and muscle ring finger protein 1 (MURF1)], pro-inflammatory cytokines [TNF-α and interleukin-1 beta (IL-1 )], and anti- inflammatory cytokines (IL-6 and IL-15).

Research Hypotheses

Our working hypotheses were as follows: 1) Aging would induce impairments in body composition and myofiber dimensions. Additionally, a HFD would intensify these impairments. A 20-week CLA/n-3 administration would alleviate these impairments with or without RET and RET will yield the greatest improvements. 2) During aging, there would be impairments in muscle strength and sensorimotor function and a HFD would produce worse outcomes. A 20-week CLA/n-3 administration would attenuate these impairments with or without RET, with greater improvements ensuing during RET. 3) Aging would negatively modulate relative biomarkers, with even greater detriments with a HFD. Results of mRNA analyses would indicate a down-regulation of regulatory factors associated with muscle regeneration [myogenic regulatory factors (MyoD and myogenin) and mitogenic factor (IGF-IEa)], protein synthesis (Akt and mTOR), and anti-inflammatory cytokines (IL-6 and IL-15). There would be up-regulation of regulatory factors associated with protein degradation (atrogin-1 and MURF1) and pro-inflammatory cytokines (TNF-α and IL-1 ). Again, HFD would augment these detriments. Twenty-week CLA/n-3 administration would positively modulate these biomarkers with or without RET and would enhance these improvements during RET.

Assumptions

1. All laboratory equipment yielded accurate measurements over the course of repeated testing. 2. All animals were free of disease and muscle wasting afflictions such as tumors and cancer. 3. All animals received similar handling and treatment during the experimental period.

Delimitations

1. There were a total of 60 male C57BL/6 mice, aged 44 weeks (8 months), (Jackson Laboratories, Bar Harbor, Maine). After ten were sacrificed for baseline measurements, the mice were randomly divided into five groups (n=10), which included 1) normal diet control (C), 2) HFD control (H), 3) HFD+RET (HE), 4) HFD+CLA/n-3 (HCN), and 5) HFD+ RET+ CLA/n-3 (HECN). 2. Animals were free of any physical illnesses; particularly those that may increase muscular malfunction regarding atrophy.

Limitations

1. Since our current MR technology is not able to evaluate human myofiber dimensions, the proposed study was constrained to C57BL/6 mice. 2. There was individual variation in daily diet, which could alter the quantity of HFD consumption, modulating the degree of inflammation. However, this issue was carefully monitored. 3. There was individual variation in daily diet which could alter the quantity of CLA/n-3 administration though it was be carefully monitored.

Definitions of Terms

Sarcopenia – The progressive loss of muscle tissue that occurs with aging, which is primarily responsible for age-related decreases in muscle strength and power [1]. Sarcopenic obesity – a recent medical term, refers to a new trend in aged individuals who simultaneously demonstrate reductions and increases in lean mass and fat mass, respectively [20]. Inflammation – a biological response of tissues to stimuli such as injury or irritants. The intracellular inflammatory response is mediated by releasing pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and Interleukin-1 (IL-1). Chronic inflammation with increased pro-inflammatory cytokines promotes sarcopenic process. Anti-inflammatory cytokines (i.e. IL-6) inhibit or attenuates activity of the pro-inflammatory cytokines [43]. Insulin like growth factor-1 (IGF-1) Ea – IGF-1Ea is released from the liver and is a 70 amino acid peptide that resembles the hormone insulin. IGF-1Ea is unique in its capacity to stimulate the expansion of existing myonuclear domains through translation initiation induced protein accretion as well as facilitate further growth through stimulation of satellite cell mitogenic and myogenic processes. Mammalian target of rapamycin (mTOR) pathway – The primary pathway responsible for exercise induced skeletal muscle protein synthesis through enhancing translation initiation [44]. Myogenic regulatory factors – A class of proteins, which mediate the commitment of somatic cells to the myogenic lineage. Myogenic regulatory factors responsible for early

5 differentiation include Myf5, and MyoD, while Myogenin and MRF4 are thought to mediate terminal differentiation of muscle stem cells to fully committed, fusion capable myoblasts [45].

CHAPTER TWO REVIEW OF RELATED LITERATURE

Age-Mediated Impairments and Sarcopenia

Hormonal changes and sarcopenia Hormonal changes might play a key role in sarcopenia, as reductions in anabolic hormones could induce this degenerative process. Anabolic hormones including insulin, GH, IGF-I, and testosterone elicit muscle hypertrophy by enhancing protein synthesis and attenuating protein breakdown, while catabolic hormones such as cortisol stimulate protein catabolism [46, 47]. Anabolic hormone activity decreases with age, likely leading to sarcopenia [48]. While the liver serves as the major source of IGF-I, local allocations of IGF-I in skeletal muscle (less than 50% of total IGF-I) primarily attribute to muscle growth and repair [49, 50]. Age-related dysregulation of GH might provoke muscle wasting during organismal senescence. A number of studies report that GH levels generally decrease with age, starting at the age of 50 years, leading to alterations in body composition. Rudman et al. [51] found an 8.8% increase in fat free mass in healthy elderly individuals (61-81 yrs) over a 6-month period of GH administration. In a follow- up study, lean body mass increased about 6% and fat mass decreased by 15% in the elderly (> 61 y) after GH injections for 18 months. Additionally, the anabolic properties of testosterone on muscle protein support the contention that impaired protein synthesis with ensuing loss of muscle mass may be accompanied with decreased testosterone levels during senescence [52]. Alterations of motor unit and sarcopenia Along with endocrinal effects, one of the major contributors to sarcopenia involves the process of motor unit remodeling. Since muscle innervations play an important role in maintaining muscle mass and strength, decline in muscle innervations during aging can induce sarcopenia [53]. Degeneration is induced when impaired neural innervations occur through various conditions such as injury, surgery, and exposure to medications (e.g. intravenous corticosteroid and neuromuscular blocking agents) [54]. Altered motor unit remodeling might influence the sarcopenic process by the selective denervation of type II fibers and collateral sprouting of axons from type I fibers [55]. This development provokes a decrease in muscle mass after the fifth decade despite adaptations resulting in changes in body mass and stature [56]. Previous findings suggested that age-related muscle wasting correlates with decreased number of

7 motor units [57]. To support this hypothesis, Doherty et al. [58] investigated the influence of motor unit loss in the biceps brachii and brachialis in young (22-38 yrs) and older (60-81 yrs) healthy individuals. Results indicated significantly reduced number of motor units in the older group compared to young individuals, suggesting an age-related decrement in motor units [58]. This finding was supported in recent evidence by McNeil et al. [59]. They substantiated these findings by comparing the number of motor units in the tibialis anterior of a young group (25 yrs) and two older (65 yrs and older than 85 yrs) groups. They also found a significant reduction in motor unit numbers in the older group when compared to the younger group and further decreases in those experiencing senescence [59]. Oxidative stress-mediated mitochondria impairments and sarcopenia While oxygen is commonly recognized as the most important element not only for human existence but also for all living organic species, it also possesses deleterious effects. Current research demonstrates that sarcopenia is at least partly explained by the aberrant up-regulation of oxidative metabolism and the ensuing increase in reactive oxygen species (ROS) [60]. ROS are generated as a natural byproduct of oxidative metabolism, reacting to oxygen molecules. Accumulation of this harmful product induces a greater rate of cellular damage to important substances [e.g. deoxyribonucleic acid (DNA), proteins, and lipid-containing structure] in humans [61-63]. A study reported that ROS appear to be linked to muscle damage and also modulate skeletal muscle contraction by influencing the functional state of calcium (Ca+2) channels [64]. Ca+2 is one of the major factors of ROS production in the mitochondria during the aging process. Overall, Ca+2 accumulation in the mitochondria may augment ROS production [65]. Reduction of Ca+2 uptake by the sarcoplasmic reticulum (SR) can increase intracellular and mitochondrial Ca+2, leading to ROS generation, muscle protein oxidation, and necrosis during aging [66]. Since SR Ca+2 channels possess high sensitivity to oxidative stress (affecting permeability capacity), increases in ROS might reduce antioxidant defense systems and cause modifications in membrane fluidity, resulting in muscle fatigue and weakness [67-69]. Previous findings reported that ryanodine receptor binding (marker of Ca+2 channel availability) and SR-Ca+2 pump (SERCA1) activity decrease in the aged muscle [70]. Moreover, progressive accumulation of Ca+2 in the mitochondria can mediate the permeability transition pore (PTP), a major contributor to cell death [71]. Specifically, the opening of PTP enhances the release of solutes in the

8 mitochondrial matrix and intermembrane space, thus inducing damage to these structures of the outer mitochondrial membrane [72]. This mitochondrial dysfunction consequently accelerates ROS production and rate of myofiber death [73]. Oxidative stress-mediated apoptosis and sarcopenia Mitochondria play crucial roles in various cellular functions, [e.g. adenosine triphosphate (ATP) generation, aerobic metabolism, and mediating ROS production] [74]. Therefore, this structure could be a major target of oxidative damage, leading to gradual cell death. During aging, alteration of mitochondria stimulated by significantly increased oxidative stress appears to attenuate protein synthesis and ATP production, elevating necrosis and apoptosis [75, 76]. The mitochondria can induce apoptosis via caspase activation [73]. Caspases, which is a group of cysteine proteases, tend to stimulate apoptotic processes. Cytochrome C released from the mitochondria in the cytosol induces formation of apoptosome via dATP and apoptosis protease activating factor-1 (Apaf-1) binding, activating Caspase-9. Caspase-9 activates caspase-3 elevating apoptosis [24]. In addition to cytochrome c release, the mitochondria releases apoptosis-inducing factor (AIF) (caspase-independent pathway of apoptosis), which acts on specific myocyte regions. This results in DNA fragmentation [77], destruction of nuclei [24], and myofiber death [73]. Recent data demonstrated a significant increase in apoptosis-inducing factors (AIF) in old rats compared to young animals, suggesting that age-related muscle wasting involves AIF [78]. The disintegrated outer membrane of mitochondria is also explained by an increased ratio of pro-apoptotic [e.g. Bcl-2-associated X protein (Bax) and BH3 interacting domain death agonist (Bid)] to anti-apoptotic [e.g. B-cell leukemia/lymphoma 2 (Bcl-2)] factors. Increased oxidative stress and other apoptotic stimuli enhance Bid to mediate structural modifications in Bax, which in turn allows its entry into the outer mitochondrial membrane, triggering increased mitochondrial permeability to apoptotic mediators [79-81]. While this harmful effect can be prevented by Bcl-2, a greater increase in Bax/Bid ratio over BCL-2 exists in both resting and overloaded myofibers during aging [80-82]. Age-induced increase in skeletal muscle apoptosis appears to be a main contributor to sarcopenia as apoptosis-stimulating factors (e.g. oxidative stress, impaired mitochondrial capacity, and denervation) increase with advanced age [80, 83, 84]. A collection of studies demonstrated an increased magnitude of oxidative damage to the mitochondria with aging [85, 86]. Findings suggested that with aging, oxidative stress from

9 mitochondria is the central cause for apoptosis, subsequent impairments in function, and increasing rate of cancer [75]. Muller et al. [87] supported the role of oxidative stress in sarcopenia by demonstrating a three-fold increase in ROS in 30 month-old mice compared to 10 month-old mice. Additionally, the older generation experienced a 30% loss in gastrocnemius mass. Concomitantly, Vasilaki et al. [88] discovered elevated ROS from isolated mitochondria in resting skeletal muscle in old mice compared to the young adults. Furthermore, the progressive decrement in muscle weight during aging accompanies increased rate of DNA fragmentation, which is related to elevated active cystolic and nuclear caspase-9 in old senescent rats [79]. In addition, ROS may induce morphological alteration to skeletal muscle function. Particularly, the sarcopenic phenotype is classified by chronic low-grade inflammation, resting muscle damage (e.g., extensive Z-band streaming or zigzagging), and complete disarray of the myofibril structure [89]. This mechanism combined with increased oxidative stress in senescent myofibers might augment muscle damage.

Inflammation-Mediated Impairments and Muscle Wasting

Pro- and anti-inflammatory cytokines Inflammation, a response of tissue to trauma or infection, can be triggered by various soluble components, [e.g. increased blood flow, vascular permeability, and inflammatory mediators (cytokines)]. These components collectively serve to induce homeostasis and tissue repair [90]. In response to injury, the local inflammatory response is accompanied by systemic inflammation, leading to production of numerous hepatocyte-derived acute phase proteins [e.g. C-reactive protein (CRP)] [91]. The clinical signs of inflammation are characterized by pain, heat, redness, swelling, and functional impairment. Intracellular molecules called cytokines, regulate various portions of cellular systems by inducing proliferative and adaptive response to stimuli [64]. Specific immune cells secrete cytokines, which carry signals to inflammation sites. These signals facilitate an influx of lymphocytes, neutrophils, and other cells involved in the healing process [91]. Similar to hormones, cytokines mediate cell communication as a signaling molecule. The term cytokine represents a diverse family of polypeptide regulators provided by cells of diverse embryological origin. There has been argument about the definition of cytokine and hormone. While classical hormones circulate in nanomolar (10-9) concentration, some cytokines such as interleukin-6 (IL- 6) circulate in picmolar (10-12) concentrations, increasing up to 1,000-fold in response to specific

10 damage [92]. All nucleated cells, particularly endo/epithelial cells and resident macrophages, stimulate production of IL-1, IL-6, and TNF-α [92]. Contrastingly, classical hormones, such as insulin, are released from discrete glands (e.g. pancreas). The inflammatory response is incorporated in the process of destroying pathogens by providing oxidant molecules and stimulation of T and B lymphocytes [93]. Moreover, endogenous substrates released through inflammatory pathways stimulate anti-oxidant systems and enhance the activation of T and B lymphocytes [94]. Pro-inflammatory cytokines, TNF-α, interleukin-1 beta (IL-1 ), and IL-6 are involved in this process as primary regulators. A number of physiologists [95-97] observed inflammatory cytokines, specifically pro- inflammatory cytokines, i.e. IL-1 (both α and isoforms) and TNF-α. TNF-α and IL-1 are classified as representative pro-inflammatory cytokines, which are locally produced. These pro- inflammatory cytokines enhance leukocyte proliferation, cytotoxicity, secretion of proteolytic enzymes, and synthesis of prostaglandins (PGs), stimulating a secondary cascade of inflammatory cytokines such as IL-6 [92]. IL-6, a secondary inflammatory cytokine serves as a counter inflammatory cytokine, preventing the overshooting of destructive inflammatory response [98]. IL-6, previously regarded as a pro-inflammatory cytokine, demonstrated anti- inflammatory properties in a recent study [99]. Although a previous finding suggested that IL-6 primarily mediates exaggerated inflammatory response in animal models [100], a number of human and animal studies demonstrated anti-inflammatory effects of IL-6 [43, 101]. Unlike IL-1 and TNF-α, IL-6 does not stimulate the up-regulation of primary inflammatory mediators [e.g. nitric oxide (NO) and metalloproteinases (MMPs)] [102]. In addition, IL-6 appears to prohibit lipopolysaccharide (LPS)-induced production of TNF-α in cultured human monocytes [103]. When endotoxin is infused in mice, TNF-α concentration is dramatically increased in both anti- IL-6 treated mice and IL-6 knockout mice compared to counterpart control groups [101]. These results indicate the TNF-α regulating property of IL-6 [104]. As observed in other anti- inflammatory mediators, IL-6 treatment in humans enhances activation of IL-1 receptor antagonist (IL-1RA), which conducts anti-inflammatory activity through inhibition of pro- inflammatory IL-1 receptor [99]. Moreover, IL-6 appears to induce the secretion of soluble TNF- α receptor (sTNFR), an anti-inflammatory component [99]. Normally, inflammatory cytokines are interwoven and regulated by feedback mechanisms mediated by concentrations of pro-inflammatory mediators (e.g. IL-10) [105]. As an

11 antagonistic factor, IL-10 attenuates release of TNF-α, IL-1, and IL-6 in diverse cell types [106], affecting other inflammatory proteins transcriptionally regulated by nuclear transcription factor (NF-kB) [107]. Reports indicated that IL-10 inhibits release of inflammatory cytokines by both T cells and NK cells via dysregulation of accessory cell (macrophage/monocyte) capacity [108, 109]. Although TNF-α and IL-1 synergistically enhance inflammatory processes themselves, they additionally activate secondary inflammatory factors [chemokines, PG, platelet-activating factor (PAF)], magnifying the inflammatory response [106]. In addition to inhibition of pro- inflammatory factors, IL-10 also stimulates production of their natural antagonist. IL-10 induces production of IL-1RA and soluble p55 and p75 TNF receptor (TNFR) [110-112]. In addition, IL- 10 down-regulates expression of IL-1RI and IL-1RII [113, 114] which are stimulated by monocytes. This demonstrates the capacity of IL-10 as it hinders monocyte activation and also enhances activation of anti-inflammatory molecules. IL-10 blocks activation of prostaglandin (PG) E2, by attenuating cyclooxygenase 2 (COX-2) expression [115]. This also reduces expression of matrix MMPs, which is mediated by a PGE-cAMP pathway [116]. The balance between pro- and anti-inflammatory mediators can diagnose inflammatory-related diseases, and treatment with anti-inflammatory components is a promising approach for a medical solution. Individuals diagnosed with cardiovascular-related disease genetically express low IL-10 production [116], theoretically suggesting a novel approach to treatment through anti- inflammatory interventions (i.e. shifting the balance towards the anti-inflammatory direction) [105, 117]. Chronic low-grade inflammation during aging Although aging is generally accepted as a major contributor to various chronic diseases, the relationship between aging and age-related chronic diseases remain unaddressed. Since chronic inflammatory exposure is closely related to various age-induced diseases, molecular inflammation might saliently contribute to chronic disease during aging [118]. Circulating inflammatory elements are elevated with increased levels of TNF-α [119], IL-1RA [120], IL-6 [121], sTNFR [122], and acute phase proteins (e.g. CRP) [123]. Increased pro-inflammatory cytokines display crucial roles in age-related pathogenesis, atherosclerosis [124], type 2 diabetes [125], osteoporosis [126], and sarcopenia [127]. During aging, impaired immune systems (e.g. dysfunctional lymphocyte) might be accompanied with chronic low-grade inflammation [128] associated with increased levels of pro-

12 inflammatory cytokines [129]. A number of early immunogerontologic studies suggested that advanced age coincides with increased levels of IL-6 expression in leukocytes of systemic circulation [119, 121, 130]. Similarly, few studies also reported increased levels of TNF-α concentration in senescent individuals [119, 125]. Previous findings indicated that old individuals experienced a greater level of asymptomatic bacteuria associated with inflammation. To support this assertion, Prio et al. [131] collected clean-catch urine samples from 40 patients (aged 70-91 years) hospitalized for functional disability. Results implied that patients with asymptomatic bacteriuria appear to exhibit substantially higher expression of soluble TNF receptor-1 (sTNFR-1) and neutrophils in blood compared to those without bacteuria. These findings substantiate the speculative link between asymptomatic bacteuria infections and low-grade immune activity in frail old individuals [131]. Inflammation is linked to pathologic lesions found with atherosclerosis [124]. Bruunsgarrd et al. [119] divided centenarians into three groups based on ankle-brachial blood pressure index (low index imply atherosclerosis). Excluding other inflammatory disorders, results denoted significantly greater TNF-α and CRP levels in those with a low ankle-brachial index, indicating that TNF-α is independently associated with atherosclerosis in the elderly. High levels of plasma TNF-α concentration also appear to correlate with impaired insulin sensitivity in the aged individuals [125]. As previously mentioned, a growing body of evidence clearly indicated that chronic low-grade inflammatory status in the elderly promotes various age-related diseases and mortality risks. Chronic inflammation-related diseases and muscular abnormalities in aged populations Progressive muscle impairment is implicated in the various inflammation-related diseases such as cancer [132], AIDS [133], sepsis [134], chronic obstructive pulmonary disease [135], and chronic heart failure (CHF) [136]. These diseases are accompanied with an increased rate of muscular catabolism and result in induced cachexia and subsequent muscle wasting [135]. Aberrant changes in skeletal muscle cause abnormally morphological alteration [137], decline in metabolism capacity [136], and impaired viability of myocyte [138]. Drexler et al. [137] used ultrastructural morphometry to compare the skeletal muscle ultrastructure between old CHF patients and healthy individuals. The density of mitochondria and the surface density of mitochondrial cristae (structurally correlated with myocyte oxidative capacity) decreased 20% in

13 the CHF patient group. In addition, the capillary length density of myocyte declined (p<0.01). Volume and surfer density of mitochondria positively correlated to both peak exercise oxygen uptake (VO2; r=0.56, p<0.001, n=60) and VO2 at anaerobic threshold (r=0.535, p<0.0001, n=60) [137], implying that intrinsic abnormalities in skeletal muscle due to chronic inflammation attenuates exercise capacity or muscle function. Intrinsic abnormalities by chronic inflammation partially contribute to impaired vitality, provoking exercise intolerance. The attenuation of skeletal muscle viability due to myocyte apoptosis appears to gradually impair cardiac function in CHF patients [139]. Adams et al. [138] collected muscle tissues from old CHF patients to investigate the effect of inflammatory disease on apoptosis and its relationship to exercise intolerance. In contrast to the healthy old control group, 47% of old CHF patient exhibited apoptosis [138]. Moreover, patients demonstrating myocyte apoptosis possess a lower maximal oxygen uptake (VO2 max) (p=0.0005) with greater nitric oxide synthase (iNOS) expression (p=0.015) [138]. The authors concluded that CHF-related apoptosis could induce functional impairment.

Sarcopenic Obesity

High fat diet-induced obesity As stated earlier, development of obesity is mediated by an imbalance between energy intake and energy expenditure. Since a high fat diet (HFD) has a greater energy density than other energy sources, HFD is considered high energy intake [140]. It has been well established that there is a positive relationship between a HFD and obesity. A HFD (60% lipid) induces an overweight phenotype, oral glucose intolerance, hyperinsulinemia, hypertrophied islets and adipocytes, stage 2 steatosis and reduced liver PPAR-alpha and GLUT-2 in C57BL/6 mice [141]. Poudyal et al. (2010) found 8-9 week old rats fed combined high carbohydrate and HFD (52% carbohydrate, 24% fat, 25% fructose in drinking water) developed signs of metabolic syndrome, including elevated abdominal and hepatic fat deposition, collagen deposition in heart and liver, cardiac stiffness, and oxidative stress markers (plasma malondialdehyde and uric acid concentrations), with diminished aortic ring reactivity, abnormal plasma lipid profile, impaired glucose tolerance, and hypertension [142]. Further, it was reported that a HFD (60%) with fructose (30%) water indicated phenotypes of metabolic complications such as insulin resistance, hypertension, dyslipidemia and fatty liver [143].

Inflammation-induced changes in body composition and disability during aging Obesity is defined as an increase in fat mass and is associated with an increase in health risk factors. Linear accumulations of adipose tissue and percent body fat during the aging process remains a well-established occurrence in human life. This increase is characterized by elevated levels of visceral adipose tissue [144] while subcutaneous fat is decreased, contributing to sarcopenia [145]. The apparent link between inflammation and obesity alterations of body composition results in physical impairments. As stated earlier, adipose tissue is the primary source of inflammatory cytokines. These harmful mediators released from adipocytes might directly mediate physical disability by altering body composition, which is typical during the aging process [146]. Taken together, the strong relationship between increased fat mass and escalated inflammatory cytokine secretion may worsen impairments in body composition, (i.e., acceleration of sarcopenic obesity) Additionally, increased fat mass, loss of lean body mass, and subsequent strength impairment might accelerate metabolic syndrome and disability [147-149]. Previous reports indicated that sarcopenic and obese individuals exhibited greater impairments in instrumental daily activities compared to normal individuals (i.e., normal body composition) and even lean sarcopenic groups (low muscle mass but normal fat mass) [149]. Therefore, it can be hypothesized that sarcopenic obesity synergistically accelerates the sarcopenic process followed by increased risk of physical disability. Sarcopenic obesity and inflammatory cytokines-mediated insulin resistance Insulin resistance associated with chronic inflammation in skeletal muscle has been well established. This relationship involves the permeability of inflammatory cells into skeletal muscle. This observation is substantiated by subsequent findings: 1) skeletal muscle in type II diabetes mellitus patients (T2DM) promotes increased expression of macrophages and CD 154 (T-cell marker) [150], 2) skeletal muscle in T2DM displayed increased inflammatory cytokines, (e.g. TNF-α and IL-6, NOS, fibrinogen, and CRP) [151], 3) skeletal muscle supplied and released various inflammatory cytokines from adipocytes [152], and 4) skeletal muscle consisted of innate immune systems, [e.g. cytokine receptors and toll-like receptors (TLRs)] [153]. The link between impaired insulin action and aging has been well accepted [154, 155]. Aging studies using animal models have demonstrated increased plasma pro-inflammatory

15 cytokine concentration (e.g. TNF-α level) during aging [156, 157]. TNF-α appeared to down- regulate glucose transporter type 4 (GLUT4) mRNA expression in cultured adipocyte and myocyte [158, 159]. Age-mediated increase in adipose tissues may play a key role in the stimulation of plasma TNF-α expression, subsequently impairing insulin sensitivity in senescent individuals [125]. A recent study indicated that adipokine (produced from visceral adipose tissue) negatively regulated multiple metabolic functions [160]. Moreover, adipokine centrally releases various inflammatory cytokines and chemokines, including TNF-α, IL-6, and monocyte chemotactic protein-1 (MCP-1) [161]. Aging accelerates the rate of increases in visceral adipose tissue and pro-inflammatory cytokines [162]. Age-associated inflammation positively correlates to visceral adipose tissue levels [163]. This theoretically implies that elevated visceral adipose tissue accounts for age-related alteration in systemic inflammation, consequently impairing insulin resistance and eliciting muscle wasting. TNF-α, recognized as a pleiotropic cytokine, produces diverse cellular changes including apoptosis, proliferation, and inflammation. Although macrophages are considered a primary source for TNF-α production, other specific cells, (e.g. myocytes) also generate this cytokine. TNF-α directly influences insulin resistance [164] when activated in human, animal, and cultured muscle cells [165]. Correspondingly, numerous studies have demonstrated that increased expression of TNF-α in skeletal muscle induces insulin resistance and/or diabetes [165-167]. In fact, TNF-α in skeletal muscle inversely correlates to maximal glucose disposal rate [165]. In an animal study, high fructose diet-mediated insulin resistance is closely linked to TNF-α level [168], while mice ablating TNF-α function and its receptors are protected from obesity-mediated insulin resistance [169, 170]. Furthermore, suppression of TNF-α by its anti-TNF-α antibodies or TNF-α converting enzyme inhibitors, improved insulin sensitivity in obese or non-obese insulin resistant models [164, 171]. As a tight association exists between skeletal muscle and glucose disposal, muscle becomes an important target tissue for therapeutic anti-TNF-α treatment. TNF-α exerts its effects via specific cell membrane receptors (TNFR-1 and TNFR-2), expressed in most nucleated cells [172]. Cross-linking these receptors enhances TNF-α signaling. TNFR-1 functions as a main receptor (more proficiently expressed) [173]. TNF-α induces its most harmful and cytotoxic effects by binding to TNFR-1 [174]. Skeletal muscle cells produce both TNFR-1 and TNFR-2 [175]. TNF-α tends to reduce tyrosin phosphorylation [176] and increase serin phosphorylation [177]. This imbalance of insulin receptor substrate-1 (IRS-1)

16 phosphorylation may cause ubiquinization/proteosomal degradation of IRS-1 or attenuate IRS-1 expression binding to the p85 subunit of phosphoinositide 3-kinase (PI3K), impairing insulin metabolic pathways [177, 178]. On the other hand, suppression of TNF-α activation, via anti- TNF-α-antibody injections, promotes insulin sensitivity by improved insulin receptor phosphorylation in skeletal muscle [178, 179]. TNF-α also appears to diminish signal transduction of protein kinase B (AKT) and consequently impairs insulin-mediated glucose uptake into skeletal muscle cells [177]. In addition, TNF-α attenuates AMP-activated protein kinase (AMPK) activation [170] which is pivotal in the regulation of intramuscular fatty-acid metabolism [180]. AMPK signaling promotes increased rate of skeletal muscle fatty-acid oxidation by stimulating phosphorylation of acetyl-CoA carboxylase (ACC). This results in decreased malonyl-CoA concentration and increased influx of long-chain fatty acyl CoA into the mitochondria by palmitoyl transferase-1 [181]. In regards to this observation, impaired intramuscular fatty-acid metabolism (decreased fatty-acid metabolism) with obesity [182, 183] is associated with attenuated skeletal muscle AMPK activity [184]. When bound to TNFR, TNF-α inhibits AMPK expression through transcriptional up-regualtion of protein phosphatase 2C, which suppresses acetyl CoA carboxylase phosphorylation and fatty-acid oxidation and increases diacylglycerol concentration. These processes result in insulin resistance [170]. IL-1B, a pro-inflammatory cytokine is described in pathogenesis [e.g. type 1 diabetes mellitus (T1DM)] and is associated with impaired pancreatic B-cell structure and function [185, 186]. IL-1 expression is also increased in -cells of T2DM patients, causing high glucose- mediated -cell deterioration and apoptosis [187, 188]. Reduced islet -cell concentration is considered a main factor of the initiation and progression of both T1DM and T2DM [189]. A clinical study using an animal model suggested IL-1 antagonism as a promising solution for improving these inflammatory-related diseases [190]. The IL-1RA, an intrinsic anti- inflammatory cytokine, prevents biologic responses to interleukin-1 [191]. While IL-1 stimulates substantial pro-inflammatory events via binding to its receptor, these harmful events are countered by IL-1RA by prohibiting IL-1 binding to type I IL-1 receptors [185]. An important effect of IL-1RA involves the improvement of glycemic profile. Specific blockade of IL-1 signal transduction mediated by IL-1RA improves glycemic profile and insulin sensitivity [192]. IL-6, recognized as both pro- and anti-inflammatory cytokine, regulates immune systems and is expressed in various cells including myocytes [43, 193]. IL-6 may promote glucose

17 disposal in skeletal muscle via insulin signaling pathways [194, 195]. Although controversial evidence exists, some postulated that IL-6 enhances glucose transport and/or glycogenesis in skeletal muscle [196, 197]. IL-6 also increases signaling transduction for AMPK activation in both skeletal muscle and adipose tissue [198]. Overexpressed IL-6 concentration can protect animals against high fat-induced obesity and insulin resistance [195]. However, conflicting data demonstrate negative effects of IL-6 on insulin resistance and glucose metabolism in skeletal muscle. Kim et al. [199] reported that acute IL-6 administration blunted insulin signaling- induced glucose uptake. This event coincides with impaired PI 3-kinase (PI3K) activity (insulin- mediated and IRS-1-related) and increased fatty acyl-CoA level in skeletal muscle. Other studies demonstrated that IL-6 attenuates IRS-I, GLUT-4, and peroxisome proliferator-activated receptor- (PPAR- ) expression [25, 200]. In addition, IL-6 appears to recruit IRS-1 to IL-6 receptor cascades in order to stimulate transient phosphorylation of IRS-1serine, resulting in IRS-1 ubiquination in myotubes [201]. IL-10, an anti-inflammatory cytokine, contributes to glucose metabolism in skeletal muscle [193]. This molecule, expressed in a wide range of immune cells including monocytes/macrophage, appears to suppress activation of pro-inflammatory cytokines and chemokines [202]. IL-10 administration enhances insulin sensitivity and protects myofibers from obese-induced increase in inflammatory cytokines, macrophage infiltration, and other harmful effects impairing insulin signaling pathways [203]. It was reported that IL-10 also highly synthesized in the adipocyte macrophages in lean mice appeared to protect adipose tissue against TNF-α-mediated insulin resistance [204]. Furthermore, cotreatment of IL-10 attenuates adverse effects related to IL-6 and hyperglycemia on insulin resistance in the liver [199]. In this respect, IL-10 might deserve great attention due to its beneficial effects, i.e. anti-inflammatory properties and insulin sensitivity improvement in both liver and skeletal muscle. Chemokines, implicated in various systemic inflammatory disorders and pathogenesis (e.g. atherosclerosis), are activated via attraction, infiltration, and activation of leukocytes [205]. The chemokines, MCP-1 and IL-8 are substantially expressed in those diagnosed with obesity and atherosclerosis [206]. Moreover, increased signaling of these molecules is implicated in T2DM [207]. Studies demonstrated the effects of MCP-1 on glucose metabolism and insulin signaling pathways. Abnormally elevated MCP-1 production in adipose tissues is highly associated with insulin resistance [208, 209]. Skeletal muscle produces MCP-1 and IL-8 and

18 their receptors including IL-8 receptor alpha and beta (CXCR1 and 2 respectively) and C-C chemokine receptor 1,2,4,5, 10 (CCR1, 2, 4, 5, and 10 respectively) [210]. Chemokines and their receptors contribute to chronic low-grade inflammation via attraction and activation of monocytes and T cells [211]. This pathway may be attributed to the insulin resistance in skeletal muscle with activated inflammatory molecules [212]. MCP-1 attenuates insulin sensitivity and considerably diminishes insulin-mediated glucose uptake in skeletal muscle [210]. The physiological mechanism of IL-8 in human skeletal muscle remains inconclusive. Toll-like receptors (TLRs) play a primary role in innate immune systems. These receptors activate both innate and adaptive immune defense systems when recognizing invading pathogens from microbe molecules [90]. Bound TLRs activate signal transduction pathways, including the mitogen-activated protein kinase (MAPK) pathway (initiates transcriptional factors like NF-kB), resulting in the transcription of multiple pro-inflammatory cytokines [213]. Skeletal muscle tissues produce various TLR family members [214]. Of these members, TLR2 induces palmitate-mediated insulin resistance in cultured myotubes by blocking both phosphorylation of an insulin receptor and AKT [215]. A previous study demonstrated positive linear correlation between increased TLR4 and severity of insulin resistance, indicating a strong link between TLR4 and insulin resistance pathways [216]. Correspondingly, other studies indicated that TLR4 is also associated with HFD-induced obesity, inflammation, and insulin resistance [217, 218]. On the other hand, TLR4 depleted mice displayed a considerably reduced capacity of saturated fatty acid (e.g. palmitate), which triggers insulin resistance in skeletal muscle [219]. Moreover, palmitate-induced NF-kB signaling pathway is inhibited by the absence of TLR4 activation (in vivo and in vitro) [219]. TLR2 and TLR4 enhance expression of MCP-1 by binding to their respective ligands, which also stimulates transcriptional pathways of NF-kB [214]. Sarcopenic obesity and adipokine-induced insulin resistance Numerous products secreted from visceral adipose tissue called adipokines appear to mediate insulin resistance through both circulating hormonal or local effects on adipose tissue [220]. A recent study indicated that adipokines negatively regulate multiple metabolic functions [160]. Moreover, adipokines centrally release various inflammatory cytokines and chemokines, including TNF-α, IL-6, and MCP-1 [161]. Increased inflammation positively correlates with visceral adipose tissue levels [163]. This theoretically implies that elevated visceral adipose

19 tissue accounts for systemic inflammation, consequently impairing insulin resistance and eliciting muscle wasting. Leptin secreted by adipocytes regulates energy balance by controlling food consumption and energy expenditure [221]. Leptin deficiency or mutation of its receptor appeared to stimulate substantial hyperphagia and obesity in both animal [222] and human studies [223]. On the other hand, it is suggested that leptin tends to stimulate pro-inflammatory cytokines, such as TNF-α and IL-6 by macrophage [224]. It is also demonstrated that elevated leptin levels are closely correlated to increased risk of coronary artery disease [225]. Adiponectin, a protein hormone largely secreted from adipose tissue into circulation, regulates metabolic processes such as fatty acid catabolism [226]. In contrast to other adipokines, circulating levels of adiponectin inversely correlated with body fat percentage, particularly abdominal fat [212]. The beneficial effects of adiponectin include improving insulin sensitivity and decreasing the risk of atherosclerosis and hypertension [220]. Adipokine stimulates energy expenditure and fatty acid oxidation via activation of AMP-activated protein kinase (AMPK) and also increases expression of PPARα target genes including CDγ6, acyl-coenzyme oxidase, and unclupling protein 2 [227]. Moreover, adiponectin exhibits anti-inflammatory effects by decreasing TNF-α secretion by macrophage and limiting activation of NF-kB [228]. Adiponectin also possess vascular-protective effects by stimulating endothelial nitric oxide production or modulating concentration of adhesion molecules and scavenger receptors [227, 229]. Resistin, a cystein-rich protein, was first identified in animal models as an adipocyte- derived factor, which decreased after treatment with thiazolidinediones (TZD) [230]. Both animal and human studies indicated that resistin from adipocytes induced insulin resistance [231, 232]. Resistin has been linked to inflammatory responses as it increases transcriptional signaling, leading to escalated expression of pro-inflammatory cytokines:IL-1, IL-6, and TNF-α [233, 234]. Moreover, resistin increases expression of intracellular adhesion molecule-1 (ICAM1), vascular cell-adhesion molecule-1 (VCAM1), and chemokine ligand-2 (CCL2), which are involved in leukocyte recruitment to infection sites [235]. A recent study demonstrated positive correlations between obesity, insulin resistance, and chronic inflammation possibly regulated partly by resistin activation [236]. Collectively, as resistin induces insulin resistance and inflammatory response, speculatively resistin might exist as a link between inflammation and insulin resistance [150].

Inflammation-Mediated Sarcopenia

Inflammatory cytokine-induced impairments in anabolic systems and sarcopenia IGF-I is known as a primary contributor to cellular growth and differentiation [237]. The anabolic effect of insulin has been well established [238]. It has been generally accepted that IGF-I promotes myogenesis in skeletal muscle by stimulating myoblast differentiation and fusion into multinucleated myotubes [239]. An early study has suggested that increased differentiation depends on the biosynthesis rate of insulin receptors [240]. As observed with insulin administration, IGF-I also enhances myonuclear accretion into existing myotubes, leading to myofiber hypertrophy [241]. Interestingly, IGF-I receptor accretion is reduced approximately 50% during fusion into myotubes compared with proliferation into myoblasts [240]. This suggests that despite similar characteristics between the two, IGF-I and insulin have divergent effects on proliferation and differentiation in skeletal muscle. IGF-I expression is elevated during regeneration of skeletal muscle, leading to the production of new muscle fibers [242]. IGF-I promotes proliferation and differentiation of satellite cells [243], and regulates the timing of myogenic regulatory factors (MRFs) and other cell cycle regulators in order to enhance muscle regeneration [244]. Moreover, it stimulates hypertrophy of existing myofibers by increasing activation of the IGF-I receptor-related pathway, resulting in increased protein synthesis and decreased protein breakdown [245, 246]. It has been demonstrated that IGF-I-mediated intracellular signaling is activated to enhance protein synthesis through increased activation of PI3K-AKT-mamalian target of rampmycin (mTOR) and reduced activation of glycogen synthase kinase 3 (GSK3 ), leading to myofiber hypertrophy [247]. In addition, as a stimulator of protein translation, PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate, a phospholipid component of the cell membrane, providing a lipid-binding platform on the cell membrane for the kinase known as AKT1 [247]. Once phosphorylation of AKT1 occurs in the membrane by 3 -phosphoinositide-dependent protein kinase (PDK), it subsequently promotes phosphorylation of diverse proteins involved in protein synthesis, gene transcription, cell proliferation, and survival [247-249]. AKT1 signaling involved in the mTOR pathway consequently stimulates expression of p70S6K, which induces protein translation and reduces activation of 4EBP-1, an inhibitor of the protein eIF4E [250, 251]. More specifically, p70S6K activation promotes translation of mRNA encoding ribosomal

21 proteins and elongation factors through the phosphorylation of the 40S ribosomal S6 protein [252]. In addition to its anabolic properties, IGF-I also has an anti-catabolic effect on skeletal muscle by way of decreasing forkhead box O (FOXO) transcription factors and muscle specific ubiquitin ligases such as atrogin-1 and muscle ring finger protein-1 (MURF1) [246]. As another pathway associated with PI3K-AKT1 phosphorlyation, glycogen synthase kinase 3 (GSK3) [248] and FOXO1 [253] have been implicated in mediating protein degradation. AKT1 phosphorylation appears to inhibit GSK activation, which in turn prevents synthesis of the translation initiation factor eIF2b. This demonstrates that AKT1 signaling can increase protein synthesis by stimulating translation initiation [247, 248]. Therefore, this pathway not only positively contributes to myofiber hypertrophy but also attenuates muscle atrophy through improvement of protein turnover. Forkhead transcriptional signaling, which promotes synthesis of both MURF1 and atrogin-1, is inhibited by decreased phosphorylation of AKT1 [246, 253]. Therefore, AKT1 inhibits the activation of FOXO1-induced muscle atrophy pathway. IGF-I has received great attention for its ability to stimulate myogenesis and the development of muscle precursor cells [254-256]. Therefore, IGF-I administration has been suggested to prevent or delay muscle wasting [257-259], improve muscle quality, and enhance muscle fatigue resistance in atrophic conditions [260]. In animal models, high expression of IGF- I stimulates myofiber hypertrophy with increased strength in mdx dystrophic mice [261] and minimizes muscle wasting during aging [262]. In this regard, IGF-I treatment could be applied as a therapeutic intervention to age-related muscle wasting conditions. Transgenic mice with overexpression of IGF-I in skeletal muscle has been shown to improve age-related declines in myoplasmic Ca2+ transient and enhance force of single intact fibers from the flexor digitorum [263, 264]. These results suggest that IGF-I treatment can improve Ca2+ restoration during excitation-contraction (EC) coupling in aged skeletal muscle. During the aging process, there are a decreased number of dihydropyridine receptors (DHPRs) and increased rates of uncoupling of DHPRs to ryanodine receptors (RyR) in skeletal muscle [265]. However, IGF-I overexpression considerably increases in the number of DHPRs approximately 52% in the extensor digitorum longus muscle [266]. Consequently, IGF-I improves restoration of specific muscle force in aged myofibers by promoting EC coupling, pathway which is heavily involved in intracellular Ca2+ regulation [264].

IGF-I has a neurogenic effect and can possibly be utilized to address age-related muscle disease. Overexpression of IGF-I appears to reduce impaired regulation of neural transmission associated with age-related motor unit remodeling [267]. Moreover, IGF-I-induced alterations of the motor neuron improve age-related impairment of specific force in single myofibers [268, 269]. These data indicate multiple advantages of IGF-I, such as improvements to age-related declines in skeletal muscle. While the effect of IGF-I on myofiber hypertrophy has been well established, efforts to ameliorate muscle atrophy may have little impact in particular conditions (e.g. aging) or disease states associated with increased pro-inflammatory cytokines. Pro-inflammatory cytokines negate the benefits of IGF-I on skeletal muscle by inhibiting the anabolic signaling pathway. Particularly, TNF-α and IL-1 have been considered primary factors, which induce IGF-I resistance, prevent protein synthesis, and impair proliferation of muscle precursor cells [270, 271], which potentially accelerate muscle wasting in aging populations. Even low concentrations of these pro-inflammatory cytokines attenuate IGF-I-induced protein synthesis in myoblast and C2C12 cells [270, 272]. These pro-inflammatory cytokines also appear to inhibit protein synthesis rate of differentiation in myogenesis, resulting in an attenuation of myogenic differentiation [270, 272]. The impaired proliferation and differentiation of muscle precursor cells exposed to inflammation are explained by pro-inflammatory cytokine-mediated suppression of MyoD [273] and myogenin [270], which consequently impairs myogenesis in skeletal muscle. The deficiency of IGF-I expression is partially explained by increased pro-inflammatory cytokines including TNF-α and IL-1 [274]. As stated earlier, since IGF-I effect is negated by increased inflammatory status [245], age-induced elevation of inflammatory cytokines might play a key role in muscle wasting in older populations. It has been suggested that increased pro- inflammatory cytokines are inversely correlated to IGF-I expression in frail elderly individuals [30]. The local expression of IGF-I is substantially decreased (p<0.01) in animals with CHF compared to a control group and this attenuation is associated with reduced myofiber cross- sectional area (r=0.62; p<0.01) [275]. This evidence suggests that increased pro-inflammatory cytokines and decreased local IGF-I synthesis provide a catabolic stimulus, which consequently leads to muscle wasting. While elevation of circulating cytokines including TNF-α, IL-6 and IL-1 in inflammation-related chronic disease has been well established [276, 277], increased

23 concentration of local pro-inflammatory cytokines and their potential role in the development of muscle wasting has been postulated and received great attention [275, 276, 278, 279]. The deficient level of circulating IGF-I appears to negatively impact muscle mass and accordingly, strength as well. Niebauer et al. [280] found a 13% decrease in leg muscle cross-sectional area, with lower absolute strength (-24%) and strength per unit area of muscle (-14%) in old CHF patients who had a lower IGF-I levels compared with patients who had normal IGF-I level [280]. TNF-α-mediated apoptosis and sarcopenia Age-related alterations in immune function trigger increased levels of catabolic cytokines [e.g. tumor necrosis factor-α (TNF-α)], resulting in sarcopenia [281]. TNF-α activates caspase-8 by binding to its receptor and upregulating apoptotic signaling protein caspase-3, causing myofiber loss during aging [26]. Oxidative stress-mediated TNF-α might deteriorate myogenic regeneration. A recent study supported this assertion by demonstrating a greater susceptibility of aged satellite cells to apoptosis with TNF-α treatment [282]. In addition, aged animals expressed attenuated levels of bcl-2 in comparison to young animals, indicating impaired regenerative capacity in response to muscle damage [282]. Also, apoptosis and inflammation closely interacts with another mechanism involving elevated oxidative stress [281]. ROS can directly damage myofibers and stimulate the expression of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1 [283]. With chronic inflammatory exposure, these mediators trigger a reduction in muscle mass and strength.

Exercise-Induced Changes in Inflammation Level and Sarcopenia

Aerobic exercise training and inflammation in aged muscle The beneficial effects of regular exercise training on biological homeostasis, including improvement in inflammatory status are well established [284]. However, the immune response to exercise may depend on various factors, including type, intensity, duration, frequency, and period of exercise and physical capacity of each individual. For example, strenuous exercise intensity is associated with increased systemic pro-inflammatory cytokines by enhanced NF-kB signaling activation via phosphorylation of IKKα and IKK [11]. On the other hand, moderate regular exercise intensity appears to reduce pro-inflammatory cytokines [285, 286]. One study demonstrated that aerobic exercise has favorable effect on inflammatory profiles, leading to an increase in anti-inflammatory cytokines and a decrease in pro-

24 inflammatory cytokines in skeletal muscle [286]. Kalani et al. [287] reported that life-long voluntary wheel running decreased systemic CRP levels without alterations in IL-6 concentration in old rats. Furthermore, Mazzetti et al. [288] reported that 4 wks of treadmill running reduced TNFR1 expression in the soleus muscle of aged rats. Moreover, they also found the age-related elevation of apoptosis signaling including caspase-8, caspase-3, and DNA fragmentation to be attenuated by exercise [288]. It has been suggested that pro-inflammatory cytokines might be attenuated with regular exercise via reduction of adipose tissue [289], which is a primary source of TNF-α and IL-6 [290]. On the other hand, body composition-independent effects of aerobic exercise have also been implicated. Seo et al. [291] suggested that chronic aerobic exercise with calorie restriction attenuated age-associated activation of NF-kB in the hepatic cells in old rats [291]. Since an increased inflammatory response is attributable to increased activation of NF-kB [292], it might be conceivable that regular exercise has a favorable effect on improving inflammation in aged muscle. Collectively, regular aerobic exercise is potentially an effective intervention to improve age-related inflammatory alterations, leading to a decrease in risk factors for sarcopenia. The mechanisms underlying the anti-inflammatory effects of aerobic exercise are not fully understood due to its complicated interplay among the cytokines. However, recently it has been suggested that there is a muscle-derived increase in plasma concentration of IL-6 in response to exercise [293]. IL-6 mRNA and protein levels increase during skeletal muscle contraction [294]. IL-6 serves as a crucial biological mediator during exercise when activated and secreted from skeletal muscle. IL-6, similar to hormone actions, influences hepatic and adipose tissues by regulating glucose homeostasis and inducing lipolysis during exercise [295]. Furthermore, elevation of IL-6 expression produces anti-inflammatory cytokines, including IL1RA, sTNFR, and IL-10 [293, 296]. Therefore, it can be implicated that muscle-derived IL-6 secretion by exercise protects skeletal muscle against pro-inflammatory cytokine-mediated impairments. Resistance exercise training and inflammation in aged muscle Resistance exercise training (RET) effectively enhances myofiber hypertrophy and functional outcomes, including strength, power, and mobility in various age groups [33]. With adequate protein consumption, RET cost-effectively attenuates muscle wasting in older individuals. Therefore, it is important to understand underlying mechanisms of RET which

25 alleviate age-induced muscle defects. Previous reports indicated that repeated loading increased number of myofibrils, strength, and power in aged muscle [297, 298]. A recent study suggests that multiple signaling intermediates (IRS-1, AKT, and mTOR) might explain adaptive responses to loading in specific muscle groups [299]. This pathway promotes anabolic responses through increased translational events and attenuates catabolic processes by down-regulating primary mediators of ubiquitination (proteasomal degradation) [246, 247]. RET increases muscle strength and mass via specific bouts of lengthening (eccentric) and shortening (concentric) contraction. Although earlier studies reported little or no differences between the responses elicited by the two contraction types, recent data contrastingly suggested that eccentric contractions optimally induces muscle hypertrophy [300]. Eccentric contraction elicits muscle damage, possibly attracting leukocytes to the injury site. Neutrophils exist in the damage site for up to 24 hours [301-303] and macrophages for 24 to 336 hours after exercise [304-306]. These substrates play a key role in the degradation of damaged myofibers by producing ROS, nitrogen species [307, 308], and pro-inflammatory cytokines (IL-1 and TNF-α) [309]. Skeletal muscle expresses IL-1 and TNF-α for up to 1β0 hours after exercise [310, 311], to promote the breakdown of injured muscle tissue [309]. A study demonstrated that a single bout of RET considerably increases mRNA expression of TNF-α, IL-1 , cyclooxygenase 2 (COX2), and IKK in skeletal muscle [298]. Correspondingly, previous evidence demonstrated transcriptional up-regulation of these pro-inflammatory cytokines [312, 313]. However, aged muscles, compared to young, display greater ultrastructural damage [34], slower recovery rate [35], and blunted inflammatory response [36] following repeated muscle loading (e.g. eccentric exercise), possibly resulting in blunted adaptation to RET. Acute inflammatory responses of immune cells to an exercise bout promotes repair [38] and regeneration of myofibers through growth factors [39]. However, a blunted inflammatory response to mechanical stimuli might interfere with an adaptive response necessary for remodeling in aged damaged muscle. A previous study demonstrated that fewer macrophages and elevated cytokine levels at rest might devastate resting muscles and attenuate the regenerative response to mechanical loading [37]. Furthermore, Hamada et al. (152) suggested that aging might impair the adaptive response of human skeletal muscle to eccentric exercise because of differential modulation of a discrete set of inflammatory and anti-inflammatory cytokine genes. Results demonstrated attenuated increases in IL-6 in aged muscle following a

26 resistance exercise bout when compared to young subjects [36]. As previously mentioned, IL-6 stimulates multiple metabolic adaptations, enhancing component delivery to working muscle, lipolysis, glycogenolysis [314], and production pro-inflammatory cytokine antagonists [296]. Sub-cellular changes, i.e., reduced muscle TNF-α mRNA and protein levels, and increased protein synthesis further illustrates the positive impact of chronic RET in the elderly [315]. In addition, mRNA expression of TNF-α, IL-6, and TLRs in skeletal muscle significantly decreased in obese elderly following 12 wks of concurrent resistance and aerobic exercise [316]. Skeletal muscle is characterized as an endocrine organ for its ability to produce inflammatory cytokines (e.g. TNF-α and IL-6) [317]. As TNF-α level negatively correlates with protein synthesis, reduced TNF-α concentration by chronic RET can improve muscle growth. Additionally, IGF-I concentration (inversely correlated with TNF-α level) increased in the frail elderly in response to repeated loading [318]. Therefore, RET might promote myofiber hypertrophy by increasing anabolic mediators and decreasing catabolic factors [319-321]. However, current research demonstrates that older adults (60 ~ 75yrs) exhibited blunted hypertrophic myofiber and molecular responses to 16 wks of a 3 days/week RET compared to young subjects (20-35 yrs) [322]. Moreover, another study demonstrated reduced muscle precursor cells in resting muscle and diminished proliferative capacity following RET in aged muscle [323]. Exercise-induced ROS generation also damages proteins, nucleic acids and lipids, impairing cellular structure and function [324, 325]. However, chronic RET can restore age- induced mitochondrial dysfunctions that cause sarcopenia [326]. IL-15 is an important contributor to RET-induced muscle hypertrophy [327]. IL-15, the most abundant cytokine in skeletal muscle [328], protects against muscle protein degradation [329, 330] and fat accumulation [331-333]. IL-15 content progressively decreases with aging [334]. IL-15 exerts its anabolic effects (muscle regeneration) in C2 and bovine by stimulating myosin heavy chain synthesis [335]. In a clinical study, IL-15 administration inhibited muscle wasting in cancer cachexia animals by attenuating protein degradation associated with the ubiquitin-proteolysis pathway [329]. Furthermore, Quinn et al. (297) demonstrated that overexpression of IL-15 in the mouse C2 myogenic cell line enhances myotube hypertrophic action, independent of IGF-I signaling. These hypertrophic actions occur through the stimulation of protein synthesis and inhibition of protein degradation, proposing the use of IL-15 administration as a novel approach to improve muscle mass in wasting conditions [330].

Collectively, RET-induced increase in IL-15 expression might maintain or even improve myofiber size under various catabolic conditions, such as age-related muscle wasting.

Anti-Inflammatory Supplements and Sarcopenia

Conjugated linoleic acid and inflammation in aged muscle Conjugated linoleic acid (CLA) contains geometric isomers of 18-carbon polyunsaturated fatty acids from linoleic acid and is generally consumed from red meat fat and dairy products [336]. Of these isomers, cis-9, trans-11 (c9, t11) and trans-10, cis-12 (t10, c12) are recognized as the most biologically active isomers. CLA has received considerable attention due to its health enhancing capacity, i.e. anti-carcinogenesis, anti-atherosclerosis, anti-hypertension effects, and reduction in body fat and diabetes [337]. In addition to these benefits, CLA consists of anti- inflammatory properties in skeletal muscle, i.e. inhibition of TNF-α production following LPS injection [338]. CLA mediates activation of both PPAR-α [339] and PPAR- [340]. CLA- mediated PPAR- exerts anti-inflammatory effects. The effects of PPAR- include: 1) attenuation of pro-inflammatory cytokine activation [341] by inhibiting NF-kB [342], JNK, and p38 MAPK [343], 2) suppression of macrophage activity by inhibiting signal transducer and activator of transcription 1 (STAT1) and NF-kB [342], and 3) inhibition of LPS-mediated synthesis of COX-2 in macrophages through decreases in NF-kB activity [344]. However, several studies suggested that these properties of CLA are exclusive to t10, c12 [345, 346]. Conflicting data suggested that this isomer (t10, c12) is rather involved in antagonizing PPAR- and impairs insulin sensitivity [347-349]. Correspondingly, this isomer has shown to be associated with increased insulin resistance, inflammatory prostaglandins (PGs), and cytokines [350, 351]. However, a more recent study reported that combining c9, t11 with t10, c12 effectively inhibits age-related muscle wasting by reducing pro-inflammatory cytokines, (e.g. TNF-α) and preserving wet muscle mass [10]. In a clinical study, a 0.5% CLA administration reduced muscle mass loss in mice bearing the colon-26 adenocarcinoma, indicating an anti- cachectic effect of CLA on muscle [352]. More recently, it has been shown that CLA treatment improved lipid metabolism and insulin sensitivity [353], which can improve body composition. CLA treatment reduced expression of lipogenic genes while increasing expression of lipolytic genes and the mRNA level of PPAR-α, which enhances lipid metabolism and insulin signaling in skeletal muscle. Moreover, TNF-α level, which induces insulin resistance, markedly decreases, indicating CLA-induced alleviation of insulin resistance [353]. Furthermore, another study

28 reported that lean body mass increased substantially with a combined diet of 1% CLA and 1% n- 3 fatty acid compared to a normal diet in mice [354]. In addition to anti-inflammatory properties, CLA also possesses antioxidant properties, which regulate oxidative stress [355]. CLA demonstrated greater antioxidant ability than α- tocopherol and similar effects exhibited by butylated hyroxytoluene (BHT) [356]. In animal liver, CLA obtained a more potent antioxidant effect compared to vitamin A, protecting microsomes and mitochondria against peroxidative damage [357]. Likewise, Rahman et al. [10] demonstrated enhanced endogenous antioxidant systems and decreased levels of oxidative stress biomarkers after CLA supplementation. While CLA has received great attention for its positive ergogenic effect on body composition, several studies hypothesized that CLA supplementation with RET might synergistically enhance strength as well. Pinkoski et al. [358] discovered that 7 wks of RET with CLA intake induces positive effects which include, 1) greater increase in lean body mass, 2) augmented fat loss, and 3) attenuated elevation of 3-methylhistidine (3MH). The authors demonstrated that CLA administration during RET induces a relatively small change in body composition accompanied by diminished training-induced catabolism. Additionally, previous evidence suggests that combined CLA with creatine supplementation effectively increased lean body mass and strength during heavy RET [359]. Tarnopolsky et al. [360] corroborated these results in an aging model investigation. Older subjects (>65 yrs) performed RET for six months with or without CLA+creatine combined supplementation. Results indicated that CLA+creatine enhanced RET-induced improvements in muscular endurance, strength, lean body mass, and lower fat free mass compared to RET alone [360]. Collectively, CLA administration during RET can facilitate RET-induced benefits for body composition, strength, and functional capacity. Omega-3 poly unsaturated fatty acid and inflammation in aged muscle Omega 3 polyunsaturated fatty acid (n-3) possesses anti-inflammatory and anti-cachetic properties [361]. Clinical research suggested that fish-oil derived n-3, particularly eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6), is widely used to manage various inflammatory diseases [362] due to its inhibitory effects on tumorigenesis and inflammation [363, 364]. The prostaglandins and leukotrients derived from EPA via COX and lipoxygenases (LOX) possess less potent pro-inflammatory effects compared to their counterpart, arachidonic acid (AA) [365]. Increasing n-3/AA ratio might cause anti-

29 inflammatory effects, since this process induces less powerful lipid inflammatory mediators. Supplementation of n-3 during inflammatory conditions demonstrates these effects [366, 367]. Moreover, n-3 also plays a crucial anti-inflammatory role by inhibiting the NF-kB pathway. Reports indicated reduced levels of TNF-α, IL-1, and IL-6 production after LPS treatment of monocytes and lymphocytes following n-3 fatty acid intake [368]. One of the primary mechanisms associated with sarcopenia is the attenuated regeneration capability of muscle precursor cells. TNF-α attributes to muscle wasting associated with inhibition of myogenesis in myoblasts and myoblast and myotube apoptosis [361]. TNF-α in human myoblasts or murine C2C12 myoblasts inhibits myosin heavy chain expression and myogenic differentiation [369]. Moreover, TNF-α-induced apoptosis impairs differentiated myotubes [370]. Elevation of TNF-α concentration increased in myoblast apoptosis and attenuated myogenic differentiation [371]. Differentiated myotubes expressed increased total myofiber protein due to elevated levels of TNF- α (via MAPK pathway) [372]. However, these anabolic effects were counteracted through TNF-α induced apoptosis pathway by enhancing caspase-3 activation [370]. While several studies suggested that dietary supplement improved muscle mass, a recent study demonstrated positive effects of n-3 consumption on muscle regenerative capacity. Magge et al. [361] reported that EPA treatment inhibited detrimental effects of TNF-α on CβC1β myogenesis. This in turn decreased TNF- α mediated loss of myosin heavy chain expression, increased myotube fusion, and restored myotube diameter indices to normal levels [361]. Furthermore, a study reported that chronic intake of long chain n-3 also stimulated activation of the Akt-mTOR-S6K1 signaling pathway [373]. Therefore, it would be suggested that n-3 can improve muscle wasting during aging by improving protein synthesis and regeneration capacity in skeletal muscle. Interestingly, when n-3 was given in combination with CLA, the adverse effects of CLA were reduced or even reversed. This reversal was associated with decreased hepatomegaly, and liver fat and improved lipodystrophy, leptin, and adiponectin levels, thus improving metabolism [374]. These supplements can attenuate RET-induced adverse effects (e.g. abnormal increases in oxidative stress and NF-kB-mediated protein degradation) with improved muscle regeneration. Therefore theoretically, consumption of these anti- inflammatory and antioxidant supplements at rest or during RET may improve sarcopenia and facilitate RET-induced hypertrophy in older individuals.

Magnetic Resonance Imaging Tracking Changes in Skeletal Muscle

Muscle architecture is defined as the primary factor associated with mechanical behavior of skeletal muscle [375, 376]. Muscle architecture, characterized as the arrangement of myofiber relative to the axis of force generation [375], is assessed by measuring various parameters, [e.g. myofiber length and cross-sectional area (CSA)] [377]. Although no technology exists for human trials, the most current magnetic resonance (MR) technology, using one of the world’s largest magnets (900 MHz) at the National High Magnetic Field Laboratory (NHMFL), provides a unique opportunity to perform longitudinal in vivo analyses on changes in muscle architecture and metabolic properties using small rodent models. The NHMFL allows researchers to utilize a non-invasive MR technique termed Diffusion Tensor Imaging (DTI) to study the microstructure of muscle tissue. DTI is based on the principle that cellular diffusion of water corresponds to cell geometry in muscle. The advantage of DTI is that the random diffusion of water molecules can probe with far greater detail than general imaging techniques [378, 379]. The main characteristic of the DTI analysis include the mean diffusion of water and the 3 principle directions of water diffusion, denoted as eigen vectors 1, 2, and 3 [378, 379]. Tseng et al. [380] suggested that eigen value 1 represented the long axis, while 2, and 3 were situated perpendicular to the 1. They demonstrated that 2 represented diffusion transport parallel to the myocardial sheets, while 3 represented diffusion within the myocardial cells (e.g. CSA). Galban et al. [381] demonstrated that in skeletal muscle tissue, 1, 2, and 3 correspond to diffusive transport along the long axis, endomysium, and CSA of a muscle fiber respectively. Diffusion-weighted MR imaging As previously mentioned, DTI is based on the principle that cellular diffusion of water corresponds to muscle cell architecture. The apparent diffusion coefficient (ADC), defined as the diffusivity of water, is restricted in muscle by the presence of cell membrane and diameter. Thus permeability of the sarcolemma following damage increases, while cell swelling enlarges the cell diameter resulting in lower restriction of water and an increase in the ADC [378, 379]. The ADC increases following ischemia reperfusion injury in mouse muscle [378, 379]. Eigen values 1, 2, and 3, represent the long axis of the muscle fiber, and the long and short cross-sectional axes of myofiber, respectively. In fact, following an ischemic event, it was reported that both 2 and 3 in mice having reperfusion injury increases while 3 has greater augmentation and far greater correlation with the tissue damage evaluated by histology [378].

Based on this assumption, the short axis would indicate the effect of cell swelling following muscle damage.

Summary of and Future Research Implications

In summary, sarcopenia is associated with a decline in muscle mass, strength, and functional capacity, consequently devastating quality of life in older individuals. Chronic low- grade systemic inflammation is closely correlated with development of a HFD-induced sarcopenic process. RET has been suggested as one of the most effective interventions for improvement in muscle mass and strength in aging populations. However, aged muscle appears to have blunted adaptation in response to RET due to undesired effects (e.g. elevated NF-kB activity, impaired myogenesis and protein turnover, and increased oxidative stress, etc.). Based on the properties of CLA and n-3, it can be hypothesized that these supplements can improve sarcopenia by attenuating resting inflammation and facilitating RET-induced improvements.

CHAPTER THREE RESEARCH DESIGIN AND METHODS

Overview of Experimental Design

A longitudinal design served to investigate the sarcopenic process utilizing repeated in vivo MR and dual energy X-ray absorptiometry (DXA) measurements in a middle aged mouse model for 20 weeks, emphasizing the importance of chronic CLA/n-3 administration at rest and with programmed long-term RET. C57BL/6 male mice [N=60, 8 month old, 50 for experimental groups (10/group, 5 groups) + 10 for in vitro baseline] were housed in a temperature controlled room on a 12:12h light-dark cycle. The present study consisted of two phases: Phase (I) 1-month acclimation period followed by a 20-week treatment with monthly in vivo MR analysis and Phase (II) in vitro molecular analysis. Animal housing and all in vivo assessments were completed at the National High Magnetic Field Laboratory (NHMFL) at the Florida State University. During 1-month acclimation period, all animals underwent the first set of in vivo MR, DXA, and functionality assessments. After acclimation, ten animals (9-month old) were randomly selected by using random number tables and sacrificed for baseline in vitro analyses. The remaining mice were then randomly assigned into five experimental groups (n=10/5 groups): 1) normal diet control (C), 2) HFD control (H), 3) HFD+RET (HE), 4) H+CLA/n-3 (HCN), and 5) HFD+RET+CLA/n-3 (HECN). Once all experimental procedures were completed, all animals underwent post-treatment functionality measurements. They were then sacrificed; skeletal muscles including the gastrocnemius, soleus, quadriceps, and hamstrings from hindlimb and triceps and forearm flexor from forelimb were isolated for all in vitro analyses. Body mass of all animals were measured every 6th day.

Resistance Exercise Training

One-Month Acclimation Period The one-month acclimation phase served to attenuate any potential differences resulting from unfamiliar environments, e.g. new housing facility, functional tests, and ladder climbing protocol, allowing animals to psychologically and physiologically acclimate to the experimental environment before the 20-week experimental period.

Ladder Climbing Resistance Exercise Training Protocol Programmed RET was conducted for RET groups [group 3 (HE) and 5 (HECN)] using the ladder climbing resistance exercise protocol. The construction of the ladder climbing apparatus included a 100 cm length at 1 cm grip grid at 85° [382]. Before the initial exercise protocol, all RET groups were familiarized with the apparatus for three days without weight. The weight was attached to the base of the tail with foam tape and clip. RET groups were trained three times per week for 20 weeks by ladder climbing. The initial load was 50% of their body weight and increased progressively (1% of body weight on a biweekly basis) through the training period. The training protocol consisted of 4 sets of 3 repetitions with 1-min rest intervals between repetitions and 2-min between sets. Two identical resistance sets and two ladders were prepared for RET, implemented by two investigators. The order of training groups was rotated to minimize any potential variation induced by each trainer’s methods. Specific distances were divided and labeled at 7 points along the ladder climbing apparatus. Climbing up to 7 point (top of the ladder) represents the completion of one repetition. Each training session was performed to exhaustion or the successful completion of 3 sets. Criteria for animal exhaustion (training cessation) included: 1) sliding down the ladder despite assistance provided by the investigator in the form of pushing at the hips twice, and 2) failure to reach the pre-labeled 4 distance points within 90 seconds. Measurements of climbing distance, climbing time, loaded weights, and other notable conditions were recorded for every training session.

CLA/n-3 Mix Administration

During the acclimation period, all animals were fed the normal lab diet. Control group was fed with a normal diet (10% fat by energy, D12450B; Research Diets, New Brunswick, NJ). The animals then were given HFD (60% fat, D12492; Research Diets) for 20 weeks. The CLA/n- 3 mix diets were administered with 1% of CLA (0.5% of c9, t11 and 0.5% t10, c12) and 1% of PUFA enriched in n-3 fatty acids donated by Ocean Nutrition. Daily portions of meals were stored in sealed plastic bags, in a -20°C to minimize oxidation of the fatty acids. This procedure, sometimes overlooked, is important since oxidized fatty acids may produce undesirable effects. A new food was served in the morning on each day throughout the experimental period and old food was discarded. Daily food consumption was measured every day. Upon termination of this study, the average amount of total and daily consumption was calculated.

Specific Aim 1

Specific Aim 1 was to determine the degrees by which CLA/n-3 mix administration for 20 weeks modulates body composition [body weight (BW), lean body mass (LBM), and fat mass (FM)] and myofiber dimensions during HFD in middle aged animals with or without programmed RET. Working Hypothesis Aging would impair body composition and myofiber dimensions and greater impairments would be accompanied with HFD. Twenty-week CLA/n-3 administration would alleviate these impairments with or without RET and would provide the greatest improvements during RET. Design for Aim 1 Changes in body composition and myofiber diameter would be measured before and after 20-week CLA/n-3 administration with or without RET. The design was a 5 (experimental conditions) x 2 (time: pre- and post-treatment) repeated measures ANOVA. In vivo Analysis Using Dual Energy X-ray Absorptiometry (DXA). Mice were anaesthetized and their whole body was assessed in vivo by means of DXA (iDXA; GE Healthcare) to determine body weight (BW)(g), lean body mass (LBM)(g), and fat mass (FM)(g). DXA measurements were performed before and after the 20-week experimental protocol. In vivo MR Diffusion Tensor Imaging (MR DTI) Technology. To investigate alterations in myofiber dimensions, in vivo MR DTI was applied to four animals in each group. This technology provided details about morphological changes in muscle fiber size and density. In vivo MR DTI procedures were performed hindlimb muscles (the gastrocnemius, plantaris and soleus) for dimensional changes. DTI is based on the principle that the cellular diffusion of water corresponds to cell geometry in muscle. The DTI protocol provided the mean diffusion of water within muscle and 3 principle directions of water diffusion denoted as eigen values 1, 2, and 3 ( 1, 2, and 3), [378, 379]. In skeletal muscle tissue 1, 2, and 3 correspond to diffusive transport along the long axis, endomysium, and cross sectional area of a muscle fiber respectively [381]. The MR probe with mice was inserted into the magnet bore and the scan performed, lasting approximately 3-4 hrs. The full MRI procedure consists of the following steps: 1) loading of the animal in the appropriate MRI coil; 2) positioning of the MRI coil in the magnet; 3) setup scans to localize the region of interest (i.e. the hind limb) and optimize signal acquisition; 4)

35 performance of the experimental scans; 5) removal of the animal from the magnet; 6) recovery of the animal from anesthesia under warming conditions (e.g. heating pad); and 7) return of the awake animal to their own cage.

Specific Aim 2

Specific Aim 2 was to examine the extent of which 20-week CLA/n-3 administration improves muscle strength and sensorimotor function during HFD in middle aged animals with or without programmed RET. Working Hypothesis During aging, there would be impairments in muscle strength and sensorimotor function. HFD would cause more of a deteriorating effect. Twenty-week CLA/n-3 administration would attenuate these impairments with or without RET, with greater improvements ensuing during RET. Design for Aim 2 Changes in muscle strength and sensorimotor function were measured before and after 20-week CLA/n-3 administration with and without RET in middle-aged animals. The design was a 5 (experimental conditions) x 2 (time: pre- and post-treatment) repeated measures ANOVA. Muscle Strength (Grip strength) Test. The purpose of this test was to evaluate the strength of the animal’s limb muscles, and to quantify the deterioration of muscular function associated with aging. In the grip strength test, the animal’s forelimbs were placed on a tension bar while it was restrained manually by the scruff of the neck and base of the tail with a towel placed over the animal. The mouse was gently pulled back until it loses its grip from the bar. The force generated as it attempted to maintain its grip was measured in grams by a strain gauge (DFS-101 Force gauge, AMETEK TCI, CA). Each animal was subjected to three trials with the greatest force of the three trials being the criterion measure. Inclined Plane (Sensorimotor Coordination) Test. The inclined plane test was previously developed by Murphy et al. [383]. Each animal was placed onto the surface of a rectangular Plexiglas plane (60 x 122 cm) inclined at a fixed angle (beginning at 50 degrees). Animals were placed facing the upper edge of the plane at a distance approximately 10 cm from the top and are released after a 5-s delay allowing stable footing. If the animal remained on the surface of the Plexiglas without sliding backwards for 5 sec, the trial was successful. The animal was given a maximum of three trials at any given angle and scored as a “fail” if all three trials

36 were unsuccessful. The angle was then declined by 2 degrees and repeated following a rest period of at least 5 min. Testing was discontinued if animals failed on two consecutive angles of inclination. Angle of first fall, total number of falls, and threshold angle (last angle at which animal succeeded at least once) was recorded for each animal.

Specific Aim 3

Specific Aim 3 was to examine the extent to which 20-week CLA/n-3 administration improves inflammation state, myogenic and mitogenic capacity, and protein turnover (synthesis and degradation) during HFD in middle aged animals with or without programmed RET. Working Hypothesis Aging would negatively modulate relative biomarkers, with even greater detriments with HFD. Results of mRNA analyses would indicate a down-regulation of regulatory factors associated with muscle regeneration [myogenic regulatory factors (MyoD and myogenin) and mitogenic factors (IGF-IEa)], protein synthesis (Akt and mTOR), and anti-inflammatory cytokines (IL-6 and IL-15). There would be up-regulation of regulatory factors associated with protein degradation (atrogin-1 and MURF1), pro-inflammatory cytokines (TNF-α and IL-1 ). Again, HFD would augment these detriments. Twenty-week CLA/n-3 administration would positively modulate these biomarkers with or without RET and would enhance these improvements during RET. Design for Aim 3 Target genes associated with muscle regeneration, protein turnover (synthesis and degradation), and pro- and anti-inflammatory cytokines pathway were analyzed before and after CLA/n-3 administration with and without RET in aged muscle. The design therefore analyzed 6 experimental conditions (B, C, H, HE, HCN, and HECN) using one-way ANOVA. If any significant group differences were found, further post hoc tests were performed to localize main or interaction effects. Muscle Tissue Collection. Once all experimental procedures were completed, all animals were sacrificed for in vitro analyses. Each mouse was anesthetized using a 4.0-4.5% v/v isoflurane gas in medical grade oxygen, followed by a continuous flow of 2% v/v isoflurane gas through a vapor system throughout surgery. An incision through the skin from the medial side of the thigh to the abdomen was made, ensuring to stay subcutaneous and to avoid any major

37 arteries or veins. The skin was then reflected to expose the muscles of the lower leg. The muscles were kept moist during the dissections with isotonic saline (0.9 % NaCl). With scissors, an incision along the white facial line demarcating lateral aspects of the lower leg was made from the ankle to 3-5mm proximal from the ankle. The Achilles tendon was then cut and the plantar flexors reflected proximally while cutting the medial fascia of the posterior compartment. All muscles (gastrocnemius, soleus, quadriceps, and hamstring) were cut from the distal aspect using forceps and scissors, weighted, and snap frozen in liquid nitrogen. Isolated tissues were stored at -80°C for target mRNA analyses. RNA Isolation. The pre-weighed muscle samples (30~50 mg) were used for RNA isolation following a method developed previously by Dr. Adams [384-387]. Once each sample was homogenized by 1ml of TRI reagent (Molecular Research Center, Inc., Cincinnati, OH), BCP reagent (Molecular Research center, Inc., Cincinnati, OH) was added to separate phase. After the homogenized-tissue solution was precipitated with isopropanol, the extracted RNA pellet was washed out with an ethanol and water solution consisting of 75% ethanol and 25% diethylpyrocarbonate (DEPC) water. The pellet was completely dried with a speed dry centrifuge (HETO power dry centrifuge) for 5min and dissolved in a pre-calculated volume of nuclease-free water. The total RNA quantification was measured by using a ND-1000 Spectrophotometer (Nanodrop products, Wilmington, DE). All RNA samples were stored at -80°C for subsequent RT-PCR procedure. Reverse Ttranscription Polymerase Chain Reaction (RT-PCR). RT-PCR procedure has been well developed [384-387]. As described, one g of RNA was used for reverse transcription with a combination of nuclease-free water (5 PRIME, Inc.), 4 l of 5x FS buffer (Invitrogen), 2 l of 100mM DTT (Invitrogen), 1 l of 10mM dNTP (Invitrogen), and 2 l of oligo-dT/random primer mix (Invitrogen). After the pre-incubation at 44 C for 5 min, 1 l of SuperScript II Reverse Transcriptase (200 U/ l) (Invitrogen) was added. The RT reaction mixture was incubated at 44 C for 50 min, denatured at 88 C for 5 min, cooled at 2 C for 1 min, spined down to collect all at the bottom, and then stored at -80 C for subsequent PCR analysis. We designed each set of forward and reverse primers using DNA Star Laser-gene 7 software. The designed primer was tested to decide optimal protocol (e.g., PCR cycles and cDNA amounts added). For each PCR reaction, 18S (324-bp product) was coamplified with each target gene (mRNA) to provide a ratio of target mRNA/18S.

PCR reactions were generated in 25 l of mixture consisting of 2.5 l of 10x PCR buffer (Invitrogen), 0.5 l of 10 mM dNTP (Invitrogen), 1 l of 50 mM MgCl2 (Invitrogen), 0.15 l of Biolase DNA polymerase (Invitrogen), a specific amount of primer set (Integrated DNA Technologies, Inc.), a specific amount of 18S primer/competimer mix (Integrated DNA Technologies, Inc.), and lastly an amount of nuclease-free water (5 Prime Inc.) necessary to keep the final volume of 25 µl of mixture. DNA Engine Peltier Thermal Cycler (BIO-RAD, Hercules, CA) was used for the denaturing step which starts for 3 min at 96°C, followed by a specific number of cycles of 1 min at 96°C, 45 sec at a specific annealing temperature, 45 sec at 72°C, and ending with 3 min at 72°C. Twenty-four l of the 25 l PCR product was separated by electrophoresis (100V) in 2 % agarose gel (BIO-RAD, Hercules, CA), pre-mixed with ethidium bromide solution (0.1 g/ml) (BIO-RAD, Hercules, CA). Gels were run with molecular weight markers (100 bp Hyper Ladder, exACTGene) to confirm the expected sizes of each mRNA. The primer and 18S bands detected were captured under UV light by Chemi Doc XRS (BIO-RAD, Hercules, CA) and density of each band was quantified with manufacturer provided software (Quantity One 4.6.5).

Statistical Analysis and Sample Size Determination

The specific ANOVA models to be used were aforementioned under each specific aim (Statistica, StarSoft, Tulsa, OK). The design was 5 (experimental conditions) x 2 (time: pre- and post-treatment) repeated measures ANOVA for in vivo measurements (i.e. DXA, MR, functionality) while one-way ANOVA was utilized to analyze in vitro data (i.e. wet weight and mRNA expression). Post hoc tests were performed to localize main or interaction effects. Muscle mass was one of the primary outcome measures in the present study and provided the basis for the determination of sample sizes. We determined effect sizes based on previous evidence showing differences in lean body mass in C57BL/6 mice under CLA and/or n-3 treatment conditions [354]. Maintaining α=0.05 and 1-=0.80, a sample size of 10 animals per experimental model for in vivo analyses was required to detect differences between the 5 conditions.

CHAPTER FOUR RESULTS

Food and CLA/n-3 Administration

All macronutrient values for food consumed are presented in Table 1. The fat content was modified to provide 60% kcal from fat mixed in semi-purified animal diet with or without 1% CLA/n-3 fatty acid. The energy consumption (Kcal/gm) differed between normal and high fat diet groups (e.g. normal diet; 3.8Kcal/gm and high fat diet; 5.2Kcal/gm). On average, all groups consumed approximately 2.9 g diet/day during the intervention period. Therefore, the normal diet group (C) consumed significantly lower calories (-4 Kcal/d, p ≤ 0.01) than the HFD groups (H, HE, HCN, and HECN) as demonstrated in Table 2.

Morbidity and Mortality

All animals in C were considered healthy (maintained body weight without hairloss) and survived the entire 20-wk experimental period. All animals in HE and HCN also survived through the entire intervention period. However, 2 mice in H died during the intervention period, and 3 animals in H exhibited detectable abnormalities (i.e. marked hairloss with reduced body mass). Two mice in HCN demonstrated similar aberrations as the two diseased mice in H (i.e. hairloss with reduced body mass) although less severe. One mouse died in HECN before the end of the intervention period. Overall, the rate of morbidity and mortality was greater in H compared to all other experimental groups.

Table 1: Profile of diets

Table 2: Daily food and energy consumption Group N Food (g/d) Energy (Kcal/d) C 9 2.90 ± 0.01 11.03 ± 0.03 H 9 2.89 ± 0.02 15.03 ± 0.06 # HE 10 2.89 ± 0.01 15.02 ± 0.06 # HCN 10 2.89 ± 0.02 15.02 ± 0.1 # HECN 9 2.89 ± 0.02 15.03 ± 0.08 # Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). # p ≤ 0.05, significantly different from C.

Dual X-ray Absorptiometry (DXA) Determined Body Composition Total body mass (TBM), lean body mass (LBM), and fat mass (FM) were determined using Dual X-ray Absorptiometry (DXA) (Table 3). There was a significant group x time interaction (F (1,4) = 6.04, p ≤ 0.01, ES = 0.37) and group effect (F (1,4) = 6.01, p ≤ 0.01, ES = 0.36) for TBM in H (+26%), HE (+35%), HECN (+22%) from baseline, while no significant change was found in C and HCN. There was a main time effect for TBM (F (1,4) = 24.20, p ≤ 0.01, ES = 0.37). Interestingly, TBM in HCN was significantly lower than both H (p = 0.024) and HE (p = 0.002) (Figure 1).

Table 3: DXA determined body composition Group N TBM-pre TBM-post LBM-pre LBM-post FM-pre FM-post (g) (g) (g) (g) (g) (g) C 9 31.6 ± 0.9 28.8 ± 0.9 13.9 ± 0.8 10.9 ± 1.1* 14.2 ± 0.7 14.6 ± 1.4

H 9 31.7 ± 0.9 39.8 ± 3.7* 14.8 ± 0.7 10.2 ± 1.1* 14.0 ± 1.2 24.3 ± 3.5* HE 10 31.5 ± 0.7 42.4 ± 1.3* 14.4 ± 0.8 6.5 ± 0.6* 13.4 ± 0.9 32.4 ± 1.6* HCN 10 31.2 ± 0.7 32.7 ± 2.0 14.5 ± 0.6 14.0 ± 1.3 14.1 ± 0.6 16.5 ± 1.4 HECN 9 31.7 ± 0.8 38.7 ± 1.6* 14.4 ± 0.7 15.3 ± 0.8 13.9 ± 1.2 19.8 ± 1.0* Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline.

Figure 1: Comparison of Total Body Mass among conditions. Comparison of TBM among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C ! p ≤ 0.05, significantly different from HCN

There was a significant group x time interaction (F (1,4) = 10.68, p ≤ 0.01, ES = 0.5) and group effect (F (1,4) = 6.74, p ≤ 0.01, ES = 0.39) for LBM in H (-31%, p = 0.0002) and HE (- 55%, p = 0.0000001) from baseline, but not in HCN and HECN. The LBM in HECN was significantly greater than C, H, and HE (p ≤ 0.05) There was a main time effect for LBM (F (1,4) = 39.51, p ≤ 0.01, ES = 0.48) in C (-27%, p = 0.009) (Figure 2).

Figure 2: Comparison of Lean Body Mass among conditions. Comparison of LBM among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C @ p ≤ 0.05, significantly different from H $ p ≤ 0.05, significantly different from HE

There was a significant group effect (F (1,4) = 11.6, p ≤ 0.01, ES = 0.53) and group x time interaction (F (1,4) = 10.7, p ≤ 0.01, ES = 0.51) in H ( +74%, p ≤ 0.01) and HE (+142%, p ≤ 0.01)but not in C and HCN. There was a main time effect (F (1,4) = 54.5, p ≤ 0.01, ES = 0.57) for FM in HECN (+43%, p ≤ 0.05). Interestingly, HCN demonstrated lower FM than both H (p = 0.01) and HE (p = 0.000002) (Figure 3).

Figure 3: Comparison of Fat Mass among conditions. Comparison of FM among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline @ p ≤ 0.05, significantly different from H $ p ≤ 0.05, significantly different from HE

Neuromuscular Functionality

Neuromuscular functionality measured included muscular strength (grip strength) and sensorimotor function (incline plane test). Values are presented in Table 4.

Table 4: Neuromuscular functionality measurements Group N Grip strength Grip strength Incline plane Incline plane (g)-pre (g)-post (°)-pre (°)-post C 9 176.6 ± 5.3 171.5 ± 3.6 33.1 ± 0.6 32.4 ± 1.2 H 9 181.3 ± 5.3 154.5 ± 3.5* 32.4 ± 0.6 28.8 ± 0.9* HE 10 182.1 ± 3.8 198.3 ± 6.6* 32.4 ± 0.7 33.8 ± 0.6 HCN 10 186.0 ± 4.7 154.0 ± 4.9* 31.8 ± 0.6 33.4 ± 0.7 HECN 9 175.1 ± 4.0 213.8 ± 5.8* 32.2 ± 0.5 37.6 ± 1.2* Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline.

There was a significant group x time interaction (F (1,4) = 21.9, p ≤ 0.01, ES = 0.68) and group effect (F (1,4) = 8.9, p ≤ 0.01, ES = 0.46) for grip strength in H (-15%, p = 0.0003) and HCN (-17%, p = 0.000005) while no signiciant change was found in C. There was a significant increase in grip strength for HE (+9%, p = 0.002) and HECN (+22%, p = 0.0000003) from baseline with no difference between HE and HECN at post (Figure 4).

Figure 4: Grip Strength Test among conditions. Comparison of grip strength among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C @ p ≤ 0.05, significantly different from H ! p ≤ 0.05, significantly different from HCN

There was a significant group effect (F (1,4) = 10.2, p ≤ 0.01, ES = 0.49) and group x time interaction (F (1,4) = 9.3, p ≤ 0.01, ES = 0.47) for senrorimotor function in H (-11%, p = 0.001) whereas no significant change was observed in C, HCN, and HE. HECN significantly increased sensorimotor function (+17%, p = 0.00002) from baseline. The increase in HECN was greater than that exhibited by HE and HCN (Figure 5).

Figure 5: Incline Plane Test among conditions (° incline). Comparison of incline plane score among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C @ p ≤ 0.05, significantly different from H $ p ≤ 0.05, significantly different from HE ! p ≤ 0.05, significantly different from HCN

Myofiber Dimensions

To investigate alterations in myofiber dimensions, in vivo MR DTI was applied to four animals in each group. There was a main time effect (F (1,4) = 13.97, p ≤ 0.01, ES = 0.48) for FA driven by significant decrease in HECN (-22%, p = 0.02) from baseline whereas no change was found in all other groups (C, H, and HE). FA in HCN decreased from baseline and approached significannce (p = 0.08) (Figure 6).

Figure 6: Fractional anisotropies among conditions. Comparison of FA values among conditions (four mice from each group) pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

There was no main time or time x group effect for λ1 from pre- to post-interveion. There was a main time effect (F (1,4) = 8.35, p ≤ 0.01, ES = 0.36) for λ2. HECN decreased λ2 from baseline (p = 0.04), but no change occurred in all other groups (C, H, HE, or HCN). There was a main time effect (F (1,4) = 7.26, p ≤ 0.05, ES = 0.33) for λ3 from pre- to post- intervention. HCN significantly increased λ3 from baseline (p = 0.05) while no significant change was found in all other groups (C, H, HE, or HECN) (Figure 7).

Figure 7: Eigenvalues (λ) 1, 2, and 3 among conditions. Comparison of eigenvalues among conditions (four mice from each group) pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

Muscle Mass

There was a significant group effect (F (1,5) = 5.3, p ≤ 0.01, ES = 0.36) for gastrocnemius wet weight driven by decreases in C (-27%, p = 0.005), H (-39%, p = 0.0002), and HCN (-35%, p = 0.0003) from baseline while not in HE and HECN. Both RET groups (HE and HECN) had significantly greater gastrocnemius wet weight than H and HCN by 41% and 34%, respectively (Figure 8).

Figure 8: Gastrocnemius muscle wet weight among conditions. Comparison of muscle wet weight among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline @ p ≤ 0.05, significantly different from H ! p ≤ 0.05, significantly different from HCN

There was a significant group effect (F (1,5) = 5.8, p ≤ 0.01, ES = 0.38) for soleus wet weight. No significant difference was found in C, HE, HCN, and HECN compared to baseline, but H significantly decreased soleus wet weight (-24%, p ≤ 0.01) at post-intervention. Soleus wet weight in both RET groups (HE and HES) were significantly greater than C, H, and HCN (Figure 9).

Figure 9: Soleus muscle wet weight among conditions. Comparison of muscle wet weight among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C @ p ≤ 0.05, significantly different from H ! p ≤ 0.05, significantly different from HCN

There was a main group effect (F (1,5) = 4.32, p ≤ 0.01, ES = 0.5) for quadriceps wet weight in C (-33%), H (-43%), HE (-23%), HCN (-43%), and HECN (-23%) from baseline. Quadriceps wet weight in H and HCN was significantly lower than HE and HECN (p ≤ 0.01) (Figure 10).

Figure 10: Quadriceps muscle wet weight among conditions. Comparison of muscle wet weight among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline @ p ≤ 0.05, significantly different from H ! p ≤ 0.05, significantly different from HCN

There was a significant group effect (F (1,5) = 11.87, p ≤ 0.01, ES = 0.55) for hamstrings wet weight, showing lower wet weight in all groups (C -43%, H -47%, HE -32%, HCN -42%, and HECN -32%) compared to baseline. Hamstrings wet weight was significantly greater in both RET groups (HE, 28% and HECN, 29%) compared to H and HCN (Figure 11).

Figure 11: Hamstrings muscle wet weight among conditions. Comparison of muscle wet weight among conditions pre and post 20-week intervention. Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline @ p ≤ 0.05, significantly different from H ! p ≤ 0.05, significantly different from HCN

Changes in Transcript Factors

Changes in relative gene expressions are presented as densitometry values of the target gene divided by the 18s rRNA as an internal control. Regulators of inflammation There was no significant group effect for TNF-α in the gastrocnemius. However, post hoc revealed that relative mRNA expression of TNF-α was significantly higher in H compared to C (p = 0.02), HE (p = 0.006), HCN (p = 0.03), and HECN (p = 0.02) (Figure 12).

Figure 12: Relative RT-PCR results for TNF-α mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). @ p ≤ 0.05, significantly different from H

There was a significant group effect (F (1,5) = 6.18, p ≤ 0.01, ES = 0.41) for TNF-α mRNA expression in the quadriceps. Significantly decreased TNF-α expression was found in C (p = 0.009), H (p = 0.0003), HCN (p = 0.000009), and HECN (p = 0.01) from baseline, but not in HE. In addition, HCN had significantly lower TNF-α levels than C (p = 0.02), HE (p = 0.002), and HECN (p = 0.02). TNF-α expression was greater in HE than H (p = 0.02) (Figure 13).

Figure 13: Relative RT-PCR results for TNF-α mRNA expression using 18S ribosomal RNA as an internal standard (quadriceps). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline @ p ≤ 0.05, significantly different from H ! p ≤ 0.05, significantly different from HCN

Although there was no significant group effect for relative IL-1 mRNA expression in gastrocnemius, our post hoc revealed that IL-1β mRNA expression in HCN was significantly greater compared to baseline (p = 0.01). Also, IL-1β levels in HCN were greater compared to H (p = 0.04), HE (p = 0.003), and HECN (p = 0.03) (Figure 14).

Figure 14: Relative RT-PCR results for IL-1β mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline ! p ≤ 0.05, significantly different from HCN

While there was a no main group effect, post hoc in the soleus showed greater IL-1β expression of in HCN (p = 0.03) from baseline. Also, HCN had significantly higher IL-1β expression than H (p = 0.04), HE (p = 0.008), and HECN (p = 0.05) (Figure 15).

Figure 15: Relative Relative RT-PCR results for IL-1β mRNA expression using 18S ribosomal RNA as an internal standard (soleus). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline ! p ≤ 0.05, significantly different from HCN

There was a significant group effect (F (1,5) = 3.15, p ≤ 0.05, ES = 0.26) for IL-1β levels in the quadriceps. The IL-1β mRNA expression in HCN was significantly greater (p = 0.003) than baseline. In addition, IL-1β levels in HCN were significantly higher than C (p = 0.02), HE, and HECN (p = 0.004) (Figure 16).

Figure 16: Relative RT-PCR results for IL-1β mRNA expression using 18S ribosomal RNA as an internal standard (quadriceps). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline ! p ≤ 0.05, significantly different from HCN

There was no significant group effect in the gastrocnemius, soleus, or quadriceps for IL- 6 mRNA expression. However, post hoc revealed markedly increased IL-6 expression in HE (p = 0.02), HCN (p = 0.01), and HECN (p = 0.04) compared to baseline in the gastrocnemius (Figure 17).

Figure 17: Relative RT-PCR results for IL-6 mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

The soleus represented that IL-6 was elevated in only HCN (p = 0.04) from baseline (Figure 18).

Figure 18: Relative RT-PCR results for IL-6 mRNA expression using 18S ribosomal RNA as an internal standard (soleus). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

In the quadriceps, HECN greatly increased IL-6 (p = 0.04) from baseline (Figure 19).

Figure 19: Relative RT-PCR results for IL-6 mRNA expression using 18S ribosomal RNA as an internal standard (quadriceps). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

A significant group effect was found (F (1,5) = 3.35, p ≤ 0.05, ES = 0.34) only in the soleus. Post hoc in the gastrocnemius showed that IL-15 levels significantly increased in HCN (p = 0.009) and HECN (p = 0.04) compared to baseline. Moreover, IL-15 levels in HCN was significantly higher (p = 0.04) than H (Figure 20).

Figure 20: Relative RT-PCR results for IL-15 mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline @ p ≤ 0.05, significantly different from H

In the soleus, IL-15 expression in C was significantly higher (p = 0.03) than baseline. IL- 15 levels were significantly lower in H (p = 0.001) and HE (p = 0.003) than C. HECN had greater IL-15 expression compared to H (p = 0.02) and HE (p = 0.05) (Figure21).

Figure 21: Relative Relative RT-PCR results for IL-15 mRNA expression using 18S ribosomal RNA as an internal standard (soleus). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C @ p ≤ 0.05, significantly different from H $ p ≤ 0.05, significantly different from HE

Regulators of Protein Synthesis There was a significant group effect (F (1,5) = 2.63, p ≤ 0.05, ES = 0.23) for the quadriceps. In the quadriceps, Akt in C was significantly higher (p = 0.001) than baseline. Also, C had greater Akt expression compared to H (p = 0.02) and HECN (p = 0.04) (Figure 22).

Figure 22: Relative Relative Relative RT-PCR results for Akt mRNA expression using 18S ribosomal RNA as an internal standard (quadriceps). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C

There was no significant group effect for the mTOR mRNA expression in the gastrocnemius, soleus, or quadriceps. In the gastrocnemius, however, post hoc showed increased mTOR expression in HCN (p = 0.03) from baseline (Figure 23).

Figure 23: Relative Relative RT-PCR results for mTOR mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

Regulators of Protein Degradation Althoug there was no significant group effect for atrogin-1 in the gastrocnemius, soleus, or quadriceps, our post hoc analysis revealed greater atrogin-1 expression in C (p = 0.009) and HCN (p = 0.009) than baseline in the gastrocnemius (Figure 24).

Figure 24: Relative Relative RT-PCR results for atrogin-1 mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

In the soleus, markedly higher atrogin-1 expression was observed in C (p = 0.03) compared to baseline. In addition, atrogin-1 in C was significantly higher (p = 0.01) than H and HECN (p = 0.05) (Figure 25).

Figure 25: Relative Relative RT-PCR results for atrogin-1 mRNA expression using 18S ribosomal RNA as an internal standard (soleus). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). # p ≤ 0.05, significantly different from C

Although there was no siginificant group effect for relative mRNA expression for MURF1 in the gastrocnemius, soleus, or quadriceps, post hoc in the gastrocnemius showd that HCN showed significantly higher MURF1 levels (p = 0.008) than baseline (Figure 26).

Figure 26: Relative Relative RT-PCR results for MURF1 mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

In the soleus, MURF1 mRNA expression in HCN was significantly higher than H (p = 0.02) and HE (p = 0.03) (Figure 27).

Figure 27: Relative Relative RT-PCR results for MURF1 mRNA expression using 18S ribosomal RNA as an internal standard (soleus). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). @ p ≤ 0.05, significantly different from H $ p ≤ 0.05, significantly different from HE

In the quadriceps, only HCN demonstrated significantly higher MURF1 levels (p = 0.009) than baseline while no change was observed in all other groups (Figure 28).

Figure 28: Relative Relative RT-PCR results for MURF1 mRNA expression using 18S ribosomal RNA as an internal standard (quadriceps). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

Regulators of Mitogenesis No group effect was observed for IGF-IEa in the gastrocnemius, soleus, or quadriceps. However, post hoc in the soleus revealed significantly lower IGF-IEa expression in C (p = 0.04) compared to baseline. The decline was even greater in both H (p = 0.008) and HE (p = 0.008) (Figure 29).

Figure 29: Relative Relative RT-PCR results for IGF-IEa mRNA expression using 18S ribosomal RNA as an internal standard (soleus). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

Regulators of Myogenesis No significant group effect was observed for MyoD in the gastrocnemius, soleus or quadriceps. However, post hoc in the soleus revealed significantly lower MyoD expression in HE (p = 0.02) compared to baseline (Figure 30).

Figure 30: Relative Relative RT-PCR results for MyoD mRNA expression using 18S ribosomal RNA as an internal standard (soleus). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline

MyoD expression in the quadriceps was significantly lower in HECN than C (p = 0.03) and HE (p = 0.02) (Figure 31).

Figure 31: Relative Relative RT-PCR results for MyoD mRNA expression using 18S ribosomal RNA as an internal standard (quadriceps). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). # p ≤ 0.05, significantly different from C $ p ≤ 0.05, significantly different from HE

There was a significant group effect for myogenin in the gastrocnemius (F (1,5) = 2.54, p ≤ 0.05, ES = 0.22), but not in either soleus or quadriceps. In the gastrocnemius, HCN exhibited significantly higher myogenin expression (p = 0.03) compared to baseline. Moreover, HCN had greatly higher myogenin expression than HE (p = 0.03) and HECN (p = 0.005) (Figure 32).

Figure 32: Relative Relative RT-PCR results for myogenin mRNA expression using 18S ribosomal RNA as an internal standard (gastrocnemius). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). * p ≤ 0.05, significantly different from baseline # p ≤ 0.05, significantly different from C ! p ≤ 0.05, significantly different from HCN

With on main effects, post hoc in the quadriceps showed that HECN had significantly lower myogenin expression compared to C (p = 0.01) and HCN (p = 0.01) (Figure 33).

Figure 33: Relative Relative RT-PCR results for myogenin mRNA expression using 18S ribosomal RNA as an internal standard (quadriceps). Values are mean ± standard errors. (normal diet control = C; high fat diet = H; high fat diet + resistance exercise = HE; high fat diet + CLA/n-3 = HCN; and high fat diet + resistance exercise + CLA/n-3 = HECN). # p ≤ 0.05, significantly different from C ! p ≤ 0.05, significantly different from HCN

CHAPTER FIVE DISCUSSION

The overarching aim of the present study was to investigate the effects of CLA/n-3 administration with or without resistance exercise training (RET) in middle aged mice on changes in body composition, myofiber dimension, and functional capacity (muscle strength and sensorimotor function) under chronic high fat diet for 20 weeks. We also sought to investigate the possible cellular and molecular mechanisms, which regulated these effects. The major findings of this study were that CLA/n-3, in part, prevented HFD-induced negative changes in body composition with improved inflammatory state. In addition, CLA/n-3 administration attenuated HFD-induced loss of soleus muscle mass with or without RET. Also, chronic RET independently or combined with CLA/n-3 administration attenuated the decline in muscle mass in gastrocnemius, soleus, quadriceps and hamstrings. CLA/n-3 administration also reduced the impairment of sensorimotor function under HFD condition and synergistically facilitated RET- mediated improvements. Strength loss during HFD was significantly improved by RET while no additive effects from CLA/n-3 were observed. CLA/n-3 administration attenuated HDF-induced impairemetnt in sensorimotor function and enhanced RET-induced improvement. Lastly, there was a significant increase in myofiber cross sectional area (i.e. λβ) in HCN and decrease in FA in HECN from baseline.

Morbidity and Mortality

There was greater incidence of morbidity and mortality in the HFD group (H) compared to all other experimental groups, indicating the harmful health effects of HFD. The obese population has been escalating gradually, leading to an increased rate of obesity-associated health problems, morbidity, and mortality [388]. Our results are consistent with previous findings in humans, which respected to obesity-related morbidity and mortality. Adams et al. [389] investigated the relationship between body mass index (BMI) and risk of death in 527,265 U.S male and female and found a greater risk of death with higher BMI independent of gender. Moreover, middle-aged individuals (age of 50 years) exhibited increased mortality rate of 20 to 40 percent among overweight while risk was less among those underweight. Thus, obesity is highly regarded as the most preventable cause of mortality [390]. Obesity is linked to the development of metabolic syndrome such as T2DM and hyperlipidemia. It has been generally

77 accepted that the primary factor mediating obesity is sedentary lifestyle accompanied with unbalanced energy consumption (i.e. high caloric diet). As excessive visceral fat serves as a large contributor to a state of chronic systemic inflammation, obesity may greatly increase the risk for inflammation-based pathologies and mortality. It was suggested that elevated inflammatory cytokine levels coincide with decreased LBM and increased FM in the older population [21]. In fact, Zimmermann et al. [391] demonstrated a close association between increased levels of CRP and cardiovascular-related mortality. Collectively, it is conceivable that HFD-induced obesity promotes an increased rate of mortality, which is possibly attributable to a chronic inflammation and co-morbid diseases.

Body Composition

As anticipated, there was a significant increase in TBM in H after 20 weeks of HFD. This increase was found in HE and HECN as well. The greater final body weight is likely associated with higher energy intake compared to the normal diet group. Our results indicated that an increase in TBM was mainly attributable to FM gains. These findings agreed with Poudyal et al. [142] who reported that rats consuming high caloric diet through high fat and carbohydrate feeding exhibited significant weight gain accompanied by greater abdominal fat pads than the normal diet-fed control group. Martins et al. [140] also demonstrated a significant increase in body weight in C57BL/6 mice under 10 weeks of HFD. Based on these findings, it is clear that greater energy intake with sedentary lifestyle results in a positive energy balance, resulting in increased TBM mainly from fat gains. Unexpectidly, we observed that RET under HFD condition induced a greater increase in FM than sedentary condition, which does not accept our hypothesis. These results are in conflict with Dunstan et al. [392] who demonstrated a marked decrease in FM of older T2DM patients following 6 months of high intensity RET. However, Krisan et al [393], in agreement with our results, reported no differences between HFD-fed animals undergoing sedentary conditions or 12 weeks of RET. They also demonstrated that epididymal fat pad mass was significantly greater in Sprague-Dawley rats fed HFD compared to the normal diet counterpart. Our data demonstrated that FM was maintained or had a relatively smaller increase in limb areas compared to the trunk region following RET. This likely occurred because our RET program utilized a ladder climbing apparatus that directly loaded the fore and hind limbs rather than the whole body (i.e. trunk). Therefore, it might be conceivable that a full body RET program loading a larger region of the body would promote

78 greater fatty acid catabolism. In support of this idea, Hunter et al. [394] reported that after 25 weeks of full body RET, older men and women showed significant decreases in FM accompanied by reductions in both subcutaneous and visceral adipose tissue. An intriguing finding of the present study was that CLA/n-3 administration attenuated or prevented the HFD-induced increase in TBM. This improvement was associated with blunted FM gain while maintaining LBM with CLA/n-3 administration. Our results concurred with previous studies demonstrating the fat-reducing effects of CLA in various rodent species, including mice [395-397] and rats [398, 399]. Also, other previous studies demonstrated the positive effects of n-3 long chain polyunsaturated fatty acids on lipid metabolism in both rodent [400] and human [401] models. In addition, Sneddon et al. [402] suggested that CLA/n-3 supplementation prevented fat mass gains in young obese humans. Although CLA/n-3 administration appears beneficial as a fat reducing agent, our results failed to display any additive effects on fat metabolism with concurrent RET administration. Although the mechanisms mediating the actions of CLA and n-3 on fat metabolism remain equivocal, activation of peroxisome proliferator activated receptor-α (PPAR-α), which enhances lipid metabolism and insulin signaling in skeletal muscle, may be associated. In fact, Inoue et al. [353] demonstrated that 8 weeks of CLA administration increased PPAR-α mRNA expression with lower triglyceride concentration in the Zucker rats compared with control groups. CLA also appeared to increase uncoupling proteins (UCPs), which are expressed in various adipose tissues and mediate lipid catabolism. Takahashi et al. [403] reported that CLA administration increased UCP2 in brown adipose tissue of C57BL/6J and ICR mice. Another plausible mechanism for lipid catabolism is apoptosis of adipocytes. TNF-α, known as a major inflammatory cytokine, appeared to upregulate apoptosis signaling in human adipose cells [404]. When CLA was given to female mice, there was increased mRNA expression of TNF-α in white adipose tissue and reduced in skeletal muscle. In addition, inhibitory effects of TNF-α on lipoprotein lipase (LPL) [405], acetyl-CoA-carboxylase (ACC) and fatty acid synthase (FAS) synthesis was exhibited in previous investigations [406]. Although it appeared that lipid catabolism was mainly driven by CLA, n-3 might also play a significant role as it has been shown to decrease systemic lipid metabolites and induce fatty acid oxidation [407]. The evidence was supported by other studies which showed a reduction in visceral adipose tissue in rats following n-3 administration under HFD condition [408, 409] as this decrease in visceral fat

79 might be closely related to declines in size [410] and number [411] of adipocytes. Based on these findings, CLA/n-3 supplementation appears to exhibit positive effects on lipid metabolism and shows promise as a fat reducing agent. In addition to an increase in FM, significantly reduced LBM was observed in H and HE. This result indicated that HFD mediated a negative alteration in LBM, and RET was not able to prevent or attenuate the impairment under HFD condition. This finding was inconsistent with our data for muscle wet weight as HE and HECN groups showed greater muscle wet weight compared to C, H, and HCN. Although speculative, possible mechanism explaining these results is decreased muscle glycogen availability during RET under HFD. One of the intracellular mechanisms implicated for cell growth in response to mechanical loading includes upregulation of Akt [412]. Akt phosphorylation is associated with growth factors such as insulin and is stimulated in response to muscle contraction [413, 414]. Akt activation mediates myofiber hypertrophy through upregulation of downstream targets such as mTOR, euckryotic initiation factor 4E-binding protein (4E-BP1), and 70-kDa S6 kinase (p70S6K) [248]. During a glygogen depleted state, mechanical loading may induce a metabolic disorder resulting in reduced glycogen breakdown [415]. Thus, other energy sources such as protein might be necessary to be provided for cellular energy homeostasis during exercise [416] and recovery [417]. In fact, Creer et al. [418] reported that a low carbohydrate (CHO) diet group (2% CHO and 77% fat) after glycogen-depletion resulted in lower muscle glycogen availability compared to high carbohydrate group (80% carbohydrate and 7% fat). They also demonstrated that after RET, Akt phosphorylation was not stimulated while 1.5-fold increase from pre-exercise (p ≤0.05) was observed in the high CHO group. Also, mTOR phosphorylation in response to exercise was similar to that of Akt. In the present study, our CHO content was lower (-61%) than the normal diet. Therefore, it might be speculated that intracellular adaptations to RET were blunted under HFD. In agreement with our hypothesis, muscle loss under HFD was attenuated with CLA/n-3 administration. Moreover, although not significant, there was an increase in LBM in HECN (+6%), which solely exhibited increased LBM over the 20-week intervention. We observed marked decreases in LBM even in the normal diet control group suggesting an age-dependent loss of muscle mass in sedentary middle-aged animals. It has been suggested that CLA/n-3 can combat muscle wasting by attenuating or maintaining negative regulators of muscle mass. For

80 example, Rahman et al [10] found that long term (24 weeks) CLA administration in middle aged mice produced greater increases in LBM and muscle wet weight compared to a control group. This was accompanied by relatively lower oxidative stress and inflammatory response in mice fed CLA. In a human study, CLA treatment (3.4g/d) over 12 weeks in young obese individuals increased LBM (+1.26kg) but slightly decreased in a placebo group (-0.05kg) [419]. Furthermore, Pinkoski et al. [358] reported additive effects of CLA supplement during RET on body composition. Young individuals who completed 7 weeks of RET with 5g/day of CLA gained greater LBM (+1.4kg) compared to a placebo group who gained only 0.2 kg. This suggests that CLA exerts some potentiating effect to RET-mediated adaptations to LBM. It also has been reported that chronic intake of n-3 stimulates the up-regulation of the Akt-mTOR-P70 ribosomal protein S6 Kinase (S6K1) signaling pathway, a prominent pathway for protein synthesis [373]. Moreover, obese individuals who consumed combined CLA/n-3 supplement for 12 weeks had increased LBM by 2.4% [402]. Corresponding with our hypotheses, chronic CLA/n-3 administration appeared to be effective in improving body composition by itself and may also facilitate RET-induced adaptations in part. Thus CLA/n-3 supplementation may possibly serve as a novel dietary strategy to combat sarcopenic obesity in older adults.

Muscle Mass and Myofiber Dimensions

Previous research with CLA or/and n-3 supplementation has been restricted to an indirect measure of muscle tissue such as DXA analysis [402]. To our knowledge, the present study is quite unique as we are the first to apply a long-term direct in vivo analysis to measure myofiber changes especially associated with CLA/n-3 administration under HFD with or without chronic RET. In particular, we utilized a non-invasive MR technique termed Diffusion Tensor Imaging (DTI) which has been validated for analysis of change in myofiber cross sectional area (CSA) [378, 381]. We found a significant decline in gastrocnemius wet weight in C, H, and HCN from baseline but not in the RET groups (HE and HECN). Quadriceps wet weight in all five groups (C, H, HE, HCN, and HECN) decreased from baseline although the rate of decline was attenuated in HE and HECN. In addition, muscle weight in the RET groups (HE and HECN) was significantly higher than H or HCN for both gastrocnemius and quadriceps. Our results were in agreement with prior evidence which demonstrated positive effects of RET [420] with or without CLA supplements on muscle mass [358]. Interestingly, soleus wet weight decreased only in H from the baseline while both RET groups (HE and HECN) exhibited greater values than C, H,

81 and HCN. Since the soleus is recognized as a red oxidative postural muscle mainly composed of type I myofibers, it is relatively sensitive to daily acitiviy or muscle contraction. Taken together, these results in agreement with our hypothesis suggest that both RET and CLA/n-3 adminisration improve muscle mass under long-term HFD. Our MR data partially corresponded with the DXA and muscle wet weight analyses. DTI values λ2 and λ3 are main indicators of myofiber dimension and directly correspond with CSA [378]. The fact that DTI value λ3 was increased in HCN indicated that chronic CLA/n-3 administration favored an increase in CSA. As mentioned ealier, CLA/n-3 administration is effective in improving body composition (i.e. decreaed FM and maintaind LBM). However, unexpectedly HES exhibited a decreased λ2 value. Paradoxically, muscle wet weights of the gastrocnemius, soleus, and quadriceps were relatively greater in HECN which conflicts with the λ3 value for CSA. This discrepancy might be due to the fact that only 4 animals were selected per group for in vivo MR DTI measurement. This is relatively a small sample size and may have impacted the statistical power.

Muscle Strength and Sensorimotor Function

The present study employed a direct measure of muscular strength and sensorimotor function by utilizing an adapted grip strength and incline plane test, respectively, for animal model experiments. As anticipated, grip strength decreased in H from baseline but not in the control group. Thus, chronic HFD might have mediated a decline in muscle strength. However, we also found a significant loss of strength in HCN. This finding was in agreement with changes in muscle wet weight in these particular groups. The decline in muscle wet weight under HFD was not improved with CLA/n-3 administration without RET. However, muscle weight was improved with RET with or without CLA/n-3 administration. It seems that the strength gains were associated with increases in muscle mass via RET, but CLA/n-3 administration failed to exert any additive effects on the muscle mass and strength. The sensorimotor function (incline plane test) data provided interesting outcomes. There was a significant decline in sensorimotor function from baseline only in H while no change in C, HCN, and HE. This indicated that CLA/n-3 administration or RET tended to attenuate the process of decline in sensorimotor function under chronic HFD condition. Intriguingly, only HECN exhibited a significant increase in sensorimotor function from baseline, which may indicate that CLA/n-3 administration enhances RET-induced improvement of sensorimotor

82 function. Overall, as we anticipated, impaired sensorimotor function under HFD was improved by either RET or CLA/n-3 administration independently and when combined.

Regulator of Inflammation

TNF-α, IL-1β, and IL-6 were measured as markers of inflammation. As generally accepted, increased pro-inflammatory cytokine concentrations along with decreased growth factor levels contribute to muscle wasting [124]. There were no main group effects for relative mRNA expression of TNF-α in gastrocnemius. However, post hoc revealed that TNF-α mRNA expression in the gastrocnemius was significantly greater in H compared to the C, HE, HCN, and HECN. This implies that chronic HFD possibly leads to increased levels of catabolic cytokines, such as TNF-α, inducing muscle wasting. Since TNF-α mediates IGF-I resistance and provokes catabolism and muscle wasting [170], increased activation of this molecule may indeed mediate loss of muscle mass. It was suggested that increased TNF-α activity might contribute to lipid- induced insulin resistance in obesity [170], which agrees with our findings. Regarding these observations, it is convincible that chronic HFD-induced obesity possibly triggers an earlier onset and aggravation of the sarcopenic process in middle aged populations. We found both RET and CLA/n-3 administration tended to reduce HFD-induced increase in TNF-α in the gastrocnemius but no synergistic effect in combined interventions. Our findings agreed with Greiwe et al. [315] who observed reduced muscle TNF-α mRNA levels as well as increased protein synthesis, illustrating the positive impact of chronic RET in the elderly. Inoue et al. [353] found a marked decrease in TNF-α levels in rats fed CLA with increased insulin sensitivity. Furthermore, Winzell et al. [354] demonstrated that a combined diet of 1% CLA and 1% n-3 substantially increased LBM compared to normal diet in mice. There were significant group effects for TNF-α levels in the quadriceps. Interestingly, TNF-α expression in the quadriceps markedly reduced in all groups from baseline except HE (p = 0.09). Also, the RET groups (HE and HECN) showed greater TNF-α expression than both sedentary HFD groups (H and HCN). This finding suggests that there was no aging-effect on TNF-α levels during our 20-week experiment period in middle aged mice. Greater TNF-α expression in both RET groups may be due to acute inflammatory response to mechanical load-induced muscle damage. In fact, it has been previously demonstrated that TNF- α is stimulated by intense exercise [295, 421]. Our RET protocol provided mechanical overloading to the quadriceps mediating an acute

83 inflammatory response, since muscles were isolated within 24 hours after the last session of RET. Intriguingly, the HCN group had significantly lower TNF-α expression than C, HE and HECN, signifying the anti-inflammatory effect of chronic CLA/n-3 administration on skeletal muscle. Our results were supported by Inoue et al. [353] which demonstrated a reduction in TNF-α expression in rat skeletal muscle with CLA administration compared to control. It also has been shown that EPA and DHA treatment attenuated LPS-induced TNF-α levels in cultured cell compared to control cell [422]. Unexpectedly, we observed a significant increase in the protein breakdown marker IL-1 only in HCN from baseline in all three muscles, while there were main group effects only in the quadriceps. Also, the HCN possessed greater IL-1 expression than H, HE, and HECN in the gastrocnemius and soleus. As for the quadriceps, HCN exhibited greater expression of IL-1 than C, HE, and HECN. Collectively, these findings indicate that chronic CLA/n-3 treatment increased a pro-inflammatory cytokine expression in skeletal muscle. These results conflicted with the previous evidence, which demonstrated the anti-inflammatory properties of both CLA and n-3 [338, 423]. On the other hand, other previous studies have reported adverse effects of CLA including insulin resistance with decreased leptin levels or fatty liver and enlarged spleen despite reduced body fat. To our knowledge, increased IL-1 expression in skeletal muscle with CLA/n-3 administration has not been reported. However, one previous study found that 12 weeks of CLA administration markedly increased CRP (110%) levels compared to the placebo group, indicating an adverse effect of CLA on the inflammatory response [423]. The increased IL-1 levels was attenuated with RET, as the HECN had lower IL-1 expression than HCN. Although the induction of IL-1 expression with CLA/n-3 administration was opposed to our hypothesis, these findings partially agree with our muscle mass data, where values were greater in HE and HECN compared to HCN. IL-6 mRNA expression was greatly elevated in the gastrocnemius of HE, HCN, and HECN from baseline. In addition, IL-6 levels in HCN and HECN greatly increased in the soleus and quadriceps from baseline, respectively, demonstrating an up-regulation of IL-6 signaling in association with RET or CLA/n-3 treatment. Although traditionally regarded as a pro- inflammatory cytokine, IL-6 has recently been shown to exhibit anti-inflammatory properties [99]. For example, IL-6 attenuated LPS-induced TNF-α expression in cultured human monocytes [347]. Moreover, IL-6 treatment in humans mediated an increase in IL-1 receptor antagonist and

84 soluble TNF-α receptor, which inhibit signaling events downstream to IL-1 and TNF-α, respectively [99]. In addition to its anti-inflammatory properties, IL-6 has been suggested to stimulate multiple metabolic adaptations which enhance substrate delivery to working muscles and increase lipolysis and glycogenolysis [314]. Based on previous evidence, it would be reasonable to conclude that increased muscle IL-6 expression inhibited or attenuated the up- regulation of pro-inflammatory cytokines by activating their antagonists with improving intracellular adaptations to exercise. It also appears that IL-6 up-regulation may possibly promote increases in either total LBM or local muscle mass. IL-15, the most abundant cytokine in skeletal muscle, has attracted attention due to its anti-inflammatory and anabolic properties. For instance, IL-15Rα has been shown to down- regulate TNF-α signaling by blocking the TNF-α 1 receptor. IL-15 is able to promote anabolism by suppressing protein degradation (347) in addition to inducing an accumulation of myosin heavy chain (MHC) in differentiated myotubes in human cell cultures [424]. In the present study, IL-15 mRNA expression in the gastrocnemius significantly increased in both CLA/n-3 treated groups (i.e. HCN and HECN) from baseline. Moreover, HCN was significantly greater than H, indicating a positive effect of the supplement on anabolic signaling. There was a main group effect for soleus IL-15 expression. The H and HE groups showed significantly lower IL-15 levels compared to C, while no differences were observed between the CLA/n-3 treated groups (i.e. HCN and HECN) and C. Further, IL-15 mRNA expression was greater in HECN than both H and HE. This indicated negative effects of chronic HFD on anabolic signaling and a blunted adaptive response to mechanical loading in this catabolic environment. CLA/n-3 administration attenuated the effects of HFD and synergistically improved the anabolic or anti-inflammatory effects as well. Corresponding with our hypothesis, HFD-induced negative alteration in inflammatory state was improved by either RET or CLA/n-3 administration independently and when combined.

Regulators of Protein Turnover

Protein kinase B (Akt) and mammalian target of rapamycin (mTOR) level were measured as biomarkers of protein synthesis. There was a significant group effect for the quadriceps. Akt mRNA expression for the quadriceps was higher in C compared to baseline. Also, Akt levels of C was markedly higher than HECN (p = 0.04) and H (p = 0.02). Significantly greater mTOR

85 mRNA expression was observed in the gastrocnemius muscle of HCN compared to baseline. This is consistent with the findings of Gingras et al. [373] who reported that chronic administration of n-3 fatty acids enhanced protein metabolism by up-regulating the Akt-mTOR- S6K1 pathway and enhancing insulin sensitivity. Moreover, Chung et al. [425] reported that cell cultures treated with trans-10, cis-12, which is an isomer of CLA, showed greater mTOR- p70S6K phosphorylation compared to the controlled counterparts. Relative mRNA expression of atrogin-1 and MURF1, which are E-3 ubiquitin ligase proteins, were analyzed as markers of protein degradation. For atrogin-1, there was a significant increase in C and HCN compared to baseline in the gastrocnemius. In the soleus muscle, C exhibited greater atrogin-1 levels than H and HECN. MURF1 expression in HCN was greater than baseline in the quadriceps. Based on our results, it seems that protein degradation through E3 ligases were more likely affected by aging as opposed to HFD. It also appears that protein degradation was not improved by RET or CLA/n-3 administration under HFD. In contrast, the activation of E-3 ligase proteins was promoted by CLA/n-3 administration. A previous study conducted by Li et al. [426] suggested that IL-1 is closely related to the activation of atrogin-1 and. Consistent with their findings, we found greater IL-1β expression in HCN compared to other experimental groups. However, the elevated protein degradation may be compensated by increased protein synthesis through the Akt-mTOR-S6K1 pathway with CLA/n-3 treatment. Also, greater IL-15 levels would facilitate this restitutive effect.

Regulators of Mitogenesis

Insulin-like growth factor-I Ea (IGF-IEa) was analyzed as a positive regulator of mitogenesis. In the soleus muscle, IGF-IEa mRNA expression was markedly decreased in C (p = 0.043) from baseline. There was much larger decrement in H (p = 0.008) and HE (p = 0.008). This suggests that aging-induced impairments of cellular anabolism are linked to reduced IGF- IEa concentration, which may be exacerbated by chronic HFD. As we hypothesized, these impairments were improved by CLA/n-3 administration. A previous study reported that IGF-IEa tended to stimulate production of IL-10, which is a representative anti-inflammatory cytokine produced by human T cells [427]. While only speculative, it might be possible that relatively higher IGF-IEa levels might produce a compensatory effect in muscle exposed to pro- inflammatory cytokines such as IL-1.

Regulators of Myogenesis

MyoD and myogenin levels were measured as markers of proliferation and differenciation of satellite cells, respectively. We observed significantly decreased MyoD expression in HE from baseline in the soleus. In the quadriceps, HECN exhibited lower MyoD levels than C and HE. Regarding Myogenin, there was a main group effect in the gastrocnemius. Myogenin expression was markedly greater in HCN compared to baseline and HE. Intriguingly, HECN had lower myogenin expression compared to C and HCN in the gastrocnemius and quadriceps. While not corresponding with the hypothesis, our findings partially agree with Larsen et al. [428] who reported that CLA mimicked the inhibitory effect of inflammatory cytokines on myogenic regulators in cultured cells, suggesting a negative effect of CLA on myogenesis. However, Magee and colleagues [361] demonstrated that EPA treatment attenuated TNF-α-induced loss of myosin heavy chain (MyHC) along with increased myogenic fusion back to control levels in C2C12 cells.

Conclusions

Environmental factors such as a HFD and physical inactivity can lead to premature morbidity and mortality. It is difficult to find the intervention, which can possibly blunt the sarcopenic process followed by functional deficit. Our findings suggest that chronic CLA/n-3 administration can partially attenuate HFD-induced alterations in body composition with aging by attenuating the loss of LBM and increase in FM. Further, chronic RET and/or CLA/n-3 administration appears to attenuate a loss of muscle mass and function under HFD environment. This may possibly be mediated by improvements in the inflammatory state (e.g. lower TNF-α and higher IL-6 and IL-15), increased growth factor expression (e.g. IGF-IEa), elevated protein synthesis activity (e.g. mTOR), and heightened muscle regeneration (e.g. myogenin). Moreover, we found that RET prevented HFD-induced deficits in strength and sensorimotor function. When administered in conjunction with CLA/n-3, it conferred the greatest degree of improvement in sensorimotor function. Finally, we are the first group to demonstrate the ability of MR DTI to detect HFD-induced and intervention-mediated alterations in myofiber dimensions with in vivo model utilizing the most powerful MR technology. Overall, the present study suggests that chronic RET and/or CLA/n-3 appears to be partially efficacious as demonstrated by improvements in body composition and anabolic signaling under HFD-induced catabolism in

87 middle aged rodents. Future research may need to be conducted in middle-aged obese populations to verify our findings.

APPENDIX A ANIMAL CARE AND USE COMMITTEE APPROVAL OF STUDY PROTOCOL

REFERENCES

1. Melton, L.J., 3rd, S. Khosla, and B.L. Riggs, Epidemiology of sarcopenia. Mayo Clin Proc, 2000. 75 Suppl: p. S10-2; discussion S12-3. 2. Morley, J.E., et al., Sarcopenia. J Lab Clin Med, 2001. 137(4): p. 231-43. 3. Carmeli, E., R. Coleman, and A.Z. Reznick, The biochemistry of aging muscle. Exp Gerontol, 2002. 37(4): p. 477-89. 4. Wolfson, L., et al., Strength is a major factor in balance, gait, and the occurrence of falls. J Gerontol A Biol Sci Med Sci, 1995. 50 Spec No: p. 64-7. 5. Aniansson, A., et al., Muscle function in 75-year-old men and women. A longitudinal study. Scand J Rehabil Med Suppl, 1983. 9: p. 92-102. 6. Tinetti, M.E. and C.S. Williams, Falls, injuries due to falls, and the risk of admission to a nursing home. N Engl J Med, 1997. 337(18): p. 1279-84. 7. Rantanen, T., et al., Midlife hand grip strength as a predictor of old age disability. Jama, 1999. 281(6): p. 558-60. 8. Giampaoli, S., et al., Hand-grip strength predicts incident disability in non-disabled older men. Age Ageing, 1999. 28(3): p. 283-8. 9. Lees, S.J., K.A. Zwetsloot, and F.W. Booth, Muscle precursor cells isolated from aged rats exhibit an increased tumor necrosis factor- alpha response. Aging Cell, 2009. 8(1): p. 26-35. 10. Rahman, M., et al., Conjugated linoleic acid (CLA) prevents age-associated skeletal muscle loss. Biochem Biophys Res Commun, 2009. 383(4): p. 513-8. 11. Kim, J.S., J.M. Wilson, and S.R. Lee, Dietary implications on mechanisms of sarcopenia: roles of protein, amino acids and antioxidants. J Nutr Biochem, 2009. 12. Bamman, M.M., et al., Cluster analysis tests the importance of myogenic gene expression during myofiber hypertrophy in humans. J Appl Physiol, 2007. 102(6): p. 2232-9. 13. Welle, S., S. Totterman, and C. Thornton, Effect of age on muscle hypertrophy induced by resistance training. J Gerontol A Biol Sci Med Sci, 1996. 51(6): p. M270-5. 14. Harridge, S.D., A. Kryger, and A. Stensgaard, Knee extensor strength, activation, and size in very elderly people following strength training. Muscle Nerve, 1999. 22(7): p. 831-9. 15. Reeves, N.D., M.V. Narici, and C.N. Maganaris, In vivo human muscle structure and function: adaptations to resistance training in old age. Exp Physiol, 2004. 89(6): p. 675- 89. 16. Roubenoff, R., et al., Cytokines, insulin-like growth factor 1, sarcopenia, and mortality in very old community-dwelling men and women: the Framingham Heart Study. Am J Med, 2003. 115(6): p. 429-35. 17. Doherty, T.J., Invited review: Aging and sarcopenia. J Appl Physiol, 2003. 95(4): p. 1717-27. 18. McGeer, P.L. and E.G. McGeer, Inflammation and the degenerative diseases of aging. Ann N Y Acad Sci, 2004. 1035: p. 104-16. 19. Roubenoff, R., Catabolism of aging: is it an inflammatory process? Curr Opin Clin Nutr Metab Care, 2003. 6(3): p. 295-9. 20. Roubenoff, R., Sarcopenic obesity: the confluence of two epidemics. Obes Res, 2004. 12(6): p. 887-8.

21. Cesari, M., et al., Sarcopenia, obesity, and inflammation--results from the Trial of Angiotensin Converting Enzyme Inhibition and Novel Cardiovascular Risk Factors study. Am J Clin Nutr, 2005. 82(2): p. 428-34. 22. Jensen, G.L., Inflammation: roles in aging and sarcopenia. JPEN J Parenter Enteral Nutr, 2008. 32(6): p. 656-9. 23. de Ferranti, S. and D. Mozaffarian, The perfect storm: obesity, adipocyte dysfunction, and metabolic consequences. Clin Chem, 2008. 54(6): p. 945-55. 24. Dirks, A.J. and C. Leeuwenburgh, The role of apoptosis in age-related skeletal muscle atrophy. Sports Med, 2005. 35(6): p. 473-83. 25. Rotter, V., I. Nagaev, and U. Smith, Interleukin-6 (IL-6) induces insulin resistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-alpha, overexpressed in human fat cells from insulin-resistant subjects. J Biol Chem, 2003. 278(46): p. 45777-84. 26. Marzetti, E. and C. Leeuwenburgh, Skeletal muscle apoptosis, sarcopenia and frailty at old age. Exp Gerontol, 2006. 41(12): p. 1234-8. 27. Mourkioti, F. and N. Rosenthal, IGF-1, inflammation and stem cells: interactions during muscle regeneration. Trends Immunol, 2005. 26(10): p. 535-42. 28. Frost, R.A. and C.H. Lang, Skeletal muscle cytokines: regulation by pathogen-associated molecules and catabolic hormones. Curr Opin Clin Nutr Metab Care, 2005. 8(3): p. 255- 63. 29. Roubenoff, R., Sarcopenia: effects on body composition and function. J Gerontol A Biol Sci Med Sci, 2003. 58(11): p. 1012-7. 30. Leng, S.X., et al., Serum levels of insulin-like growth factor-I (IGF-I) and dehydroepiandrosterone sulfate (DHEA-S), and their relationships with serum interleukin-6, in the geriatric syndrome of frailty. Aging Clin Exp Res, 2004. 16(2): p. 153-7. 31. Roth, S.M., et al., Inflammatory factors in age-related muscle wasting. Curr Opin Rheumatol, 2006. 18(6): p. 625-30. 32. Cappola, A.R., et al., Insulin-like growth factor I and interleukin-6 contribute synergistically to disability and mortality in older women. J Clin Endocrinol Metab, 2003. 88(5): p. 2019-25. 33. Kim, J.S., et al., Resting and load-induced levels of myogenic gene transcripts differ between older adults with demonstrable sarcopenia and young men and women. J Appl Physiol, 2005. 99(6): p. 2149-58. 34. Manfredi, T.G., et al., Plasma creatine kinase activity and exercise-induced muscle damage in older men. Med Sci Sports Exerc, 1991. 23(9): p. 1028-34. 35. Roth, S.M., et al., High-volume, heavy-resistance strength training and muscle damage in young and older women. J Appl Physiol, 2000. 88(3): p. 1112-8. 36. Hamada, K., et al., Senescence of human skeletal muscle impairs the local inflammatory cytokine response to acute eccentric exercise. Faseb J, 2005. 19(2): p. 264-6. 37. Przybyla, B., et al., Aging alters macrophage properties in human skeletal muscle both at rest and in response to acute resistance exercise. Exp Gerontol, 2006. 41(3): p. 320-7. 38. Dennis, R.A., et al., Aging alters gene expression of growth and remodeling factors in human skeletal muscle both at rest and in response to acute resistance exercise. Physiol Genomics, 2008. 32(3): p. 393-400. 39. Tidball, J.G., Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol, 2005. 288(2): p. R345-53.

40. Akima, H., et al., The use of magnetic resonance images to investigate the influence of recruitment on the relationship between torque and cross-sectional area in humans. European Journal of Applied Physiology, 2000. 83: p. 475-480. 41. Volpi, E., et al., Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults. Am J Clin Nutr, 2003. 78(2): p. 250-8. 42. Janssen, I., et al., The healthcare costs of sarcopenia in the United States. J Am Geriatr Soc, 2004. 52(1): p. 80-5. 43. Steensberg, A., et al., Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6. J Physiol, 2000. 529 Pt 1: p. 237-42. 44. Eley, H.L., et al., Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli. Am J Physiol Endocrinol Metab, 2007. 293(4): p. E923-31. 45. Sabourin, L.A. and M.A. Rudnicki, The molecular regulation of myogenesis. Clin Genet, 2000. 57(1): p. 16-25. 46. Gelfand, R.A., et al., Role of counterregulatory hormones in the catabolic response to stress. J Clin Invest, 1984. 74(6): p. 2238-48. 47. Sheffield-Moore, M., et al., Short-term oxandrolone administration stimulates net muscle protein synthesis in young men. J Clin Endocrinol Metab, 1999. 84(8): p. 2705-11. 48. Urban, R.J., et al., Testosterone administration to elderly men increases skeletal muscle strength and protein synthesis. Am J Physiol, 1995. 269(5 Pt 1): p. E820-6. 49. Leung, K., et al., Insulin-like growth factor I and insulin down-regulate growth hormone (GH) receptors in rat osteoblasts: evidence for a peripheral feedback loop regulating GH action. Endocrinology, 1996. 137(7): p. 2694-702. 50. Goldspink, G., Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload. J Anat, 1999. 194 ( Pt 3): p. 323-34. 51. Rudman, D., et al., Effects of human growth hormone in men over 60 years old. N Engl J Med, 1990. 323(1): p. 1-6. 52. Bhasin, S., et al., Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. J Clin Endocrinol Metab, 1997. 82(2): p. 407-13. 53. Lynch, G.S., J.D. Schertzer, and J.G. Ryall, Therapeutic approaches for muscle wasting disorders. Pharmacol Ther, 2007. 113(3): p. 461-87. 54. Thomas, C.K. and I. Zijdewind, Fatigue of muscles weakened by death of motoneurons. Muscle Nerve, 2006. 33(1): p. 21-41. 55. Webber, S.C., M.M. Porter, and P.F. Gardiner, Modeling age-related neuromuscular changes in humans. Appl Physiol Nutr Metab, 2009. 34(4): p. 732-44. 56. Gallager, D.W., K. Heninger, and G. Heninger, Periodic benzodiazepine antagonist administration prevents benzodiazepine withdrawal symptoms in primates. Eur J Pharmacol, 1986. 132(1): p. 31-8. 57. Campbell, M.J., A.J. McComas, and F. Petito, Physiological changes in ageing muscles. J Neurol Neurosurg Psychiatry, 1973. 36(2): p. 174-82. 58. Doherty, T.J., et al., Effects of motor unit losses on strength in older men and women. J Appl Physiol, 1993. 74(2): p. 868-74.

59. McNeil, C.J., et al., Motor unit number estimates in the tibialis anterior muscle of young, old, and very old men. Muscle Nerve, 2005. 31(4): p. 461-7. 60. Mosoni, L., et al., Age-related changes in glutathione availability and skeletal muscle carbonyl content in healthy rats. Exp Gerontol, 2004. 39(2): p. 203-10. 61. Fulle, S., et al., The contribution of reactive oxygen species to sarcopenia and muscle ageing. Exp Gerontol, 2004. 39(1): p. 17-24. 62. Wei, Y.H. and H.C. Lee, Oxidative stress, mitochondrial DNA mutation, and impairment of antioxidant enzymes in aging. Exp Biol Med (Maywood), 2002. 227(9): p. 671-82. 63. Fibach, E. and E. Rachmilewitz, The role of oxidative stress in hemolytic anemia. Curr Mol Med, 2008. 8(7): p. 609-19. 64. Reid, M.B. and Y.P. Li, Cytokines and oxidative signalling in skeletal muscle. Acta Physiol Scand, 2001. 171(3): p. 225-32. 65. Cadenas, E. and A. Boveris, Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria. Biochem J, 1980. 188(1): p. 31-7. 66. Wrogemann, K. and S.D. Pena, Mitochondrial calcium overload: A general mechanism for cell-necrosis in muscle diseases. Lancet, 1976. 1(7961): p. 672-4. 67. Belia, S., et al., Sodium nitroprusside, a NO donor, modifies Ca2+ transport and mechanical properties in frog skeletal muscle. J Muscle Res Cell Motil, 1998. 19(8): p. 865-76. 68. Charette, S.L., et al., Muscle hypertrophy response to resistance training in older women. Journal of Applied Physiology, 1991. 70(5): p. 1912-6. 69. Fulle, S., et al., Specific oxidative alterations in vastus lateralis muscle of patients with the diagnosis of chronic fatigue syndrome. Free Radic Biol Med, 2000. 29(12): p. 1252-9. 70. Fulle, S., et al., Modification of the functional capacity of sarcoplasmic reticulum membranes in patients suffering from chronic fatigue syndrome. Neuromuscul Disord, 2003. 13(6): p. 479-84. 71. Basso, E., et al., Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. J Biol Chem, 2005. 280(19): p. 18558-61. 72. Green, D.R. and G. Kroemer, The pathophysiology of mitochondrial cell death. Science, 2004. 305(5684): p. 626-9. 73. Hajnoczky, G., et al., Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium, 2006. 40(5- 6): p. 553-60. 74. Chen, Q., et al., Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem, 2003. 278(38): p. 36027-31. 75. Ishii, N., Role of oxidative stress from mitochondria on aging and cancer. Cornea, 2007. 26(9 Suppl 1): p. S3-9. 76. Pollack, M., et al., The role of apoptosis in the normal aging brain, skeletal muscle, and heart. Ann N Y Acad Sci, 2002. 959: p. 93-107. 77. Daugas, E., et al., Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett, 2000. 476(3): p. 118-23. 78. Dirks, A.J. and C. Leeuwenburgh, Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor, apoptosis-inducing factor, X-linked inhibitor of apoptosis, caspase-3, and caspase-12. Free Radic Biol Med, 2004. 36(1): p. 27-39.

79. Marzetti, E., et al., Age-related activation of mitochondrial caspase-independent apoptotic signaling in rat gastrocnemius muscle. Mech Ageing Dev, 2008. 129(9): p. 542-9. 80. Alway, S.E., et al., Potential role for Id myogenic repressors in apoptosis and attenuation of hypertrophy in muscles of aged rats. Am J Physiol Cell Physiol, 2002. 283(1): p. C66-76. 81. Chung, L. and Y.C. Ng, Age-related alterations in expression of apoptosis regulatory proteins and heat shock proteins in rat skeletal muscle. Biochim Biophys Acta, 2006. 1762(1): p. 103-9. 82. Song, W., H.B. Kwak, and J.M. Lawler, Exercise training attenuates age-induced changes in apoptotic signaling in rat skeletal muscle. Antioxid Redox Signal, 2006. 8(3- 4): p. 517-28. 83. Alway, S.E., et al., Denervation stimulates apoptosis but not Id2 expression in hindlimb muscles of aged rats. J Gerontol A Biol Sci Med Sci, 2003. 58(8): p. 687-97. 84. Cortopassi, G.A. and A. Wong, Mitochondria in organismal aging and degeneration. Biochim Biophys Acta, 1999. 1410(2): p. 183-93. 85. Sohal, R.S., et al., Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev, 1994. 74(1-2): p. 121-33. 86. Lee, H.C., et al., Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence. J Biomed Sci, 2002. 9(6 Pt 1): p. 517-26. 87. Muller, F.L., et al., Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production. Am J Physiol Regul Integr Comp Physiol, 2007. 293(3): p. R1159-68. 88. Vasilaki, A., et al., Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity. Aging Cell, 2006. 5(2): p. 109-17. 89. Singh, M.A., et al., Insulin-like growth factor I in skeletal muscle after weight-lifting exercise in frail elders. American Journal of Physiology, 1999. 277(1 Pt 1): p. E135-43. 90. Vasto, S., et al., Inflammatory networks in ageing, age-related diseases and longevity. Mech Ageing Dev, 2007. 128(1): p. 83-91. 91. Bruunsgaard, H. and B.K. Pedersen, Age-related inflammatory cytokines and disease. Immunol Allergy Clin North Am, 2003. 23(1): p. 15-39. 92. Cannon, J.G., Inflammatory Cytokines in Nonpathological States. News Physiol Sci, 2000. 15: p. 298-303. 93. Grimble, R.F., Inflammatory response in the elderly. Curr Opin Clin Nutr Metab Care, 2003. 6(1): p. 21-9. 94. Grimble, R.F., Nutritional modulation of immune function. Proc Nutr Soc, 2001. 60(3): p. 389-97. 95. Cao, J., et al., Activation of human bronchial epithelial cells by inflammatory cytokines IL-27 and TNF-alpha: Implications for immunopathophysiology of airway inflammation. J Cell Physiol. 96. Ficarra, G., G. Baroni, and D. Massi, Pyostomatitis Vegetans: Cellular Immune Profile and Expression of IL-6, IL-8 and TNF-alpha. Head Neck Pathol. 4(1): p. 1-9. 97. Ong, Z.Y., et al., Pro-inflammatory cytokines play a key role in the development of radiotherapy-induced gastrointestinal mucositis. Radiat Oncol. 5(1): p. 22.

98. Xing, Z., et al., IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest, 1998. 101(2): p. 311-20. 99. Tilg, H., C.A. Dinarello, and J.W. Mier, IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol Today, 1997. 18(9): p. 428-32. 100. Ikizler, T.A., Nutrition, inflammation and chronic kidney disease. Curr Opin Nephrol Hypertens, 2008. 17(2): p. 162-7. 101. Matthys, P., et al., Anti-gamma interferon and anti-interleukin-6 antibodies affect staphylococcal enterotoxin B-induced weight loss, hypoglycemia, and cytokine release in D-galactosamine-sensitized and unsensitized mice. Infect Immun, 1995. 63(4): p. 1158- 64. 102. Mastorakos, G., G.P. Chrousos, and J.S. Weber, Recombinant interleukin-6 activates the hypothalamic-pituitary-adrenal axis in humans. J Clin Endocrinol Metab, 1993. 77(6): p. 1690-4. 103. Schindler, R., et al., Correlations and interactions in the production of interleukin-6 (IL- 6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood, 1990. 75(1): p. 40-7. 104. Mizuhara, H., et al., T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6. J Exp Med, 1994. 179(5): p. 1529-37. 105. Yamaoka, M., et al., Anti-inflammatory cytokine profile in human heart failure: behavior of interleukin-10 in association with tumor necrosis factor-alpha. Jpn Circ J, 1999. 63(12): p. 951-6. 106. Moore, K.W., et al., Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol, 2001. 19: p. 683-765. 107. Schottelius, A.J., et al., Interleukin-10 signaling blocks inhibitor of kappaB kinase activity and nuclear factor kappaB DNA binding. J Biol Chem, 1999. 274(45): p. 31868- 74. 108. de Waal Malefyt, R., et al., Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J Exp Med, 1991. 174(4): p. 915-24. 109. Ding, L. and E.M. Shevach, IL-10 inhibits mitogen-induced T cell proliferation by selectively inhibiting macrophage costimulatory function. J Immunol, 1992. 148(10): p. 3133-9. 110. Hart, P.H., et al., Regulation of surface and soluble TNF receptor expression on human monocytes and synovial fluid macrophages by IL-4 and IL-10. J Immunol, 1996. 157(8): p. 3672-80. 111. Linderholm, M., et al., Elevated plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6, and IL-10 in patients with hemorrhagic fever with renal syndrome. J Infect Dis, 1996. 173(1): p. 38-43. 112. Dickensheets, H.L., et al., Interleukin-10 upregulates tumor necrosis factor receptor type-II (p75) gene expression in endotoxin-stimulated human monocytes. Blood, 1997. 90(10): p. 4162-71. 113. Jenkins, J.K., M. Malyak, and W.P. Arend, The effects of interleukin-10 on interleukin-1 receptor antagonist and interleukin-1 beta production in human monocytes and neutrophils. Lymphokine Cytokine Res, 1994. 13(1): p. 47-54.

114. Dickensheets, H.L. and R.P. Donnelly, IFN-gamma and IL-10 inhibit induction of IL-1 receptor type I and type II gene expression by IL-4 and IL-13 in human monocytes. J Immunol, 1997. 159(12): p. 6226-33. 115. Niiro, H., et al., IL-10 inhibits prostaglandin E2 production by lipopolysaccharide- stimulated monocytes. Int Immunol, 1994. 6(4): p. 661-4. 116. Mertz, P.M., et al., Interleukin 10 suppression of monocyte prostaglandin H synthase-2. Mechanism of inhibition of prostaglandin-dependent matrix metalloproteinase production. J Biol Chem, 1994. 269(33): p. 21322-9. 117. Stumpf, C., et al., Decrease of serum levels of the anti-inflammatory cytokine interleukin- 10 in patients with advanced chronic heart failure. Clin Sci (Lond), 2003. 105(1): p. 45- 50. 118. Chung, H.Y., et al., The molecular inflammatory process in aging. Antioxid Redox Signal, 2006. 8(3-4): p. 572-81. 119. Bruunsgaard, H., et al., A high plasma concentration of TNF-alpha is associated with dementia in centenarians. J Gerontol A Biol Sci Med Sci, 1999. 54(7): p. M357-64. 120. Catania, A., et al., Cytokine antagonists in aged subjects and their relation with cellular immunity. J Gerontol A Biol Sci Med Sci, 1997. 52(2): p. B93-7. 121. Wei, J., et al., Increase of plasma IL-6 concentration with age in healthy subjects. Life Sci, 1992. 51(25): p. 1953-6. 122. Hasegawa, Y., et al., Increased soluble tumor necrosis factor receptor levels in the serum of elderly people. Gerontology, 2000. 46(4): p. 185-8. 123. Ballou, S.P., et al., Quantitative and qualitative alterations of acute-phase proteins in healthy elderly persons. Age Ageing, 1996. 25(3): p. 224-30. 124. Ross, R., Atherosclerosis--an inflammatory disease. N Engl J Med, 1999. 340(2): p. 115- 26. 125. Paolisso, G., et al., Advancing age and insulin resistance: role of plasma tumor necrosis factor-alpha. Am J Physiol, 1998. 275(2 Pt 1): p. E294-9. 126. Pacifici, R., Aging and cytokine production. Calcif Tissue Int, 1999. 65(5): p. 345-51. 127. Kotler, D.P., Cachexia. Ann Intern Med, 2000. 133(8): p. 622-34. 128. Miller, R.A., The aging immune system: primer and prospectus. Science, 1996. 273(5271): p. 70-4. 129. Cohen, H.J., et al., The association of plasma IL-6 levels with functional disability in community-dwelling elderly. J Gerontol A Biol Sci Med Sci, 1997. 52(4): p. M201-8. 130. Ershler, W.B., et al., Interleukin-6 and aging: blood levels and mononuclear cell production increase with advancing age and in vitro production is modifiable by dietary restriction. Lymphokine Cytokine Res, 1993. 12(4): p. 225-30. 131. Prio, T.K., et al., Asymptomatic bacteriuria in elderly humans is associated with increased levels of circulating TNF receptors and elevated numbers of neutrophils. Exp Gerontol, 2002. 37(5): p. 693-9. 132. Costelli, P., et al., Activation of Ca(2+)-dependent proteolysis in skeletal muscle and heart in cancer cachexia. Br J Cancer, 2001. 84(7): p. 946-50. 133. Valdez, H. and M.M. Lederman, Cytokines and cytokine therapies in HIV infection. AIDS Clin Rev, 1997: p. 187-228. 134. Tiao, G., et al., Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle. J Clin Invest, 1994. 94(6): p. 2255-64.

135. Franssen, F.M., E.F. Wouters, and A.M. Schols, The contribution of starvation, deconditioning and ageing to the observed alterations in peripheral skeletal muscle in chronic organ diseases. Clin Nutr, 2002. 21(1): p. 1-14. 136. Anker, S.D., et al., Hormonal changes and catabolic/anabolic imbalance in chronic heart failure and their importance for cardiac cachexia. Circulation, 1997. 96(2): p. 526-34. 137. Drexler, H., et al., Alterations of skeletal muscle in chronic heart failure. Circulation, 1992. 85(5): p. 1751-9. 138. Adams, V., et al., Apoptosis in skeletal myocytes of patients with chronic heart failure is associated with exercise intolerance. J Am Coll Cardiol, 1999. 33(4): p. 959-65. 139. Narula, J., et al., Apoptosis in myocytes in end-stage heart failure. N Engl J Med, 1996. 335(16): p. 1182-9. 140. Martins, M.A., et al., High fat diets modulate nitric oxide biosynthesis and antioxidant defence in red blood cells from C57BL/6 mice. Arch Biochem Biophys. 141. Souza-Mello, V., et al., Comparative effects of telmisartan, sitagliptin and metformin alone or associated on obesity, insulin resistance, liver and pancreas remodelling in C57BL/6 mice fed a very high-fat diet. Clin Sci (Lond). 142. Poudyal, H., F. Campbell, and L. Brown, Olive leaf extract attenuates cardiac, hepatic, and metabolic changes in high carbohydrate-, high fat-fed rats. J Nutr. 140(5): p. 946-53. 143. Wada, T., et al., Spironolactone improves glucose and lipid metabolism by ameliorating hepatic steatosis and inflammation and suppressing enhanced gluconeogenesis induced by high-fat and high-fructose diet. Endocrinology. 151(5): p. 2040-9. 144. Borkan, G.A., et al., Age changes in body composition revealed by computed tomography. J Gerontol, 1983. 38(6): p. 673-7. 145. Hughes, V.A., et al., Anthropometric assessment of 10-y changes in body composition in the elderly. Am J Clin Nutr, 2004. 80(2): p. 475-82. 146. Ferrucci, L., et al., Serum IL-6 level and the development of disability in older persons. J Am Geriatr Soc, 1999. 47(6): p. 639-46. 147. Baumgartner, R.N., Body composition in healthy aging. Ann N Y Acad Sci, 2000. 904: p. 437-48. 148. Zamboni, M., et al., Health consequences of obesity in the elderly: a review of four unresolved questions. Int J Obes (Lond), 2005. 29(9): p. 1011-29. 149. Baumgartner, R.N., et al., Sarcopenic obesity predicts instrumental activities of daily living disability in the elderly. Obes Res, 2004. 12(12): p. 1995-2004. 150. Wellen, K.E. and G.S. Hotamisligil, Inflammation, stress, and diabetes. J Clin Invest, 2005. 115(5): p. 1111-9. 151. Perreault, M. and A. Marette, Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle. Nat Med, 2001. 7(10): p. 1138-43. 152. Bruunsgaard, H., Physical activity and modulation of systemic low-level inflammation. J Leukoc Biol, 2005. 78(4): p. 819-35. 153. Lang, C.H., et al., Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, -6, and high-mobility-group protein-1 in skeletal muscle. Shock, 2003. 19(6): p. 538-46. 154. Ferrannini, E., et al., Insulin action and age. European Group for the Study of Insulin Resistance (EGIR). Diabetes, 1996. 45(7): p. 947-53.

155. Paolisso, G., A. Scheen, and P. Lefebvre, Glucose handling, diabetes and ageing. Horm Res, 1995. 43(1-3): p. 52-7. 156. Chorinchath, B.B., et al., Age-associated differences in TNF-alpha and nitric oxide production in endotoxic mice. J Immunol, 1996. 156(4): p. 1525-30. 157. Han, D., et al., Age-related enhancement of tumor necrosis factor (TNF) production in mice. Mech Ageing Dev, 1995. 84(1): p. 39-54. 158. Liu, L.S., et al., Tumor necrosis factor-alpha acutely inhibits insulin signaling in human adipocytes: implication of the p80 tumor necrosis factor receptor. Diabetes, 1998. 47(4): p. 515-22. 159. Cornelius, P., et al., Monokine regulation of glucose transporter mRNA in L6 myotubes. Biochem Biophys Res Commun, 1989. 165(1): p. 429-36. 160. Greenberg, A.S. and M.S. Obin, Obesity and the role of adipose tissue in inflammation and metabolism. Am J Clin Nutr, 2006. 83(2): p. 461S-465S. 161. Wang, P., et al., The secretory function of adipocytes in the physiology of white adipose tissue. J Cell Physiol, 2008. 216(1): p. 3-13. 162. Vick, M.M., et al., Relationships among inflammatory cytokines, obesity, and insulin sensitivity in the horse. J Anim Sci, 2007. 85(5): p. 1144-55. 163. Xu, H., et al., Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest, 2003. 112(12): p. 1821-30. 164. Hotamisligil, G.S., N.S. Shargill, and B.M. Spiegelman, Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science, 1993. 259(5091): p. 87-91. 165. Saghizadeh, M., et al., The expression of TNF alpha by human muscle. Relationship to insulin resistance. J Clin Invest, 1996. 97(4): p. 1111-6. 166. Hotamisligil, G.S., et al., Increased adipose tissue expression of tumor necrosis factor- alpha in human obesity and insulin resistance. J Clin Invest, 1995. 95(5): p. 2409-15. 167. Bednarik, T. and H. Cajthamlova, Splenectomy as a stimulus of antibody formation. Folia Biol (Praha), 1971. 17(3): p. 196-9. 168. Togashi, N., et al., The contribution of skeletal muscle tumor necrosis factor-alpha to insulin resistance and hypertension in fructose-fed rats. J Hypertens, 2000. 18(11): p. 1605-10. 169. Uysal, K.T., et al., Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function. Nature, 1997. 389(6651): p. 610-4. 170. Steinberg, G.R., et al., Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling. Cell Metab, 2006. 4(6): p. 465- 74. 171. Togashi, N., et al., Effect of TNF-alpha--converting enzyme inhibitor on insulin resistance in fructose-fed rats. Hypertension, 2002. 39(2 Pt 2): p. 578-80. 172. von Haehling, S., E.A. Jankowska, and S.D. Anker, Tumour necrosis factor-alpha and the failing heart--pathophysiology and therapeutic implications. Basic Res Cardiol, 2004. 99(1): p. 18-28. 173. Bolger, A.P. and S.D. Anker, Tumour necrosis factor in chronic heart failure: a peripheral view on pathogenesis, clinical manifestations and therapeutic implications. Drugs, 2000. 60(6): p. 1245-57. 174. Beutler, B. and C. van Huffel, Unraveling function in the TNF ligand and receptor families. Science, 1994. 264(5159): p. 667-8.

175. Zhang, Y., et al., Cytokines and endotoxin induce cytokine receptors in skeletal muscle. Am J Physiol Endocrinol Metab, 2000. 279(1): p. E196-205. 176. del Aguila, L.F., K.P. Claffey, and J.P. Kirwan, TNF-alpha impairs insulin signaling and insulin stimulation of glucose uptake in C2C12 muscle cells. Am J Physiol, 1999. 276(5 Pt 1): p. E849-55. 177. Bouzakri, K. and J.R. Zierath, MAP4K4 gene silencing in human skeletal muscle prevents tumor necrosis factor-alpha-induced insulin resistance. J Biol Chem, 2007. 282(11): p. 7783-9. 178. Hotamisligil, G.S., et al., Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha. J Clin Invest, 1994. 94(4): p. 1543-9. 179. Borst, S.E., et al., Neutralization of tumor necrosis factor-alpha reverses insulin resistance in skeletal muscle but not adipose tissue. Am J Physiol Endocrinol Metab, 2004. 287(5): p. E934-8. 180. Steinberg, G.R., et al., AMP-activated protein kinase--the fat controller of the energy railroad. Can J Physiol Pharmacol, 2006. 84(7): p. 655-65. 181. Ruderman, N.B., et al., Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol, 1999. 276(1 Pt 1): p. E1-E18. 182. Gaster, M., et al., Reduced lipid oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin: evidence from cultured myotubes. Diabetes, 2004. 53(3): p. 542-8. 183. Hulver, M.W., et al., Skeletal muscle lipid metabolism with obesity. Am J Physiol Endocrinol Metab, 2003. 284(4): p. E741-7. 184. Bandyopadhyay, G.K., et al., Increased malonyl-CoA levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects. Diabetes, 2006. 55(8): p. 2277-85. 185. Dinarello, C.A., The role of the interleukin-1-receptor antagonist in blocking inflammation mediated by interleukin-1. N Engl J Med, 2000. 343(10): p. 732-4. 186. Bendtzen, K., et al., Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans. Science, 1986. 232(4757): p. 1545-7. 187. Maedler, K., et al., Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest, 2002. 110(6): p. 851-60. 188. Maedler, K., et al., Glucose induces beta-cell apoptosis via upregulation of the Fas receptor in human islets. Diabetes, 2001. 50(8): p. 1683-90. 189. Donath, M.Y. and T. Mandrup-Poulsen, The use of interleukin-1-receptor antagonists in the treatment of diabetes mellitus. Nat Clin Pract Endocrinol Metab, 2008. 4(5): p. 240-1. 190. Donath, M.Y., et al., Cytokines and beta-cell biology: from concept to clinical translation. Endocr Rev, 2008. 29(3): p. 334-50. 191. Dinarello, C.A. and S.M. Wolff, The role of interleukin-1 in disease. N Engl J Med, 1993. 328(2): p. 106-13. 192. Ehses, J.A., et al., IL-1 antagonism reduces hyperglycemia and tissue inflammation in the type 2 diabetic GK rat. Proc Natl Acad Sci U S A, 2009. 106(33): p. 13998-4003. 193. Hacham, M., et al., Interleukin-6 and interleukin-10 are expressed in organs of normal young and old mice. Eur Cytokine Netw, 2004. 15(1): p. 37-46.

194. Carey, A.L., et al., Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase. Diabetes, 2006. 55(10): p. 2688-97. 195. Ruderman, N.B., et al., Interleukin-6 regulation of AMP-activated protein kinase. Potential role in the systemic response to exercise and prevention of the metabolic syndrome. Diabetes, 2006. 55 Suppl 2: p. S48-54. 196. Weigert, C., et al., Palmitate, but not unsaturated fatty acids, induces the expression of interleukin-6 in human myotubes through proteasome-dependent activation of nuclear factor-kappaB. J Biol Chem, 2004. 279(23): p. 23942-52. 197. Weigert, C., et al., Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt. Am J Physiol Endocrinol Metab, 2005. 289(2): p. E251-7. 198. Kelly, M., et al., AMPK activity is diminished in tissues of IL-6 knockout mice: the effect of exercise. Biochem Biophys Res Commun, 2004. 320(2): p. 449-54. 199. Kim, H.J., et al., Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo. Diabetes, 2004. 53(4): p. 1060-7. 200. Cai, D., et al., Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB. Nat Med, 2005. 11(2): p. 183-90. 201. Weigert, C., et al., Direct cross-talk of interleukin-6 and insulin signal transduction via insulin receptor substrate-1 in skeletal muscle cells. J Biol Chem, 2006. 281(11): p. 7060-7. 202. Akdis, C.A. and K. Blaser, Mechanisms of interleukin-10-mediated immune suppression. Immunology, 2001. 103(2): p. 131-6. 203. Hong, E.G., et al., Interleukin-10 prevents diet-induced insulin resistance by attenuating macrophage and cytokine response in skeletal muscle. Diabetes, 2009. 58(11): p. 2525- 35. 204. Lumeng, C.N., J.L. Bodzin, and A.R. Saltiel, Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest, 2007. 117(1): p. 175-84. 205. Luster, A.D., Chemokines--chemotactic cytokines that mediate inflammation. N Engl J Med, 1998. 338(7): p. 436-45. 206. Charo, I.F. and M.B. Taubman, Chemokines in the pathogenesis of vascular disease. Circ Res, 2004. 95(9): p. 858-66. 207. Herder, C., et al., Chemokines and incident coronary heart disease: results from the MONICA/KORA Augsburg case-cohort study, 1984-2002. Arterioscler Thromb Vasc Biol, 2006. 26(9): p. 2147-52. 208. Kamei, N., et al., Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance. J Biol Chem, 2006. 281(36): p. 26602-14. 209. Kanda, H., et al., MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest, 2006. 116(6): p. 1494- 505. 210. Sell, H., et al., Monocyte chemotactic protein-1 is a potential player in the negative cross-talk between adipose tissue and skeletal muscle. Endocrinology, 2006. 147(5): p. 2458-67. 211. Charo, I.F. and R.M. Ransohoff, The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med, 2006. 354(6): p. 610-21.

100

212. Bastard, J.P., et al., Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw, 2006. 17(1): p. 4-12. 213. Trinchieri, G. and A. Sher, Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol, 2007. 7(3): p. 179-90. 214. Boyd, J.H., et al., Toll-like receptors differentially regulate CC and CXC chemokines in skeletal muscle via NF-kappaB and calcineurin. Infect Immun, 2006. 74(12): p. 6829-38. 215. Senn, J.J., Toll-like receptor-2 is essential for the development of palmitate-induced insulin resistance in myotubes. J Biol Chem, 2006. 281(37): p. 26865-75. 216. Reyna, S.M., et al., Elevated toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects. Diabetes, 2008. 57(10): p. 2595-602. 217. Tschop, M. and G. Thomas, Fat fuels insulin resistance through Toll-like receptors. Nat Med, 2006. 12(12): p. 1359-61. 218. Kim, F., et al., Toll-like receptor-4 mediates vascular inflammation and insulin resistance in diet-induced obesity. Circ Res, 2007. 100(11): p. 1589-96. 219. Radin, M.S., et al., Inhibition or deletion of the lipopolysaccharide receptor Toll-like receptor-4 confers partial protection against lipid-induced insulin resistance in rodent skeletal muscle. Diabetologia, 2008. 51(2): p. 336-46. 220. Rasouli, N. and P.A. Kern, Adipocytokines and the metabolic complications of obesity. J Clin Endocrinol Metab, 2008. 93(11 Suppl 1): p. S64-73. 221. Zhang, Y., et al., Positional cloning of the mouse obese gene and its human homologue. Nature, 1994. 372(6505): p. 425-32. 222. Friedman, J.M. and J.L. Halaas, Leptin and the regulation of body weight in mammals. Nature, 1998. 395(6704): p. 763-70. 223. Farooqi, I.S., et al., Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor. N Engl J Med, 2007. 356(3): p. 237-47. 224. Loffreda, S., et al., Leptin regulates proinflammatory immune responses. Faseb J, 1998. 12(1): p. 57-65. 225. Wallace, A.M., et al., Plasma leptin and the risk of cardiovascular disease in the west of Scotland coronary prevention study (WOSCOPS). Circulation, 2001. 104(25): p. 3052-6. 226. Diez, J.J. and P. Iglesias, The role of the novel adipocyte-derived hormone adiponectin in human disease. Eur J Endocrinol, 2003. 148(3): p. 293-300. 227. Kadowaki, T. and T. Yamauchi, Adiponectin and adiponectin receptors. Endocr Rev, 2005. 26(3): p. 439-51. 228. Ode, K.L., B.I. Frohnert, and B.M. Nathan, Identification and treatment of metabolic complications in pediatric obesity. Rev Endocr Metab Disord, 2009. 10(3): p. 167-88. 229. Zhu, W., et al., Vascular effects of adiponectin: molecular mechanisms and potential therapeutic intervention. Clin Sci (Lond), 2008. 114(5): p. 361-74. 230. Steppan, C.M., et al., The hormone resistin links obesity to diabetes. Nature, 2001. 409(6818): p. 307-12. 231. Degawa-Yamauchi, M., et al., Serum resistin (FIZZ3) protein is increased in obese humans. J Clin Endocrinol Metab, 2003. 88(11): p. 5452-5. 232. Levy, J.R., et al., Lipid metabolism and resistin gene expression in insulin-resistant Fischer 344 rats. Am J Physiol Endocrinol Metab, 2002. 282(3): p. E626-33. 233. Milan, G., et al., Resistin and adiponectin expression in visceral fat of obese rats: effect of weight loss. Obes Res, 2002. 10(11): p. 1095-103.

101

234. Silswal, N., et al., Human resistin stimulates the pro-inflammatory cytokines TNF-alpha and IL-12 in macrophages by NF-kappaB-dependent pathway. Biochem Biophys Res Commun, 2005. 334(4): p. 1092-101. 235. Verma, S., et al., Resistin promotes endothelial cell activation: further evidence of adipokine-endothelial interaction. Circulation, 2003. 108(6): p. 736-40. 236. Wulster-Radcliffe, M.C., et al., Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun, 2004. 316(3): p. 924-9. 237. Loughna, P.T., P. Mason, and P.C. Bates, Regulation of insulin-like growth factor 1 gene expression in skeletal muscle. Symp Soc Exp Biol, 1992. 46: p. 319-30. 238. Fulks, R.M., J.B. Li, and A.L. Goldberg, Effects of insulin, glucose, and amino acids on protein turnover in rat diaphragm. J Biol Chem, 1975. 250(1): p. 290-8. 239. Mandel, J.L. and M.L. Pearson, Insulin stimulates myogenesis in a rat myoblast line. Nature, 1974. 251(5476): p. 618-20. 240. Brunetti, A., et al., Muscle cell differentiation is associated with increased insulin receptor biosynthesis and messenger RNA levels. J Clin Invest, 1989. 83(1): p. 192-8. 241. Vandenburgh, H.H., et al., Insulin and IGF-I induce pronounced hypertrophy of skeletal myofibers in tissue culture. Am J Physiol, 1991. 260(3 Pt 1): p. C475-84. 242. Jennische, E. and H.A. Hansson, Regenerating skeletal muscle cells express insulin-like growth factor I. Acta Physiol Scand, 1987. 130(2): p. 327-32. 243. Barton-Davis, E.R., D.I. Shoturma, and H.L. Sweeney, Contribution of satellite cells to IGF-I induced hypertrophy of skeletal muscle. Acta Physiol Scand, 1999. 167(4): p. 301- 5. 244. Engert, J.C., E.B. Berglund, and N. Rosenthal, Proliferation precedes differentiation in IGF-I-stimulated myogenesis. J Cell Biol, 1996. 135(2): p. 431-40. 245. Frost, R.A., C.H. Lang, and M.C. Gelato, Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis. Endocrinology, 1997. 138(10): p. 4153-9. 246. Stitt, T.N., et al., The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy- induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell, 2004. 14(3): p. 395-403. 247. Rommel, C., et al., Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol, 2001. 3(11): p. 1009- 13. 248. Bodine, S.C., et al., Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol, 2001. 3(11): p. 1014- 9. 249. Pallafacchina, G., et al., A protein kinase B-dependent and rapamycin-sensitive pathway controls skeletal muscle growth but not fiber type specification. Proc Natl Acad Sci U S A, 2002. 99(14): p. 9213-8. 250. Nave, B.T., et al., Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J, 1999. 344 Pt 2: p. 427-31. 251. Lai, K.M., et al., Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy. Mol Cell Biol, 2004. 24(21): p. 9295-304. 252. Jefferies, H.B., et al., Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. Embo J, 1997. 16(12): p. 3693-704.

102

253. Sandri, M., et al., Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell, 2004. 117(3): p. 399-412. 254. Semsarian, C., et al., Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model. Biochem J, 1999. 339 ( Pt 2): p. 443-51. 255. Jacquemin, V., et al., IGF-1 induces human myotube hypertrophy by increasing cell recruitment. Exp Cell Res, 2004. 299(1): p. 148-58. 256. Machida, S. and F.W. Booth, Insulin-like growth factor 1 and muscle growth: implication for satellite cell proliferation. Proc Nutr Soc, 2004. 63(2): p. 337-40. 257. Barton, E.R., et al., Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice. J Cell Biol, 2002. 157(1): p. 137-48. 258. Shavlakadze, T., et al., Targeted expression of insulin-like growth factor-I reduces early myofiber necrosis in dystrophic mdx mice. Mol Ther, 2004. 10(5): p. 829-43. 259. Schertzer, J.D., et al., Modulation of insulin-like growth factor (IGF)-I and IGF-binding protein interactions enhances skeletal muscle regeneration and ameliorates the dystrophic pathology in mdx mice. Am J Pathol, 2007. 171(4): p. 1180-8. 260. Gregorevic, P., D.R. Plant, and G.S. Lynch, Administration of insulin-like growth factor-I improves fatigue resistance of skeletal muscles from dystrophic mdx mice. Muscle Nerve, 2004. 30(3): p. 295-304. 261. Lynch, G.S., et al., IGF-I treatment improves the functional properties of fast- and slow- twitch skeletal muscles from dystrophic mice. Neuromuscul Disord, 2001. 11(3): p. 260-8. 262. Barton-Davis, E.R., et al., Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci U S A, 1998. 95(26): p. 15603-7. 263. Gonzalez, E., et al., Insulin-like growth factor-1 prevents age-related decrease in specific force and intracellular Ca2+ in single intact muscle fibres from transgenic mice. J Physiol, 2003. 552(Pt 3): p. 833-44. 264. Wang, Z.M., M.L. Messi, and O. Delbono, Sustained overexpression of IGF-1 prevents age-dependent decrease in charge movement and intracellular Ca(2+) in mouse skeletal muscle. Biophys J, 2002. 82(3): p. 1338-44. 265. Renganathan, M., M.L. Messi, and O. Delbono, Dihydropyridine receptor-ryanodine receptor uncoupling in aged skeletal muscle. J Membr Biol, 1997. 157(3): p. 247-53. 266. Renganathan, M., et al., Overexpression of hIGF-1 exclusively in skeletal muscle increases the number of dihydropyridine receptors in adult transgenic mice. FEBS Lett, 1997. 417(1): p. 13-6. 267. Messi, M.L. and O. Delbono, Target-derived trophic effect on skeletal muscle innervation in senescent mice. J Neurosci, 2003. 23(4): p. 1351-9. 268. Payne, A.M., et al., Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle. J Physiol, 2006. 570(Pt 2): p. 283-94. 269. Payne, E.T., et al., Nutritional therapy improves function and complements corticosteroid intervention in mdx mice. Muscle Nerve, 2006. 33(1): p. 66-77. 270. Broussard, S.R., et al., IL-1beta impairs insulin-like growth factor i-induced differentiation and downstream activation signals of the insulin-like growth factor i receptor in myoblasts. J Immunol, 2004. 172(12): p. 7713-20.

103

271. Strle, K., et al., Proinflammatory cytokine impairment of insulin-like growth factor I- induced protein synthesis in skeletal muscle myoblasts requires ceramide. Endocrinology, 2004. 145(10): p. 4592-602. 272. Broussard, S.R., et al., Cytokine-hormone interactions: tumor necrosis factor alpha impairs biologic activity and downstream activation signals of the insulin-like growth factor I receptor in myoblasts. Endocrinology, 2003. 144(7): p. 2988-96. 273. Langen, R.C., et al., Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization. Faseb J, 2004. 18(2): p. 227-37. 274. Levine, B., et al., Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med, 1990. 323(4): p. 236-41. 275. Schulze, P.C., et al., Muscular levels of proinflammatory cytokines correlate with a reduced expression of insulinlike growth factor-I in chronic heart failure. Basic Res Cardiol, 2003. 98(4): p. 267-74. 276. Anker, S.D. and S. von Haehling, Inflammatory mediators in chronic heart failure: an overview. Heart, 2004. 90(4): p. 464-70. 277. Gielen, S., et al., Exercise training in chronic heart failure: correlation between reduced local inflammation and improved oxidative capacity in the skeletal muscle. Eur J Cardiovasc Prev Rehabil, 2005. 12(4): p. 393-400. 278. Lunde, P.K., et al., Contractile properties of in situ perfused skeletal muscles from rats with congestive heart failure. J Physiol, 2002. 540(Pt 2): p. 571-80. 279. Hambrecht, R., et al., Reduction of insulin-like growth factor-I expression in the skeletal muscle of noncachectic patients with chronic heart failure. J Am Coll Cardiol, 2002. 39(7): p. 1175-81. 280. Niebauer, J., et al., Deficient insulin-like growth factor I in chronic heart failure predicts altered body composition, anabolic deficiency, cytokine and neurohormonal activation. J Am Coll Cardiol, 1998. 32(2): p. 393-7. 281. Chung, H.Y., et al., Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev, 2009. 8(1): p. 18-30. 282. Jejurikar, S.S., et al., Aging increases the susceptibility of skeletal muscle derived satellite cells to apoptosis. Exp Gerontol, 2006. 41(9): p. 828-36. 283. Semba, R.D., F. Lauretani, and L. Ferrucci, Carotenoids as protection against sarcopenia in older adults. Arch Biochem Biophys, 2007. 458(2): p. 141-5. 284. Wannamethee, S.G., et al., Physical activity and hemostatic and inflammatory variables in elderly men. Circulation, 2002. 105(15): p. 1785-90. 285. Colbert, L.H., et al., Physical activity, exercise, and inflammatory markers in older adults: findings from the Health, Aging and Body Composition Study. J Am Geriatr Soc, 2004. 52(7): p. 1098-104. 286. Lira, F.S., et al., Chronic exercise decreases cytokine production in healthy rat skeletal muscle. Cell Biochem Funct, 2009. 27(7): p. 458-61. 287. Kalani, R., et al., Effects of caloric restriction and exercise on age-related, chronic inflammation assessed by C-reactive protein and interleukin-6. J Gerontol A Biol Sci Med Sci, 2006. 61(3): p. 211-7. 288. Marzetti, E., et al., Effects of short-term GH supplementation and treadmill exercise training on physical performance and skeletal muscle apoptosis in old rats. Am J Physiol Regul Integr Comp Physiol, 2008. 294(2): p. R558-67.

104

289. Coppack, S.W., Pro-inflammatory cytokines and adipose tissue. Proc Nutr Soc, 2001. 60(3): p. 349-56. 290. Du Clos, T.W., Function of C-reactive protein. Ann Med, 2000. 32(4): p. 274-8. 291. Seo, A.Y., et al., Hepatic oxidative stress during aging: effects of 8% long-term calorie restriction and lifelong exercise. Antioxid Redox Signal, 2006. 8(3-4): p. 529-38. 292. Kumar, A., et al., Nuclear factor-kappaB: its role in health and disease. J Mol Med, 2004. 82(7): p. 434-48. 293. Pedersen, B.K., IL-6 signalling in exercise and disease. Biochem Soc Trans, 2007. 35(Pt 5): p. 1295-7. 294. Hiscock, N., et al., Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: evidence of fiber type specificity. Faseb J, 2004. 18(9): p. 992-4. 295. Febbraio, M.A. and B.K. Pedersen, Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. Faseb J, 2002. 16(11): p. 1335-47. 296. Pretolani, M., Interleukin-10: an anti-inflammatory cytokine with therapeutic potential. Clin Exp Allergy, 1999. 29(9): p. 1164-71. 297. Fiatarone, M.A., et al., Exercise training and nutritional supplementation for physical frailty in very elderly people. N Engl J Med, 1994. 330(25): p. 1769-75. 298. Grimby, G., et al., Training can improve muscle strength and endurance in 78- to 84-yr- old men. J Appl Physiol, 1992. 73(6): p. 2517-23. 299. Glass, D.J., Molecular mechanisms modulating muscle mass. Trends Mol Med, 2003. 9(8): p. 344-50. 300. Kraemer, W.J., et al., American College of Sports Medicine position stand. Progression models in resistance training for healthy adults. Med Sci Sports Exerc, 2002. 34(2): p. 364-80. 301. Beaton, L.J., M.A. Tarnopolsky, and S.M. Phillips, Contraction-induced muscle damage in humans following calcium channel blocker administration. J Physiol, 2002. 544(Pt 3): p. 849-59. 302. MacIntyre, D.L., et al., Different effects of strenuous eccentric exercise on the accumulation of neutrophils in muscle in women and men. Eur J Appl Physiol, 2000. 81(1-2): p. 47-53. 303. MacIntyre, D.L., et al., Presence of WBC, decreased strength, and delayed soreness in muscle after eccentric exercise. J Appl Physiol, 1996. 80(3): p. 1006-13. 304. Beaton, L.J., et al., Contraction-induced muscle damage is unaffected by vitamin E supplementation. Med Sci Sports Exerc, 2002. 34(5): p. 798-805. 305. Gleeson, M., et al., Haematological and acute-phase responses associated with delayed- onset muscle soreness in humans. Eur J Appl Physiol Occup Physiol, 1995. 71(2-3): p. 137-42. 306. Malm, C., et al., Immunological changes in human skeletal muscle and blood after eccentric exercise and multiple biopsies. J Physiol, 2000. 529 Pt 1: p. 243-62. 307. Nguyen, H.X. and J.G. Tidball, Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro. J Physiol, 2003. 547(Pt 1): p. 125-32. 308. Nguyen, H.X. and J.G. Tidball, Null mutation of gp91phox reduces muscle membrane lysis during muscle inflammation in mice. J Physiol, 2003. 553(Pt 3): p. 833-41.

105

309. Cannon, J.G. and B.A. St Pierre, Cytokines in exertion-induced skeletal muscle injury. Mol Cell Biochem, 1998. 179(1-2): p. 159-67. 310. Cannon, J.G., et al., Increased interleukin 1 beta in human skeletal muscle after exercise. Am J Physiol, 1989. 257(2 Pt 2): p. R451-5. 311. Fielding, R.A., et al., Acute phase response in exercise. III. Neutrophil and IL-1 beta accumulation in skeletal muscle. Am J Physiol, 1993. 265(1 Pt 2): p. R166-72. 312. Louis, E., et al., Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle. J Appl Physiol, 2007. 103(5): p. 1744-51. 313. Akerstrom, T., et al., Exercise induces interleukin-8 expression in human skeletal muscle. J Physiol, 2005. 563(Pt 2): p. 507-16. 314. Petersen, A.M. and B.K. Pedersen, The role of IL-6 in mediating the anti-inflammatory effects of exercise. J Physiol Pharmacol, 2006. 57 Suppl 10: p. 43-51. 315. Greiwe, J.S., et al., Resistance exercise decreases skeletal muscle tumor necrosis factor alpha in frail elderly humans. Faseb J, 2001. 15(2): p. 475-82. 316. Lambert, C.P., et al., Exercise but not diet-induced weight loss decreases skeletal muscle inflammatory gene expression in frail obese elderly persons. J Appl Physiol, 2008. 105(2): p. 473-8. 317. Plomgaard, P., M. Penkowa, and B.K. Pedersen, Fiber type specific expression of TNF- alpha, IL-6 and IL-18 in human skeletal muscles. Exerc Immunol Rev, 2005. 11: p. 53-63. 318. Singh, M.A., et al., Insulin-like growth factor I in skeletal muscle after weight-lifting exercise in frail elders. Am J Physiol, 1999. 277(1 Pt 1): p. E135-43. 319. Suetta, C., et al., Coordinated increase in skeletal muscle fiber area and expression of IGF-I with resistance exercise in elderly post-operative patients. Growth Horm IGF Res, 2009. 320. Dennis, R.A., et al., Muscle expression of genes associated with inflammation, growth, and remodeling is strongly correlated in older adults with resistance training outcomes. Physiol Genomics, 2009. 38(2): p. 169-75. 321. Prescott, E., et al., Exercise training in older patients with systolic heart failure: adherence, exercise capacity, inflammation and glycemic control. Scand Cardiovasc J, 2009. 43(4): p. 249-55. 322. Kosek, D.J., et al., Efficacy of 3 days/wk resistance training on myofiber hypertrophy and myogenic mechanisms in young vs. older adults. J Appl Physiol, 2006. 101(2): p. 531-44. 323. Renault, V., et al., Regenerative potential of human skeletal muscle during aging. Aging Cell, 2002. 1(2): p. 132-9. 324. Bloomer, R.J., A.H. Goldfarb, and M.J. McKenzie, Oxidative stress response to aerobic exercise: comparison of antioxidant supplements. Med Sci Sports Exerc, 2006. 38(6): p. 1098-105. 325. Gomez-Cabrera, M.C., E. Domenech, and J. Vina, Moderate exercise is an antioxidant: upregulation of antioxidant genes by training. Free Radic Biol Med, 2008. 44(2): p. 126- 31. 326. Melov, S., et al., Resistance exercise reverses aging in human skeletal muscle. PLoS One, 2007. 2(5): p. e465. 327. Riechman, S.E., et al., Association of interleukin-15 protein and interleukin-15 receptor genetic variation with resistance exercise training responses. J Appl Physiol, 2004. 97(6): p. 2214-9.

106

328. Grabstein, K.H., et al., Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science, 1994. 264(5161): p. 965-8. 329. Carbo, N., et al., Interleukin-15 antagonizes muscle protein waste in tumour-bearing rats. Br J Cancer, 2000. 83(4): p. 526-31. 330. Quinn, L.S., et al., Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders. Exp Cell Res, 2002. 280(1): p. 55-63. 331. Alvarez, B., et al., Effects of interleukin-15 (IL-15) on adipose tissue mass in rodent obesity models: evidence for direct IL-15 action on adipose tissue. Biochim Biophys Acta, 2002. 1570(1): p. 33-7. 332. Carbo, N., et al., Interleukin-15 mediates reciprocal regulation of adipose and muscle mass: a potential role in body weight control. Biochim Biophys Acta, 2001. 1526(1): p. 17-24. 333. Quinn, L.S., Interleukin-15: a muscle-derived cytokine regulating fat-to-lean body composition. J Anim Sci, 2008. 86(14 Suppl): p. E75-83. 334. Quinn, L.S., et al., Serum and muscle interleukin-15 levels decrease in aging mice: Correlation with declines in soluble interleukin-15 receptor alpha expression. Exp Gerontol, 2009. 335. Nieman, D.C., et al., Carbohydrate ingestion influences skeletal muscle cytokine mRNA and plasma cytokine levels after a 3-h run. J Appl Physiol, 2003. 94(5): p. 1917-25. 336. Banni, S., Conjugated linoleic acid metabolism. Curr Opin Lipidol, 2002. 13(3): p. 261-6. 337. Nagao, K., et al., The 10trans, 12cis isomer of conjugated linoleic acid promotes energy metabolism in OLETF rats. Nutrition, 2003. 19(7-8): p. 652-6. 338. Yang, M. and M.E. Cook, Dietary CLA decreased weight loss and extended survival following the onset of kidney failure in NZB/W F1 mice. Lipids, 2003. 38(1): p. 21-4. 339. Moya-Camarena, S.Y., et al., Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARalpha. J Lipid Res, 1999. 40(8): p. 1426-33. 340. Houseknecht, K.L., et al., Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat. Biochem Biophys Res Commun, 1998. 244(3): p. 678-82. 341. Jiang, C., A.T. Ting, and B. Seed, PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature, 1998. 391(6662): p. 82-6. 342. Ricote, M., et al., The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature, 1998. 391(6662): p. 79-82. 343. Desreumaux, P., et al., Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies. J Exp Med, 2001. 193(7): p. 827-38. 344. Inoue, H., T. Tanabe, and K. Umesono, Feedback control of cyclooxygenase-2 expression through PPARgamma. J Biol Chem, 2000. 275(36): p. 28028-32. 345. Brown, J.M., et al., Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue. J Nutr, 2001. 131(9): p. 2316-21. 346. Brown, J.M., et al., Isomer-specific regulation of metabolism and PPARgamma signaling by CLA in human preadipocytes. J Lipid Res, 2003. 44(7): p. 1287-300.

107

347. Suzawa, M., et al., Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB1/NIK cascade. Nat Cell Biol, 2003. 5(3): p. 224-30. 348. Kennedy, A., et al., Trans-10, cis-12 conjugated linoleic acid antagonizes ligand- dependent PPARgamma activity in primary cultures of human adipocytes. J Nutr, 2008. 138(3): p. 455-61. 349. Purushotham, A., et al., Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice. J Lipid Res, 2007. 48(2): p. 444-52. 350. Riserus, U., et al., Supplementation with conjugated linoleic acid causes isomer- dependent oxidative stress and elevated C-reactive protein: a potential link to fatty acid- induced insulin resistance. Circulation, 2002. 106(15): p. 1925-9. 351. Riserus, U., et al., Effects of cis-9,trans-11 conjugated linoleic acid supplementation on insulin sensitivity, lipid peroxidation, and proinflammatory markers in obese men. Am J Clin Nutr, 2004. 80(2): p. 279-83. 352. Graves, E., et al., Conjugated linoleic acid preserves gastrocnemius muscle mass in mice bearing the colon-26 adenocarcinoma. Res Nurs Health, 2005. 28(1): p. 48-55. 353. Inoue, N., et al., Dietary conjugated linoleic acid lowered tumor necrosis factor-alpha content and altered expression of genes related to lipid metabolism and insulin sensitivity in the skeletal muscle of Zucker rats. J Agric Food Chem, 2006. 54(20): p. 7935-9. 354. Winzell, M.S., G. Pacini, and B. Ahren, Insulin secretion after dietary supplementation with conjugated linoleic acids and n-3 polyunsaturated fatty acids in normal and insulin- resistant mice. Am J Physiol Endocrinol Metab, 2006. 290(2): p. E347-54. 355. Andreoli, M.F., et al., Effects of dietary conjugated linoleic acid at high-fat levels on triacylglycerol regulation in mice. Nutrition, 2009. 25(4): p. 445-52. 356. Ha, Y.L., J. Storkson, and M.W. Pariza, Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res, 1990. 50(4): p. 1097-101. 357. Palacios, A., V. Piergiacomi, and A. Catala, Antioxidant effect of conjugated linoleic acid and vitamin A during non enzymatic lipid peroxidation of rat liver microsomes and mitochondria. Mol Cell Biochem, 2003. 250(1-2): p. 107-13. 358. Pinkoski, C., et al., The effects of conjugated linoleic acid supplementation during resistance training. Med Sci Sports Exerc, 2006. 38(2): p. 339-48. 359. Cornish, S.M., et al., Conjugated linoleic acid combined with creatine monohydrate and whey protein supplementation during strength training. Int J Sport Nutr Exerc Metab, 2009. 19(1): p. 79-96. 360. Tarnopolsky, M., et al., Creatine monohydrate and conjugated linoleic acid improve strength and body composition following resistance exercise in older adults. PLoS One, 2007. 2(10): p. e991. 361. Magee, P., S. Pearson, and J. Allen, The omega-3 fatty acid, eicosapentaenoic acid (EPA), prevents the damaging effects of tumour necrosis factor (TNF)-alpha during murine skeletal muscle cell differentiation. Lipids Health Dis, 2008. 7: p. 24. 362. Portnoi, V.A., The concept of pseudodementia. J Am Geriatr Soc, 1983. 31(5): p. 321-2. 363. Jho, D., et al., Omega-3 fatty acids: implications for the treatment of tumor-associated inflammation. Am Surg, 2003. 69(1): p. 32-6. 364. Jho, D.H., et al., Eicosapentaenoic acid supplementation reduces tumor volume and attenuates cachexia in a rat model of progressive non-metastasizing malignancy. JPEN J Parenter Enteral Nutr, 2002. 26(5): p. 291-7.

108

365. Singer, P., et al., Anti-inflammatory properties of omega-3 fatty acids in critical illness: novel mechanisms and an integrative perspective. Intensive Care Med, 2008. 34(9): p. 1580-92. 366. Wanten, G.J. and P.C. Calder, Immune modulation by parenteral lipid emulsions. Am J Clin Nutr, 2007. 85(5): p. 1171-84. 367. Calder, P.C., Polyunsaturated fatty acids, inflammation, and immunity. Lipids, 2001. 36(9): p. 1007-24. 368. Browning, L.M., n-3 Polyunsaturated fatty acids, inflammation and obesity-related disease. Proc Nutr Soc, 2003. 62(2): p. 447-53. 369. Szalay, K., Z. Razga, and E. Duda, TNF inhibits myogenesis and downregulates the expression of myogenic regulatory factors myoD and myogenin. Eur J Cell Biol, 1997. 74(4): p. 391-8. 370. Tolosa, L., et al., IFN-gamma prevents TNF-alpha-induced apoptosis in C2C12 myotubes through down-regulation of TNF-R2 and increased NF-kappaB activity. Cell Signal, 2005. 17(11): p. 1333-42. 371. Stewart, C.E., P.V. Newcomb, and J.M. Holly, Multifaceted roles of TNF-alpha in myoblast destruction: a multitude of signal transduction pathways. J Cell Physiol, 2004. 198(2): p. 237-47. 372. Alvarez, B., et al., Direct effects of tumor necrosis factor alpha (TNF-alpha) on murine skeletal muscle cell lines. Bimodal effects on protein metabolism. Eur Cytokine Netw, 2001. 12(3): p. 399-410. 373. Gingras, A.A., et al., Long-chain omega-3 fatty acids regulate bovine whole-body protein metabolism by promoting muscle insulin signalling to the Akt-mTOR-S6K1 pathway and insulin sensitivity. J Physiol, 2007. 579(Pt 1): p. 269-84. 374. Ide, T., Interaction of fish oil and conjugated linoleic acid in affecting hepatic activity of lipogenic enzymes and gene expression in liver and adipose tissue. Diabetes, 2005. 54(2): p. 412-23. 375. Lieber, R.L. and J. Friden, Functional and clinical significance of skeletal muscle architecture. Muscle Nerve, 2000. 23(11): p. 1647-66. 376. Burkholder, T.J., et al., Relationship between muscle fiber types and sizes and muscle architectural properties in the mouse hindlimb. J Morphol, 1994. 221(2): p. 177-90. 377. Heemskerk, A.M., et al., Determination of mouse skeletal muscle architecture using three-dimensional diffusion tensor imaging. Magn Reson Med, 2005. 53(6): p. 1333-40. 378. Heemskerk, A.M., et al., DTI-based assessment of ischemia-reperfusion in mouse skeletal muscle. Magn Reson Med, 2006. 56(2): p. 272-81. 379. Heemskerk, A.M., et al., Skeletal muscle degeneration and regeneration after femoral artery ligation in mice: monitoring with diffusion MR imaging. Radiology, 2007. 243(2): p. 413-21. 380. Tseng, W.Y., et al., Diffusion tensor MRI of myocardial fibers and sheets: correspondence with visible cut-face texture. J Magn Reson Imaging, 2003. 17(1): p. 31- 42. 381. Galban, C.J., et al., A diffusion tensor imaging analysis of gender differences in water diffusivity within human skeletal muscle. NMR Biomed, 2005. 18(8): p. 489-98. 382. Lee, S., et al., Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats. J Appl Physiol, 2004. 96(3): p. 1097-104.

109

383. Murphy, M.P., et al., A simple and rapid test of sensorimotor function in the aged rat. Neurobiol Learn Mem, 1995. 64(2): p. 181-6. 384. Adams, G.R., F. Haddad, and K.M. Baldwin, Time course of changes in markers of myogenesis in overloaded rat skeletal muscles. J Appl Physiol, 1999. 87(5): p. 1705-12. 385. Adams GR, C.V., Haddad F, and Baldwin KM, Cellular and molecular responses to increased skeletal muscle loading after irradiation. Am J Physiol Cell Physiol 2002: p. 283: C1182–C1195. 386. Haddad, F. and G.R. Adams, Selected contribution: acute cellular and molecular responses to resistance exercise. J Appl Physiol, 2002. 93(1): p. 394-403. 387. Haddad, F. and G.R. Adams, Aging-sensitive cellular and molecular mechanisms associated with skeletal muscle hypertrophy. J Appl Physiol, 2006. 100(4): p. 1188-203. 388. Olshansky, S.J., Projecting the future of U.S. health and longevity. Health Aff (Millwood), 2005. 24 Suppl 2: p. W5R86-9. 389. Adams, K.F., et al., Overweight, obesity, and mortality in a large prospective cohort of persons 50 to 71 years old. N Engl J Med, 2006. 355(8): p. 763-78. 390. Barness, L.A., J.M. Opitz, and E. Gilbert-Barness, Obesity: genetic, molecular, and environmental aspects. Am J Med Genet A, 2007. 143A(24): p. 3016-34. 391. Zimmermann, J., et al., Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int, 1999. 55(2): p. 648-58. 392. Dunstan, D.W., et al., High-intensity resistance training improves glycemic control in older patients with type 2 diabetes. Diabetes Care, 2002. 25(10): p. 1729-36. 393. Krisan, A.D., et al., Resistance training enhances components of the insulin signaling cascade in normal and high-fat-fed rodent skeletal muscle. J Appl Physiol, 2004. 96(5): p. 1691-700. 394. Hunter, G.R., et al., Resistance training and intra-abdominal adipose tissue in older men and women. Med Sci Sports Exerc, 2002. 34(6): p. 1023-8. 395. Park, Y., et al., Effect of conjugated linoleic acid on body composition in mice. Lipids, 1997. 32(8): p. 853-8. 396. West, D.B., et al., Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse. Am J Physiol, 1998. 275(3 Pt 2): p. R667-72. 397. DeLany, J.P., et al., Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake. Am J Physiol, 1999. 276(4 Pt 2): p. R1172-9. 398. Ryder, J.W., et al., Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression. Diabetes, 2001. 50(5): p. 1149-57. 399. Yamasaki, M., et al., Modulation of body fat and serum leptin levels by dietary conjugated linoleic acid in Sprague-Dawley rats fed various fat-level diets. Nutrition, 2003. 19(1): p. 30-5. 400. Ikemoto, S., et al., High-fat diet-induced hyperglycemia and obesity in mice: differential effects of dietary oils. Metabolism, 1996. 45(12): p. 1539-46. 401. Lombardo, Y.B. and A.G. Chicco, Effects of dietary polyunsaturated n-3 fatty acids on dyslipidemia and insulin resistance in rodents and humans. A review. J Nutr Biochem, 2006. 17(1): p. 1-13. 402. Sneddon, A.A., et al., Effect of a conjugated linoleic acid and omega-3 fatty acid mixture on body composition and adiponectin. Obesity (Silver Spring), 2008. 16(5): p. 1019-24.

110

403. Takahashi, Y., et al., Dietary conjugated linoleic acid reduces body fat mass and affects gene expression of proteins regulating energy metabolism in mice. Comp Biochem Physiol B Biochem Mol Biol, 2002. 133(3): p. 395-404. 404. Prins, J.B., et al., Tumor necrosis factor-alpha induces apoptosis of human adipose cells. Diabetes, 1997. 46(12): p. 1939-44. 405. Semb, H., et al., Multiple effects of tumor necrosis factor on lipoprotein lipase in vivo. J Biol Chem, 1987. 262(17): p. 8390-4. 406. Pekala, P.H., et al., Selective inhibition of synthesis of enzymes for de novo fatty acid biosynthesis by an endotoxin-induced mediator from exudate cells. Proc Natl Acad Sci U S A, 1983. 80(9): p. 2743-7. 407. Halminski, M.A., J.B. Marsh, and E.H. Harrison, Differential effects of fish oil, safflower oil and palm oil on fatty acid oxidation and glycerolipid synthesis in rat liver. J Nutr, 1991. 121(10): p. 1554-61. 408. Hainault, I., et al., Fish oil in a high lard diet prevents obesity, hyperlipemia, and adipocyte insulin resistance in rats. Ann N Y Acad Sci, 1993. 683: p. 98-101. 409. Baillie, R.A., et al., Coordinate induction of peroxisomal acyl-CoA oxidase and UCP-3 by dietary fish oil: a mechanism for decreased body fat deposition. Prostaglandins Leukot Essent Fatty Acids, 1999. 60(5-6): p. 351-6. 410. Belzung, F., T. Raclot, and R. Groscolas, Fish oil n-3 fatty acids selectively limit the hypertrophy of abdominal fat depots in growing rats fed high-fat diets. Am J Physiol, 1993. 264(6 Pt 2): p. R1111-8. 411. Parrish, C.C., D.A. Pathy, and A. Angel, Dietary fish oils limit adipose tissue hypertrophy in rats. Metabolism, 1990. 39(3): p. 217-9. 412. Sakamoto, K. and L.J. Goodyear, Invited review: intracellular signaling in contracting skeletal muscle. J Appl Physiol, 2002. 93(1): p. 369-83. 413. Sakamoto, K., et al., Exercise regulates Akt and glycogen synthase kinase-3 activities in human skeletal muscle. Biochem Biophys Res Commun, 2004. 319(2): p. 419-25. 414. Bolster, D.R., et al., Immediate response of mammalian target of rapamycin (mTOR)- mediated signalling following acute resistance exercise in rat skeletal muscle. J Physiol, 2003. 553(Pt 1): p. 213-20. 415. Hargreaves, M., G. McConell, and J. Proietto, Influence of muscle glycogen on glycogenolysis and glucose uptake during exercise in humans. J Appl Physiol, 1995. 78(1): p. 288-92. 416. Pilegaard, H., et al., Influence of pre-exercise muscle glycogen content on exercise- induced transcriptional regulation of metabolic genes. J Physiol, 2002. 541(Pt 1): p. 261- 71. 417. Pilegaard, H., et al., Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab, 2000. 279(4): p. E806-14. 418. Creer, A., et al., Influence of muscle glycogen availability on ERK1/2 and Akt signaling after resistance exercise in human skeletal muscle. J Appl Physiol, 2005. 99(3): p. 950-6. 419. Blankson, H., et al., Conjugated linoleic acid reduces body fat mass in overweight and obese humans. J Nutr, 2000. 130(12): p. 2943-8. 420. Verdijk, L.B., et al., Skeletal muscle hypertrophy following resistance training is accompanied by a fiber type-specific increase in satellite cell content in elderly men. J Gerontol A Biol Sci Med Sci, 2009. 64(3): p. 332-9.

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421. Febbraio, M.A. and B.K. Pedersen, Contraction-induced myokine production and release: is skeletal muscle an endocrine organ? Exerc Sport Sci Rev, 2005. 33(3): p. 114-9. 422. Weldon, S.M., et al., Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid. J Nutr Biochem, 2007. 18(4): p. 250-8. 423. Moloney, F., et al., Antidiabetic effects of cis-9, trans-11-conjugated linoleic acid may be mediated via anti-inflammatory effects in white adipose tissue. Diabetes, 2007. 56(3): p. 574-82. 424. Furmanczyk, P.S. and L.S. Quinn, Interleukin-15 increases myosin accretion in human skeletal myogenic cultures. Cell Biol Int, 2003. 27(10): p. 845-51. 425. Chung, S., et al., Trans-10,cis-12 CLA increases adipocyte lipolysis and alters lipid droplet-associated proteins: role of mTOR and ERK signaling. J Lipid Res, 2005. 46(5): p. 885-95. 426. Li, W., et al., Interleukin-1 stimulates catabolism in C2C12 myotubes. Am J Physiol Cell Physiol, 2009. 297(3): p. C706-14. 427. Kooijman, R. and A. Coppens, Insulin-like growth factor-I stimulates IL-10 production in human T cells. J Leukoc Biol, 2004. 76(4): p. 862-7. 428. Larsen, A.E., D. Cameron-Smith, and T.C. Crowe, Conjugated linoleic acid suppresses myogenic gene expression in a model of human muscle cell inflammation. J Nutr, 2008. 138(1): p. 12-6.

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BIOGRAPHICAL SKETCH

SANG-ROK LEE

Sang-Rok Lee was born in Incheon in South Korea to Kwan-hae Lee and Bok-ae Kim. From fourth grade, Sang-Rok played soft tennis over ten years as a varsity player with a number of titles in the national competitions. He entered the Daejeon University for his bachelor’s degree in Physical Education. During his time as an undergraduate, he served as the student representative in his department. Following his bachelor’s degree, he entered the Kookmin University to obtain a master’s degree in Measurement and Evaluation in Physical Education. Realizing the importance of exercise as one of the key factors for human health, he joined Southern Illinois University in Carbondale, IL for his master’s degree with an emphasis in exercise physiology. After completing his master’s degree, Sang-rok started the PhD program in exercise physiology and joined the Muscle Physiology lab working with Dr. Jeong-Su Kim at The Florida State University (FSU). For five years, he served as lab manager and research assistant in the Muscle Physiology lab and collaborated with the National High Magnetic Field Laboratory where he was tasked with conceiving and implementing projects on age-induced muscle wasting, termed sarcopenia. During his Ph.D work, Sang-Rok has published 5 peer reviewed manuscripts and presented or co presented nearly 30 abstracts at regional, national, and international conferences. Sang-Rok’s primary goal as an exercise scientist is to contribute to our society by conducing research projects that can improve quality of life.

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