Copyright by Geoffrey Josef Solares 2019

The Dissertation Committee for Geoffrey Josef Solares Certifies that this is the approved version of the following Dissertation:

ACUTE AND CHRONIC EFFECTS OF b-HYDROXY-b- METHYLBUTYRATE (b-HMB) ON GLUCOSE TOLERANCE, INSULIN SENSITIVITY AND MUSCLE ADAPTATION FOLLOWING CHRONIC RESISTANCE TRAINING

Committee:

Roger P. Farrar, Co-Supervisor

Laura J. Suggs, Co-Supervisor

Molly S. Bray

Richard E. Wilcox

Janice S. Todd

ACUTE AND CHRONIC EFFECTS OF b-HYDROXY-b- METHYLBUTYRATE (b-HMB) ON GLUCOSE TOLERANCE, INSULIN SENSITIVITY AND MUSCLE ADAPTATION FOLLOWING CHRONIC RESISTANCE TRAINING

by

Geoffrey Josef Solares

Dissertation

Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

The University of Texas at Austin May 2019

Acknowledgements

There have been many people who have contributed to my dissertation for whom I owe a debt of gratitude.

First and foremost, I would like to thank my advisor Dr. Roger Farrar. Roger, you have without a doubt been an invaluable asset to me as a researcher and as a young man, husband, and father, who had a mentor which sought to ensure that his mentee was able to feed their growing family with a steady supply of good teaching assistant jobs. No amount of words here can express that fact, Rog, I owe more than I could ever repay you.

I would also like to thank my initial advisor, Dr. John Ivy for his wisdom and perspective relating to my development as a more robust researcher. Also, you have taught me a mentoring style that forces the person to persevere and develop internal reflections paramount to a quality researcher. In essence John, you re-enforced the idea of “grit” an idea that appears to be waning nowadays.

I would also like to thank Dr. Richard Wilcox for your endless critiques to this manuscripts and encouragement to “go big or go home” when designing my experiment. I would like to thank Dr. Molly Bray. Molly, working with you over the past years as your teaching assistant and in your lab, you graciously welcomed me to a different environment and fostered a profound amount of critical thinking in me, I thank you.

iv To Wenbai Huang for is constant aid and support throughout all of our early mornings and long afternoons as we worked to complete my resistance exercise protocol and animal surgeries, I thank you.

To my siblings, Giorgio, Krystal, and Briahna, thank you for making me feel less disconnected from life back in California. I would especially like to thank my parents, who over the years have greatly helped me establish myself and my wife in Austin and who been a bastion of unconditional love throughout my entire life.

To my parents who have countless times provoked my thinking, re-shaped my viewpoints, as well as forever placing on me the emphasis that knowledge opens doors and instilling “grit” at an early age. I would not be here without your help. I love you both!

I would also like to express my gratitude to my extended family. Chuck and Sue, you have always been there for me and even hosted my family for weeks at a time whenever we arrived in town in addition to the support you gave Amy when she needed a call or longed for home you both were always there, I am eternally grateful.

Finally, to my wife and sons. Amy, I know I dragged you out here right after our wedding and the years have been a roller-coaster of a ride, but it has been a ride I would have shared with no one else. For making our apartments and house a home, warm meals, cuddles on the couch, giggles, and for growing, having, and teaching our three beautiful sons with endless love, these words do not scratch the surface of how I feel for you, I love you. While this dissertation represents my work at UT, it fails to reflect all the work you did to make this dissertation complete. Your name belongs aside mine on the diploma for all the exams, presentations, and work you helped me with throughout our time at UT. v Jackson, my son, your smiles, laughter, and innocence are infectious and have been a blessing since the moment I got to hold you and say “my boy”. Getting to hear every single time I come through the door, “Momma, Dada’s home”, you will never know how much that makes my heart swell. Never change my son. Kylar, your serenity in looking at the raucousness that can be our home life reminds me to live life in the moment and take it all in. Thank you for choosing our family and letting me be your dad. Logan, while you arrived late in the game, you have been awaited with as much joy as a father could have for their son. Your coos and giggles while I wrote this dissertation are heartwarming memories and remind me why I went through all of this - for each of my boys to be inspired. Jackson,

Kylar, and Logan, your growth in our home has been a joy I cannot imagine living without, never forget my sons, Dada loves you.

vi ACUTE AND CHRONIC EFFECTS OF b-HYDROXY-b- METHYLBUTYRATE (b-HMB) ON GLUCOSE TOLERANCE, INSULIN SENSITIVITY AND MUSCLE ADAPTATION FOLLOWING CHRONIC RESISTANCE TRAINING Geoffrey Josef Solares, Ph.D. The University of Texas at Austin, 2019 Supervisors: Roger P. Farrar & Laura J. Suggs

Maximizing protein accretion and mitigating protein degradation is a major goal for resistance training regimens and intervention therapies. The branch chain amino acid leucine has historically demonstrated a significant role in the activation of protein synthesis via the activation of the mammalian target of rapamycin complex 1 (mTORC1). A derivative of leucine metabolism, beta-hydroxy-beta-methylbutyrate (HMB) has shown similar effects on mTORC1 with recent literature suggesting adverse effects of HMB on glucose homeostasis and regulation. Herein, we used animal models to test the effects of varying doses of HMB on glucose homeostasis during a novel chronic resistance whole body training model. These data suggest that HMB effects cause acute modulation to the Akt/mTOR signaling pathway proteins, with minimal contributions to strength gains during chronic resistance whole body exercise. Our novel whole body exercise technique revealed a significant increase in strength gains with no differences in in situ force production in quadriceps and triceps surae muscle groups, but did show increased force per unit mass in both the triceps surae and quadriceps muscle groups. These data suggest an increase in whole body muscular coordination and/or synchronicity in force production that promotes increases in overall total strength. Furthermore, our data suggests that the overload placed on each individual contributing muscle to force

vii output was not significant enough to induce a hypertrophic response. We conclude that HMB ingestion provides minimal benefit during prolonged exercise regimens and the effects of HMB on blood glucose and insulin sensitivity are not adverse. Finally, our whole body resistance model presents a novel paradigm for increasing work output that can be a model of whole body resistance training.

viii Table of Contents

List of Tables ...... xii

List of Figures ...... xiii

CHAPTER I:GENERAL INTRODUCTION ...... 1

Blood glucose regulation...... 1

Signaling pathways which control blood glucose ...... 1

Resistance exercise increases hypertrophy via mTOR activation ...... 3

Timely nutritional intervention reduces proteolysis ...... 4

OBJECTIVES ...... 4

HYPOTHESES ...... 5

Study 1 ...... 5

Study 2 ...... 5

Study 3 ...... 6

SIGNIFICANCE ...... 6

LIMITATIONS ...... 7

CHAPTER II: LITERATURE REVIEW ...... 9

BETA-HYDROXY-BETA-METHYLBUTYRATE (b-HMB) ...... 9

Safety of ß-HMB ...... 10

INSULIN SIGNALING ...... 11

Akt/PKB activation, targets, and downstream effects ...... 13

MUSCLE PROTEIN SYNTHESIS SIGNALING PATHWAYS...... 14

How resistance exercise affects protein turnover ...... 17

How b-HMB interacts with protein turnover ...... 21

ix MUSCLE PROTEIN DEGRADATION SIGNALING PATHWAYS ...... 23

How b-HMB interacts with the muscle protein degradation pathways ...... 25

MUSCLE ADAPTATION TO RESISTANCE EXERCISE ...... 27

Muscle response to acute resistance exercise ...... 27

Muscle adaptations to chronic resistance exercise ...... 30

EXERCISE EFFECT ON GLUCOSE UPTAKE & GLUCOSE TOLERANCE ...... 33

AMPK involvement in glucose homeostasis within skeletal muscle .. 35

NEGATIVE REGULATION OF INSULIN SIGNALING ...... 36

b-HMB interactions with blood glucose homeostasis ...... 37

SUMMARY ...... 39

CHAPTER III: THE ACUTE EFFECTS OF BETA-HYDROXY-BETA- METHYLBUTYRATE ON GLUCOSE HOMEOSTASIS , INSULIN SENSITIVITY, AND AKT/MTOR PATHWAY PROTEINS ...... 45

ABSTRACT ...... 45

INTRODUCTION ...... 46

METHODS ...... 47

RESULTS...... 50

DISCUSSION ...... 71

CHAPTER IV: HYDROXY-BETA-METHYBUTYRATE CHRONIC SUPPLEMENTATION ON BLOOD GLUCOSE, STRENGTH, AND HYPERTROPHY IN A NOVEL RESISTANCE METHOD FOR INDUCING MUSCULAR STRENGTH ...... 76

ABSTRACT ...... 76

INTRODUCTION ...... 77

x METHODS ...... 79

RESULTS – FOUR TREATMENT ANALYSES ...... 89

RESULTS – TWO TREATMENTS ANALYSES...... 97

DISCUSSION ...... 101

CHAPTER V: GENERAL DISCUSSION ...... 116

Summary of Results ...... 116

Conclusions ...... 117

Future Directions ...... 117

APPENDICES ...... 119

Appendix A: Expanded Methods ...... 119

Appendix B: Raw Data ...... 140

REFERENCES ...... 186

VITA ...... 216

xi List of Tables

Table 4.1 Wet weights of muscles harvested from sedentary animals...... 95 Table 4.2 Wet weights of muscles harvested from resistance trained animals...... 98 Table 4.3 Body and Muscle Weights...... 103 Table 4.4 Ancillary Tissue Weights ...... 103 Table 4.5 Quadriceps Contractile Properties ...... 103 Table 4.6 Triceps Surae Contractile Properties...... 104

Table 4.7 Effect of Resistance Exercise on Tissue Dry Weight/Wet Weight Ratio ..... 115

xii List of Figures

Figure 2.1. Insulin, IGF-1 and AMPK signaling pathways ...... 41 Figure 2.2. Negative modulators of insulin and IGF-1 signaling pathways ...... 42 Figure 2.3. Interactions of b-HMB with the Akt/PI-3K signaling pathway and protein

degradation pathways ...... 43 Figure 2.4. Resistance exercise effects on subcellular signaling pathways...... 44 Figure 3.1. The blood glucose concentrations (A) and plasma insulin concentrations

(B) during an OGTT when b-HMB was provided 15 minutes prior to the

glucose bolus...... 50 Figure 3.2. The blood glucose concentrations (A) and plasma insulin concentrations

(B) during an OGTT when b-HMB was provided 150 minutes prior to

the glucose bolus...... 52 Figure 3.3. The blood glucose concentrations (A) and plasma insulin concentrations

(B) during an OGTT when b-HMB was provided 150 minutes prior to

the glucose bolus...... 53 Figure 3.4 – Glycogen Synthase Kinase 3b (GSK3b) total protein (A) and total phosphorylated protein (B) quantity as determined by median

fluorescence intensity...... 54 Figure 3.5 – Insulin Like Growth Factor 1 (IGF1R) total protein (A) & total phosphorylated protein (B) quantity as determined by median

fluorescence intensity...... 55 Figure 3.6 – Insulin receptor subunit-1 total protein (A) and total phosphorylated

protein (B) quantity as determined by median fluorescence intensity...... 57 Figure 3.7 – Akt total protein (A) and total phosphorylated protein (B) quantity as

determined by median fluorescence intensity...... 58 xiii Figure 3.8 – Mammalian target of rapamycin (mTOR) total protein (A) and total phosphorylated protein (B) quantity as determined by median

fluorescence intensity...... 59 Figure 3.9 – p70S6K total protein (A) and total phosphorylated protein (B) quantity

as determined by median fluorescence intensity...... 60 Figure 3.10 – Insulin receptor (IR) total protein (A) & total phosphorylated protein

(B) quantity as determined by median fluorescence intensity...... 62 Figure 3.11 – Phosphatase and Tensin Homolog (PTEN) total protein (A) and total phosphorylated protein (B) quantity as determined by median

fluorescence intensity...... 63 Figure 3.12 – Glycogen Synthase Kinase 3 a (GSK3a) total protein (A) & total phosphorylated protein (B) quantity as determined by median

fluorescence intensity...... 64 Figure 3.13 – Tuberous sclerosis complex 2 (TSC2) total protein (A) and total phosphorylated protein (B) quantity as determined by median

fluorescence intensity...... 65 Figure 3.14 – Ribosomal protein S6 (rpS6) total protein (A) & total phosphorylated

protein (B) quantity as determined by median fluorescence intensity...... 66 Figure 4.1: Weight attachment points...... 80 Figure 4.2: Training apparatus...... 81 Figure 4.3: In situ setup of rodent hind limb for quadricep testing...... 84 Figure 4.4 Blood glucose during oral glucose tolerance tests which occurred prior to HMB administration (A), 5 weeks (B), and 11 weeks (C) with chronic

HMB supplementation...... 91

xiv Figure 4.5 Blood glucose during oral glucose tolerance tests which occurred prior to

training (A), 5 weeks (B), and 11 weeks (C) into resistance training...... 92 Figure 4.6 Area under the curve (AUC) for blood glucose during tolerance tests which occurred prior to HMB administration (A), 5 weeks (B), and 11

weeks (C) with chronic HMB supplementation...... 93 Figure 4.7 Area under the curve (AUC) for blood glucose during tolerance tests which occurred prior to resistance exercise (A), 5 weeks (B), and 11

weeks (C) with into resistance exercise...... 94 Figure 4.8 Total Force (N) measured in Triceps Surae (A) and Quadriceps (B)

muscle groups by treatment...... 94 Figure 4.9: Total Force (N) measured in Triceps Surae (A) and Quadriceps (B)

muscle groups by treatment...... 95 Figure 4.10: Maximal carrying load per training week...... 98 Figure 4.11: Force (N) / Muscle Weight (g) in both the Triceps Surae and Quadriceps

muscle groups by treatment...... 99 Figure 4.12: Western blotting was performed on sedentary (CON) and resistance trained (EX) animal gastrocnemius medial heads (A) and quadricep

muscles (B) of the non-force measured hindlimb...... 102 Figure 4.13: Oral glucose tolerance test which occurred prior to training (A) , 5

weeks (B), and 11 weeks (C) into training...... 113 Figure 4.14: AUC calculations from oral glucose tolerance test which occurred prior

to training (A) , 5 weeks (B), and 11 weeks (C) into training...... 114

xv CHAPTER I

GENERAL INTRODUCTION

Blood glucose regulation

Blood glucose is a tightly regulated physiological parameter in humans. Entrance of glucose into a cell occurs either using a sodium – glucose transporter or by a protein- mediated glucose transporter (GLUT). In skeletal muscle, GLUT4 has been shown to mediate both insulin and contraction-induced glucose transport (138). GLUT4 proteins are sequestered within vesicles located in the cytoplasm. Stimulation by insulin or skeletal muscle contraction will translocate GLUT 4 to the plasma membrane (PM) facilitating glucose diffusion into the cell (138). Two separate GLUT4 pools have been identified, which respond to either insulin or contraction-induced stimulation (254, 311), and these pools are not absolutely independent of each other. GLUT4 protein has been shown to increase following exercise in rats (45, 100, 242, 255) and humans (62, 120, 121). These data demonstrate the conclusion that exercise can increase skeletal muscle insulin sensitivity; in part by increasing increased GLUT4 protein concentration, resulting in parallel increases in glucose uptake (253). When there is dysregulation in the clearance of glucose following a meal, elevated levels of circulating glucose will stimulate an additional release of insulin from the beta cells. If left uncorrected, the dysregulation will result in glucose toxicity, beta cell fatigue/apoptosis, and the development of T2D (67).

Signaling pathways which control blood glucose

The insulin signaling pathway begins at the insulin receptor (IR), when insulin binds to the IR alpha subunit. A conformational change in the beta subunits activates tyrosine residues permitting insulin receptor substrate (IRS) proteins 1 and 2 to be recruited 1 and will themselves phosphorylate tyrosine forming binding sites for molecules containing Src-homology 2 (SH2) domains (289). Phosphoinositide 3´-kinase (PI3-K) will then associate with IRS-1, thus activating it. PI3-K is comprised of a p85 regulatory subunit and a p110 catalytic subunit. When the p110 subunit is activated it will translocate to the plasma membrane (PM). PI3-K activation is required for insulin-stimulated glucose uptake as studies using wortmannin (170, 333), a known PI3-K inhibitor, have shown wortmannin abolishes insulin stimulated glucose uptake. At the PM the p110 catalytic subunit will phosphorylate the inositol phospholipid (PI) (23, 27) bisphosphate (PIP2) to PI (3,4,5) triphosphate (PIP3). Subsequently, PIP3 will activate PDK-1 and PDK-2 (phosphoinositide dependent kinases) thereby activating protein kinase B (Akt/PKB), atypical protein kinase C lambda and zeta (aPKC, λ/ζ) and p70 ribosomal S6 kinase (S6K1) proteins on serine/threonine residues (39, 235). Akt/PKB is involved in insulin mediated glucose transport by the GTPase activation of TBC1D4 (9, 265, 267, 296). When TBC1D4 is activated, a Rab protein in a GDP-bound state will convert to a GTP-bound state thereby releasing GLUT-4, which will then translocate to the PM to facilitate diffusion of glucose into the cell (317). The contraction-induced glucose transport, identified by 5´-AMP-activated protein kinase (AMPK) activity, was shown to elicit a separate GLUT4 pool when there was an additive amount of glucose transport in the presence of insulin (132). AMPK activation, as a direct consequence of exercise, was first reported in rodents by Winder and Hardie (329) and later confirmed in humans (52, 102, 286, 336). It has been concluded that ATP/AMP and ATP/ADP ratio reductions due to high cellular energy turnover (302) are responsible for AMPK activation. It has subsequently been demonstrated that AMPK inhibits TBC1D4 action, would inhibit GLUT4 translocation (51, 172). AMPK has been shown to be increased following exercise, as well as increasing insulin sensitivity long after the acute 2 post exercise effects have disappeared (165, 332). Moreover, chronic exercise has been shown to increase insulin sensitivity as well as basal AMPK concentration and activity in humans (101, 196, 312). It has been observed that increased basal AMPK concentrations resulted in increased basal TBC1D4 phosphorylation in both humans and rats (44, 106, 300, 312). Another mechanism by which AMPK can control ATP consumption is by regulating protein synthesis (36) by phosphorylating tuberous sclerosis complex 2 (TSC2) thus inhibiting mTOR activation (154, 279). By phosphorylating eukaryotic elongation factor 2α (eEF2), AMPK can inhibit the elongation phase of protein synthesis (141). Likewise, Akt/PKB will affect proteolytic processes via phosphorylating forkhead box O (FoxO3A), resulting in FoXO3A being excluded from the nucleus thereby preventing transcriptional activity (304). Recently in unpublished data from our laboratory, AMPKα phosphorylation and mTOR signaling proteins were elevated post exercise in animals provided a mixture of HMB, leucine, and whey protein following endurance exercise. This finding suggests that AMPKα phosphorylation can occur from protein enriched supplementation independent of exercise.

Resistance exercise increases hypertrophy via mTOR activation

Hypertrophy will occur if RE is performed chronically with sufficient stimulus to skeletal muscle, resulting in increased lean body mass and decreased fat mass. It has been shown that RE induces mTOR activation and that mTOR activation is required for muscle protein synthesis (MPS) (96, 113). mTOR regulates translational initiation of MPS in humans and rats (113, 147, 299, 316). Moreover, mTOR has been shown to be convergent point for several factors responsible for MPS such as amino acid, muscle contraction, and hormones (113, 147, 299). mTOR can be activated by Akt which will inhibit proline rich Akt substrate at 40kDa (PRAS-40) and (TSC2) thereby activating mTOR (153). mTOR is

3 phosphorylated on serine residue 2448 and when activated will subsequently phosphorylate S6K1 and eukaryotic initiation factor 4E binding protein (4E-BP1) (96, 147, 162). Over a chronic period of activation, the accretion of skeletal muscle will occur provide sufficient nutrient intake occurs.

Timely nutritional intervention reduces proteolysis

However, MPS stimulations post exercise coincides with an increased rate of proteolysis in the absence of nutritional intervention. Proteolysis is generally reliant upon the ubiquitin-proteosome pathway. The two most important ubiquitin E3 ligases are muscle atrophy F-box (MAF-bx or atrogin-1) and muscle ring-finger protein-1 (MuRF-1), which are upregulated during proteolysis (16). Both of these E3 ligases are directly influenced by regulatory transcription factors such as FOXO3A. Protein degradation has been shown to be decreased in cultured muscles in the presence of bHMB (86, 283). This finding has also been found to hold true in in vivo models (137). Conflicting findings in MPS exist with some researchers reporting bHMB increases protein synthesis (17, 86, 114, 179, 283) while others report no effect (137). It has been shown however that b HMB is capable of increasing phosphorylation of mTOR and S6K1, suggesting HMB can increase skeletal muscle protein translation via these mechanisms (17, 86, 241).

OBJECTIVES

The broad objective of this study was to identify a dose response of b-HMB on glucose homeostasis and insulin signaling in sedentary rats and the effects of b-HMB on skeletal muscle during a novel whole body resistance training regimen. Our objectives were set as follows:

4 1) Determine the varying dose effect of b-HMB on plasma glucose and the Akt subcellular protein concentrations, during oral glucose tolerance testing and an insulin sensitivity test.

2) Determine b-HMB hypertrophic effects in sedentary control animal skeletal muscle

3) Determine b-HMB hypertrophic effects in resistance trained animal skeletal muscle

4) Characterize the b-HMB effects on Akt subcellular protein concentrations in sedentary and resistance exercise animals

HYPOTHESES

Study 1

1) ß-HMB will decrease glucose clearance in a dose dependent manner when provided at either 15 or 150 minutes prior to an OGTT 2) ß-HMB will decrease insulin sensitivity when provided 150 minutes prior to an ITT. 3) ß-HMB provided 150 minutes prior to an ITT will modulated phosphorylation of signaling proteins the Akt/mTOR signaling pathway in a dose dependent manner.

Study 2

1) The addition of ß-HMB to sedentary animals will increase glucose clearance during an OGTT 2) ß-HMB will increase the phosphorylation of muscle protein synthesis signaling proteins in the Akt/mTOR signaling pathway

5 3) ß-HMB will induce a greater modulation on the phosphorylation status of muscle degradation signaling proteins in the FOXO3A pathway 4) ß-HMB supplementation in sedentary animals will increase force production in situ and cross-sectional area of hindlimb muscles

Study 3

1) The addition of ß-HMB to RT will increase glucose clearance during an OGTT 2) The addition of ß-HMB to RT will increase the phosphorylation of muscle protein synthesis signaling proteins in the Akt/mTOR pathways 3) The addition of ß-HMB to RT will induce a greater modulation on the

phosphorylation status of muscle degradation signaling proteins in the FOXO3A pathway 4) The addition of ß-HMB to RT will increase force production in situ and cross- sectional area of hindlimb muscles

SIGNIFICANCE

The following work provides evidence that b-HMB supplementation does not result in long term adverse effects in glucose homeostasis. While b-HMB does appear to affect glucose regulation, the effects appear transitory. Therefore, chronic ingestion of b-HMB can be supplemented by populations concerned with glucose homeostasis, such as diabetics, without worry. b-HMB effects on glucose homeostasis herein, can lead to designs and supplementations strategies in which including b-HMB can aid in the re- synthesis of glycogen storage post exercise, but additional studies are needed to confirm this hypothesis. Our novel whole body resistance training regimen has demonstrated a significant increase in measurable strength without increasing muscle mass or force

6 production in the quadriceps and triceps surae muscle groups, but which does increase force per gram muscle in the triceps surae.muscle group. These data suggest an increase in whole body muscular coordination and/or synchronicity in force contribution is responsible for increased overall total strength. The lack in observable hypertrophy across all measured muscles is likely due to insufficient direct overload placed on each individual contributing force generating muscle. Consequently, the insufficient stimulus was not significant enough to induce an observable hypertrophic response. We conclude that b-HMB ingestion provides no benefit during prolonged exercise regimens and the previously reported adverse effects of b-HMB are transient and non-apparent in chronically supplemented animals. Finally, our whole body resistance model presents a novel application for inducing strength gains which may be beneficial if adapted to humans.

LIMITATIONS

This study was performed using a rodent animal model. Further testing using mammalian species are necessary to confirm these findings. Study 1 examined the acute effects of b-HMB and we did not observe benefits when applied chronically. A limitation could be the fact that phosphorylation of proteins occurs transiently in response to both nutritional supplementation and muscle contraction stimulus. Therefore it is reasonable that our procurement of tissue samples occurred outside the responsive window, resulting in the findings we observed. Additional studies are warranted to investigate the time course of protein phosphorylation in response to a resistance training program combined with b- HMB supplementation.

Study 3 examined the effects of chronic b-HMB supplementation during a resistance training regimen lasting 12-14 weeks. The animals were permitted to consume both food and water ad libitum. We observed reduced skeletal muscle mass in both exercise

7 treatment groups suggesting inadequate caloric consumption when compared to control animals. This observation has impacted the potential hypertrophic effects of our training regimen as the exercising animals did possess smaller skeletal masses compared to sedentary animals. It is reasonable to expect that if our animals had been iso-calorically fed to sedentary animals, our results may have resulted in different observations. Therefore, additional studies are needed to confirm our findings and the impact our training protocol would have in iso-calorically fed animals.

8 CHAPTER II

LITERATURE REVIEW

BETA-HYDROXY-BETA-METHYLBUTYRATE (b-HMB)

ß-HMB is derived from leucine catabolism, which can occur in liver cytosol, skeletal muscle mitochondria, and liver mitochondria (186). When leucine is catabolized, the initial step is a reversible transaminase reaction which forms α-ketoisocaproic acid (KIC) (186). At this point further catabolism of leucine branches; in liver mitochondria KIC is irreversibly oxidized via an enzyme called branched-chain ketoacid dehydrogenase (BCKD) forming isovaleryl-CoA (186), whereas in skeletal muscle mitochondria KIC is converted into ß-HMB by KIC dioxygenase (261-

263). When ß-HMB was first discovered it was identified as beta-hydroxyisovaleric acid. The earliest identification and mention of this molecule was in a report by Tanaka et al. in 1968 who discovered ß-HMB in the urine of a patient suffering from isovaleric academia (292). When scientists were first postulating the synthesis of cholesterol, it was revealed that carbons derived from leucine were incorporated into newly formed cholesterol (209, 258) and was further postulated that a direct unknown precursor (later identified as ß-HMB) was responsible for cholesterol synthesis (6, 21, 32). It was not until Sanbourn (263), some 30 years after the proposal of a leucine metabolite precursor responsible for cholesterol synthesis, that the pathway of ß-HMB formation was identified. HMB itself can be catabolized by two different pathways, either via a direct conversion or by a carboxylation, both of which result in the formation of beta-hydroxy-beta- methylglutaryl-coenzyme A (HMG-CoA). Aside from condensation of acetyl-CoA or acetoacetyl-

CoA to form cholesterol, ß-HMB catabolism to HMG-CoA is the only other way to form cholesterol in the body (6, 21, 32). Ultimately the majority of leucine is catabolized to isovaleryl-

CoA which continues to be metabolized until it forms acetoacetate and acetyl-coenzyme A (CoA)

(4). In fact, only 5-10% of all leucine is catabolized to ß-HMB in the liver cytosol (222, 325). For

9 example, a 70kg individual will produce approximately 0.2 – 0.4g of ß-HMB/day, the range being influenced by the amount of daily dietary protein intake. Furthermore, approximately 10-40% of all ß-HMB produced would be excreted via urine (222). Nissen and Abumrad observed differences in ß-HMB half-life (unpublished) in the urine of rats, pigs, and sheep which differed from 1, 2, and

3 hours respectively (222). Therefore, Nissen and Abumrad suggested that the difference in clearance could be attributable to the quantity of skeletal muscle each animal possessed.

Safety of ß-HMB

In an unpublished toxicity study three pigs weighing ~20kg were fed calcium salt-HMB

(Ca- ß-HMB) at 100g/day over 4 day and 2 control pigs given an un-supplemented diet. After the four days the animals were killed and blood and tissues were collected and analyzed. The investigators examined blood chemistry, hematology, gross organ pathology, and histology which revealed that when doses of ß-HMB ~100 times greater than typically provided to humans is consumed, no differences in any parameter existed, suggesting that at least acute supplementation does not elicit any adverse effects (222).

Several studies in humans have also concluded that ß-HMB supplementation does not possess any adverse events. For instance in one study when ß-HMB was provide for 8 days in doses of either 0.5g, 1.0g, 2.0g, or 4.0g (evenly divided in 250 mg capsules) provided throughout the day, there were no changes in liver enzymes or kidney function (222). Another study using varying doses of ß-HMB in 41 male volunteers while performing a RE training program revealed a dose response percentage decrease in proteolysis. In this study subjects were given either 0g, 1.5g, or

3.0g of ß-HMB and their urine content was measured for 3-methyl-histidine, a marker of myofibrillar protein degradation, with the decreases in proteolysis percentage reported as 6.0%,

5.5%, and 4.5%, respectively. Moreover, these same subjects had decreases in creatine kinase (666,

388, and 304 µ/mL, respectively) and lactate dehydrogenase (187, 171, 169 µ/mL, respectively)

10 indicative of attenuated muscle damage (222). To determine the long term effects of ß-HMB supplementation, Baier et al. (22) provided 2 – 3 g/day for 1 year to elderly subjects. They found no changes in blood and urine for markers of renal or hepatic dysfunction. It has been concluded that within human studies ß-HMB supplementation elicits increased lean body mass, decreased fat content, and increases in strength (222), with no adverse effects whatsoever (326).

INSULIN SIGNALING

Insulin is a peptide hormone, thus requiring a receptor. The receptor contains two sets of subunits bound to the PM, an alpha located externally and a beta located internally. Upon insulin binding to the insulin receptor alpha subunit, a conformational change which activates the homologous tyrosine kinase receptors within the beta subunits will occur. Following activation, insulin receptor substrate (IRS) proteins will be recruited to the PM via pleckstrin homology and phosphotyrosine binding domains within their amino terminus (313). Upon recruitment the IRS proteins 1 and 2 (found in skeletal muscle) are phosphorylated on tyrosine residues which form binding sites for molecules containing Src-homology 2 (SH2) domains (289). While both IRS 1 and 2 proteins may be tyrosine phosphorylated they can also be serine phosphorylated which terminates the insulin signal transduction. Furthermore, IRS-1 knockout mice have demonstrated retarded growth, impaired insulin signaling especially within muscle while maintaining glucose tolerance

(13) compared to IRS-2 knockout mice which present with growth retardation in pancreatic beta islet cells and defective hepatic insulin signaling (331). The pivotal interaction between IRS-1 and IRS-2 within insulin signaling is the propagation of the signal to phosphoinositide 3´-kinase (PI3-K) and subsequently protein kinase B (Akt/PKB). PI3-K is comprised of a p85 regulatory subunit and a p110 catalytic subunit. There are typically more regulatory subunits compared to catalytic

11 subunits within PI3-kinase, this engenders competition between the heterodimer subunits. Upon IRS-1 interaction with the p85 regulatory subunit the p110 catalytic subunit will be activated and then disassociate from p85 and translocate to the PM. The translocation of the p110 subunit to the PM will propagate the insulin signal to proteins bound to the PM. The activation of PI3-K has been shown to be absolutely necessary for insulin-stimulated glucose uptake by investigations which have employed wortmannin, a fungus. The mechanism for wortmannin inhibition of PI3-K is due to a conformational change in PI3- K when wortmannin binds to lysine residue 833. Wojtaszewski et al. and others (170, 333) reported a dose response reduction in glucose uptake due to wortmannin attenuating PI3- K activation while insulin binding to its receptor and subsequent tyrosine phosphorylation of IRS-1/2 was maintained. These findings suggest that wortmannin interacts with PI3-K alone and that the activation of PI3-K mediates GLUT-4 translocation to the PM. PI3-K, specifically the p110 catalytic subunit will phosphorylate the inositol phospholipid (PI) (23, 27) bisphosphate (PIP2) to PI (3,4,5) triphosphate (PIP3). The phosphorylation of PIP3 is required for activation of pleckstrin homology domains within downstream proteins such as phosphoinositide dependent kinases 1 and 2 (PDK1, and

PDK-2). Once PDK1 and PDK2 are activated by PIP3 they can activate via serine/threonine kinases (235), protein kinase B (Akt/PKB), atypical protein kinase C (aPKC) and p70 ribosomal S6 kinase (S6K1) (39). PDK1 will activate Akt/PKB on threonine residue 308 whereas serine residue 473 will be activated by PDK-2, which has been identified by Sarbassov et al. and others (229, 268) as mTOR-RICTOR complex. Within skeletal muscle there are three isoforms of expressed Akt/PKB (1, 2, and 3), however only Akt2 has been shown to be involved with insulin stimulated glucose uptake. Additionally, aPKC in skeletal muscle is expressed with two isoforms, lambda (λ) and zeta (ζ), of which PDK-2 phosphorylates aPKC- ζ upon threonine residue 410 and aPKC- λ on threonine residue 403. 12 Observations have been made which have demonstrated the involvement of both Akt/PKB and aPKC λ/ζ within skeletal muscle insulin stimulated glucose transport (39).

Akt/PKB activation, targets, and downstream effects

Akt/PKB is involved in insulin action through several mechanisms, for example, through the GTPase activation of Akt substrate of 160 kDa (AS160), also known as TBC1D4 (9, 265, 267, 296). Upon AS160activation, a Rab protein in a GDP-bound state will convert to a GTP-bound state thereby releasing GLUT-4, which will then translocate to the PM (317). Akt/PKB is also involved in the activation of several targets aside from those responsible for glucose uptake. Akt/PKB will also phosphorylate tuberous sclerosis complex protein 2 (TSC-2), subsequently altering and deactivating the TSC-2 and TSC-1 complex that is responsible for inhibiting mTORC1. Akt/PKB can also directly phosphorylate proline rich Akt substrate 40 kDa (PRAS40) which deactivates PRAS40 thereby alleviating its inhibition upon mTORC1. Upon its activation, mTORC1 (mTOR), will inhibit 4E-BP1 via direct phosphorylation, thereby activating S6K1. This process will then permit protein synthesis to occur via the subsequent gene alterations (83). Furthermore, Akt/PKB will affect proteolytic processes via phosphorylating forkhead box O (FOXO), resulting in FOXO being excluded from the nucleus thereby preventing transcriptional activity (304). Finally, Akt/PKB can phosphorylate glycogen synthases kinase-3 (GSK-3) which deactivates it and permits glycogen synthase activity to increase which results in hepatic glycogen stores to increase (176).

13 MUSCLE PROTEIN SYNTHESIS SIGNALING PATHWAYS

Muscle protein turnover regulation is a complex process involving interactions from gene transcription, mRNA translation, and protein degradation. The three components of translation are, translation initiation, elongation, and termination. A rate limiting step in muscle protein synthesis has been found to exist at during the initiation step (18, 99). It is universally understood that that mammalian target of rapamycin (mTOR) complex 1 is a major regulator on muscle protein synthesis with protein accretion occurring when there is prolonged positive nitrogen balance (273). mTOR consists of two separate complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). Of the two complexes, mTORC1 is sensitive to as well as responds to the presence of AAs, muscle contraction, and anabolic hormone (113) signaling. The chronic effect of continued activation of the mTORC1-dependent signaling pathway will result in skeletal muscle fiber hypertrophy (118). The constituent components of mTORC1 aside from mTOR are the G protein β- subunit-like protein (GβL), and Raptor which is the “regulatory associated protein of mTOR” (73, 76, 131). Activating mTORC1 is a multi-step sequence requiring mTOR alterations via formation, localization, and phosphorylation, of its subunits. Furthermore, mTOR itself possesses several Ser/Thr kinase domains, such as HEAT, FKBP12-

Rapamycin Binding (FRB) domain, and its regulatory domain (RD), (131, 344). Its subunit, GβL is responsible for increasing mTORC1 kinase activity and the stabilization via mTOR- raptor association (131). Finally, the Raptor subunit, is fundamental for mTOR binding to substrates and kinase phosphorylation. Localized subcellular components such as homologously enriched in brain (Rheb), Proline-rich Akt substrate 40kDa (PRAS40), and phosphatidic acid (PA) (76, 158) have demonstrated interactions with mTOR which can further promote or inhibit mTORC1 14 activation. Both Rheb-GTP and PA have shown the capability to activate mTOR kinase domain directly (142, 273). Rheb-GTP achieves activation via stimulation of mTORC1 phosphorylation at Ser2448 site, whereas PA binds the FRB domain (143, 347). Conversely, PRAS40 inhibits mTOR activity by directly impeding the interaction of the mTOR kinase domain and its downstream substrates. Finally, the importance of mTORC1 activation as a regulator of mRNA translation initiation and elongation processes is evident in both rodent and human skeletal muscle (33, 316). Activation of mTOR phosphorylation at Ser2448, creates a cascading effect through the phosphorylation of two downstream proteins, 70 kDa ribosomal protein S6 kinase (p70S6k) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) (95, 96). p70S6k will then activate ribosomal protein S6 (rpS6), a component of eukaryotic 40S ribosomal subunit (162). Once activation of rpS6 occurs, this event results in translation of specific mRNA to increase protein synthesis of ribosomal proteins (eIF4G), elongation factors (eEF1/2), and poly A binding proteins (259). In contrast, 4E-BP1 regulates the binding of mRNA to the 40S ribosomal subunit (97). 4E-BP1 will compete with eukaryotic initiation factor 4G (eIF4G) resulting in the disassociation of the eIF4G and eukaryotic initiation factor 4E (eIF-4E) complex. The 4E-BP1-eIF-4E formation is capable of interaction with mRNA but cannot interact with the 43S preinitiation complex. Yet, when 4E-BP1 is hyperphosphorylated at the Thr37/46 site, it dissociates from eIF-4E thereby permitting eIF-4E to bind with eIF4G and eIF4A and form a eIF4F complex (234, 316). The eIF4F complex is a bridge linking mRNA to the 43S preinitiation complex increasing rates of mRNA translation. Thus, it is evident that both downstream pathways of mTORC1 activation result in increased protein synthesis.

15 Upstream of mTOR is a key regulator known as Akt/PKB. Inoki et al (153) demonstrated that Akt phosphorylation at the Ser473 site inhibited the activity of both PRAS40 and tuberous sclerosis complex 1/2 (TSC1/2). TSC1/2 activates the GTPase protein (GAP) converting Rheb-GTP into Rheb-GDP whereby mTOR signaling is blunted or even blocked. Yet, when Akt is phosphorylated, it in turn phosphorylates TSC2 separating it from TSC1, thus inhibiting GAP activity. Consequently, Rheb-GTP can interact with mTORC1. Therefore, the phosphorylation of Akt appears to be a critical component to the activation of mTORC1 and the chronic effect of protein accretion. The phosphorylation and activation of Akt, critical for mTOR activation, can occur via two separate mechanisms. The anabolic hormones insulin and insulin-like growth factor (IGF-1) can interact with the homologous plasma membrane bound receptor which initiates a cascading activation sequence (40). Once initiated, activation of phosphatidylinositol 3–kinase (PI3k) will lead to the phosphorylation and activation of Akt (194, 195). An balance in the amount of AMP-to-ATP ratio is constantly monitoring intracellularly by the AMP-activated protein kinase (AMPK). During resistance exercise, there is a shift in the amount of the AMP-to-ATP ratio resulting in the phosphorylation of AMPK at its Thr172 site (74). AMPK activation has been shown to reduce muscle protein synthesis by directly inhibiting mTOR and decreasing 4E-BP1 phosphorylation (36, 75). An alternative method of initiating muscle protein synthesis, is by the eukaryotic initiation factor 2– (eIF2) forming a tertiary complex with GTP. The complex formed will enable met-tRNA to bind with the 40S ribosomal subunit (rp40S). This process is another mediated initiation pathway that utilizes the process of eIF2 binding to GTP, the subsequent hydrolysis to GDP causing the release of GDP from the newly formed eIF2- GDP complex which enables eIF2-to bind another GTP thereby permitting an additional cycle of initiation (37). This process itself is mediated by eIF2B whose activity is regulated 16 by glycogen synthase kinase 3 (GSK3). When GSK3 phosphorylates serine reside 535 on eIF2Bε the activity of eIF2B is reduced (163). Interestingly, both the phosphorylation of Akt or p70S6k are able to inhibit GSK-3ɑ/β by directly phosphorylating Ser21/9 site, leading to an activation of eIF2B (163, 178).

How resistance exercise affects protein turnover

Chesley et al. (56), investigated both the magnitude and time course of muscle protein synthesis in human biceps brachii muscles at 4 hrs and 24 hrs following a single bout of heavy resistance exercise. They found that while muscle protein synthesis rates were elevated in the exercising group, the enhanced rate consisted of posttranscriptional rather than regulation. Furthermore, RNA capacity was no different between groups but RNA activity was significantly elevated in the exercising group compared to none exercising muscle. The researchers defined total RNA concentration as RNA capacity whereas RNA activity was defined as muscle protein synthesis rate per unit time per unit RNA. Interestingly, a limitation to the findings of Chesley et al. is that the assessment of muscle protein synthesis could not occur prior to 4 hours, as this was the minimum time required to reach the a[13C]-ketoisocaproic acid isotopic plateau. Thus, the possibility of elevated muscle protein synthesis rates prior to the 4 hr post resistance exercise time point Chesley et al. analyzed is probable, but yet to be performed in humans. When examining for a potential effect resistance exercise would have on signaling proteins, Bolster et al. (38) conducted a study examining the changes in signaling proteins Akt/PKB, 4E-BP1, and S6K1 over 1h post resistance exercise. The researchers identified peak concentrations of these proteins at 10 minutes post RE with rpS6 peaking at 15 minutes post RE. Furthermore, eIF4E association with eIF4G increased by 10 minutes post

17 exercise as well. This rapid response suggests potent sensitivity by muscle to exercise stimuli. Surprisingly, Bolster et al. observed no changes in eIF2Be activity or eIF2a phosphorylation within 1 hr post resistance exercise. However, in a separate study comparing 9 men in the fasted or fed state with or without performing unilateral leg resistance exercise, Glover et al. (115) found that 6 hours post resistance exercise eIF2Bε was dephosphorylated and that the de-phosphorylation eIF2Bε potentiated the response of S6K1 and rpS6 in the fed state with RE compared to either resistance exercise alone or fasted state with resistance exercise. Within animal studies this same effect was observed 16 hours post resistance exercise and was concluded to be the cause of the increased muscle protein synthesis (92) and not the disassociation of eIF4E from 4E-BP1(93). Confirmation of mTOR involvement within these findings was determined when rapamycin inhibited the post RE eIF2Bε de-phosphorylation. It is known that the organic anti-fungal bacterial product, rapamycin can inhibit mTOR activity through integration with FKBP12. This inhibition is due to the binding of the FRB domain on mTOR by the rapamycin-FKBP12 complex. As a consequence, attenuation in muscle protein synthesis when rapamycin has been given allows for the conclusion that mTOR in involved mechanistically (147, 190, 299). Specifically, Kubica et al. (190) reports resistance exercise induced elevated muscle protein synthesis rates are abolished when rapamycin was administered. Thus, mTORC1 activation is necessary for muscle protein synthesis to occur. In conjunction with the activation of the mTOR signaling pathway, Goldspink hypothesized IGF-1 release, which occurs via an autocrine/endocrine, would further activate both mTORC1 and p70S6K using the Akt signaling pathway. However, Hornberger et al. (146) observed that even when IGF-1 was pharmacologically blocked the exercise induced activation of p70S6K persisted. Collectively, these observations suggest IGF-1 likely contributes to the activation of mTOR but is not required for mTOR 18 activation. As briefly mentioned previously, PA possesses the ability to directly interact with mTORC1. O’Neil et al. (227) tested the hypothesis that resistance exercise activates mTOR signaling via a PI-3K/Akt pathway. They reported three key findings, 1) when the PI-3K/Akt pathway was blocked, it did not stop mTOR signaling and 2) eccentric contractions caused phospholipase D (PLD) to synthesize PA, and 3) if PA is blocked mTOR signaling activation is prevented. To better understand how PA affects mTOR, the Hornberger laboratory using in vitro and in vivo study designs investigated the PA / PLD relationship, including its inhibition with 1-butanol. They discovered that PLD was localized to the sarcomere z-line, an ideal location, since this is the main site for contractile force transmission (143). As an enzyme, PLD, synthesizes PA from phosphotidylcholine, and due to its location, PLD can immediately respond to sarcomere activity by activating mTOR. Furthermore, mechanical loading of sarcomeres can increase both PA accumulation and subsequent mTOR signaling (143). As stated above, the blocking of PLD/PA can occur with 1-butanol and as Hornberger et al. (143) demonstrated the activation of mTOR via exercise is abolished when PA formation is blocked. Collectively, resistance exercise induces muscle protein synthesis through mTORC1 activation and yet a disassociation exists between protein phosphorylation and muscle protein synthesis rates (116, 119, 216). Exercise is a catabolic process, it will continually break down existing energy stores and re-synthesize the needed substrates so long as the exercise persists or until substrate stores are exhausted. As stated above, AMPK is sensitive the concentration of ATP within the cell and it is responsible for both inhibiting protein synthesis an anabolic process (requiring additional ATP) while promoting the generation of ATP formation via catabolic processes. Inoki et al. (154) has shown that AMPK affects mTORC1 activation via its interaction with TSC2 during and immediately post resistance exercise. The observation 19 made by Inoki et a. (154) is reasonable since logically the activation of AMPK is due to a fall in stored cellular ATP levels and the return to homeostasis supersedes the need to induce skeletal muscle hypertrophy. Interestingly, Cheng et al. (54) demonstrated that AMPK possesses the ability to direct phosphorylate mTOR on Thr2446. They found phosphorylation on Thr2446 site of mTOR will decrease the ability of insulin, specifically Akt, to phosphorylate mTOR at the Ser2448 site, which subsequently leads to decreased phosphorylation of p70S6K. Conversely, Cheng et al. (54) also demonstrated the ability of AMPK to phosphorylate mTOR on Thr2446 is reduced when Ser2448 is already phosphorylated. Hence, Cheng et al. (54) demonstrated that there exists a switch like mechanism on mTOR which can be affected by the ATP concentration of the cell. Dreyer et al. (75) and Koopman et al. (180) both concluded that the effects of AMPK activation during and immediately post exercise was likely contributing to the lack of muscle protein synthesis they observed. However, their observations also presented a time course for AMPK inhibitory actions. The time course for AMPK inhibiting muscle protein synthesis was indirectly observed by Wilkinson et al. (324) who saw a return to AMPK baseline levels when food was given to subjects post exercise and by Rasmussen et al. (248) who observed a significant elevated in AMPK levels for 10 mins post exercise. The resultant negative nitrogen balance post exercise is also in part due to the presence of catabolic hormones. As Kraemer and Ratamess (184) previously discussed, 95% of glucocorticoid activity is comprised of either corticosterone found in rats or cortisol found in humans both of which are released from the adrenal cortex. Considering that Seene et al. (271) demonstrated elevated glucocorticoid levels during atrophic conditions and reduced muscle protein synthesis rates were observed by Shah et al. (274) and McGhee et al. (212) under energy deficit conditions with a concomitant rise in muscle protein breakdown post exercise seen by McGrath and Goldspink (213), the negative effects of 20 glucocorticoids is without question. Furthermore, both Zafeiridis et al. (349) and Tarpenning et al. (295) present clear evidence that high intensity resistance exercise results in elevated leptin and cortisol levels, respectively, and which both hormones remain elevated for a minimum of 30 mins post resistance exercise. Taken together these findings make perfect sense. During high intensity resistance exercise the demand for ATP is great. Therefore, it does not make logical sense to prioritize anabolic mechanisms which require additional ATP, a commodities the cell is using during the exercise itself. Instead, the cell prioritizes catabolic mechanisms to supply the needed ATP. However, the findings of the researchers also demonstrate that the catabolic activity within the cell is both transient and able to be reduced in the presence of nutrient intake which modulates mTOR via Ser 2448 site phosphorylation by Akt. Since muscle protein synthesis rates remain elevated for 24-48 hrs post resistance exercise, the goal should be to maximize the duration of a potential positive nitrogen balance state; since resistance exercise also increases muscle protein breakdown rates, the goal should be to mitigate the duration of elevated muscle protein breakdown. This circumstance can be positively affected by nutritional interventions, which would result in both a faster recovery and a greater protein accretion post resistance exercise by consuming a nutritional supplement which can affect mTOR signaling.

How b-HMB interacts with protein turnover

Examining the effect of b-HMB on cultured myotubes, Eley and colleagues (86) incubated their cultured myotubes with 50 µM HMB and found an increased rate of muscle protein synthesis in b-HMB cultures compared to control. Specifically, it was observed that HMB increased mTOR and 4EBP-1 phosphorylation within the cultured myotubes.

21 This effect was confirmed when rapamycin abolished the b-HMB effect (86). To test the hypothesis that b-HMB can affect hypertrophy in sedentary Wistar rats through the mTOR signaling pathway proteins, Pimentel et al. (241) gavaged Wistar rats with 320mg/kg of b- HMB over a 4 week period and found increased hypertrophy in the extensor digitorum longus (EDL) and soleus muscles, increased insulin levels, increased phosphorylation of S6K1, and increased liver insulin receptor expression compared to control. Therefore,

Pimentel et al. (241) concluded that b-HMB could increase muscle protein synthesis by directly interacting with mTOR and phosphorylation of S6K1. Wilkinson et al. (2018)

13 2 (323) directly infused 8 healthy young males 1,2 C2 leucine and H5 phenylalanine to assess muscle protein synthesis via tracer incorporation into myofibrils and muscle protein breakdown via arterio-venous (A-V) dilution by comparing its baseline rate to post provision of approximately 3 g of Ca-HMB. They found an elevated muscle protein synthesis rates with a reduced muscle protein breakdown rates in conjunction with increased phosphorylation of p70S6K1 and rpsS6 proteins. Wilkinson et al. thus concluded that Ca-HMB contained pro-anabolic properties through activation of mTORC1 (323). It is known that resistance exercise will also activate an autocrine/paracrine release of IGF-1 (25, 43, 61, 124, 217). This release was thought to promote mTOR activation and its downstream proteins through the PI3-K-Akt signaling pathway (117). However, when IGF-1 was inhibited, exercise induced S6K1 activation remained elevated, suggesting IGF- 1 capable of activating the mTOR pathway but it is not required for S6K1 activation.

Interestingly, b-HMB has been shown to increase hepatic IGF-1 synthesis. To test the hypothesis that chronic HMB supplementation would affect IGF-1 concentrations in various tissues as well as insulin and glucose serum concentrations, Gerlinger-Romero et al. administered 320 mg/kg-1 BW/day-1 daily for 4 weeks to Wistar rats and reported HMB consumption increased mRNA expression and serum levels of IGF-1 as well as 22 increased insulin concentrations (112). Incidentally, studies in transgenic mice which over express IGF-1 in skeletal muscle present with increased hypertrophy, increased strength, and reduced age-related atrophy (25, 98). Thus, the ability of b-HMB to increase IGF-1 may facilitate an increase in skeletal muscle accretion via muscle protein synthesis. Collectively, acute resistance exercise possesses the capabilities of activating multiple pathways which result in muscle protein synthesis. However, while the changes described above exist within the acute application of resistance exercise, some of the changes do not persist during chronic resistance training (72, 160). The lack of changes during chronic resistance exercise may stem for one central lacking quality of a chronic resistance exercise program, yet manifest as what appears to be a multifaceted issue. The over-arching quality which may fail to be adequately address is, sufficient stimuli of the skeletal tissue.

MUSCLE PROTEIN DEGRADATION SIGNALING PATHWAYS

Nitrogen balance, which results in protein accretion or degradation, is the result of both muscle protein synthesis and muscle protein breakdown activities summed together. If the result is positive, protein accretion occurs, if negative, protein breakdown will occur. Therefore, understanding proteolytic pathways and its activation is necessary. Proteolytic pathways in skeletal muscle are divided into one of three classifications: ubiquitin- proteasome system, lysosomal proteolysis, and/or the Ca2+-activated proteases (i.e. calpain) (159, 245). Degradation of cellular components is done by the membrane-bound lysosomes, which degrade certain ligands, receptors, channels, and transporters (28). Proteolysis via calpain occurs via sarcomere degradation, cleaving the cytoskeletal proteins titin, nebulin, and C-protein, while not degrading either myosin or action (246).

23 Conversely, caspase 3 has been shown to directly affect the actin-myosin cross-linking while also being capable of interacting with the calpain pathway (77). The primary pathway for skeletal muscle degradation, is the ubiquitin-proteasome pathway, as this pathway directly mediates myofibrillar protein degradation (159). Containing a proteasome complex (26S) that consists of a core subunit (20S) and two separate regulatory subunits (19S), this complex only degrades recognized ubiquinated proteins that possess specific ligases. The ligases required are ubiquitin: the ubiquitin- activating enzyme (E1), conjugation enzymes (E2s), and specialized ligases (E3s). Interestingly, there is a rate limiting step to the entire process that occurs at the point of E3 enzyme mediated ubiquitin ligation (35, 290). Regulation of E3 ligases occurs via transcription factors, such as, forkhead box O (FoxO) 3A found in the nucleus . FoxO3A has been shown to directly affect both muscle atrophy F-box (MAFbx/atrogin-1) and muscle ring-finger protein 1 (MuRF1) which are both enriched in skeletal, cardiac, and smooth muscle and have individually been shown to be elevated under atrophy conditions (34). Reduced expression of both MuRF1 and MAFbx/atrogin-1 occurs when their regulator FoxO3A is phosphorylated causing it to be trapped in the cytosol. In opposition to one another, Akt and AMPK have been shown to interact with FoxO3A. Specifically, Akt possesses the capability of phosphorylating FoxO3A (46) thereby inhibiting its activation while AMPK directly stimulates FoxO3A to translocate into the nucleus thus increasing the expression of MuRF1 and MAFbx/atrogin- 1 transcription (219). Tangential to AMPK affecting FoxO3A and the E3 ligase expression,

NF-kB expression is elevated during atrophy and is associated with increased expression of MuRF1 and MAFbx/atrogin-1 (48, 341). Interestingly, like FoxO3A, when NF-kB is in the cytosol, it cannot induce expression of either MuRF1 and MAFbx/atrogin-1.

24 How b-HMB interacts with the muscle protein degradation pathways

Whitehouse and Tisdale (321) demonstrated in murine myotubes that expression of the ubiquitin proteasome pathway was increased via proteolysis-inducing factor (PIF) and that it was associated with NFkB activation. PIF has been shown inhibiting protein synthesis via auto-phosphorylation of an RNA-dependent protein kinase (PKR) subsequently inducing phosphorylation eIF2. Furthermore, activation of this pathway has been observed to occur due to the presence of lipopolysaccharides (LPS), tumor necrosis factor-α (TNF-α), and angiotensin II (ANGII). To test the hypothesis that HMB would affect this pathway, Eley et al. (86, 87) and others (260) exposed murine myotubes to 50µM HMB and found that HMB was capable of attenuating the phosphorylation of PKR and eIF2 in the presence of PIF, LPS, TNF-α, and ANGII.

In both cultured muscles (86, 283) and in vivo models (137) the presence of b-HMB resulted in decreased protein degradation. Conflicting findings in muscle protein synthesis exist with some researchers reporting b-HMB increases protein synthesis (17, 86, 114, 179, 283) while others report no effect (137). Initial investigations of HMB performed by Nissen and colleagues (222) exposed isolated muscle strips of chickens and rats to various concentrations of HMB (as high as 1,000 µM); found that b-HMB had a more pronounced effect on the rate of proteolysis (reduction of 80%) compared to the rate of protein synthesis

(enhancement of 20%). It has been shown however that b-HMB is capable of increasing phosphorylation of mTOR and proteins downstream from mTOR, suggesting b-HMB can increase skeletal muscle protein translation via these mechanisms (17, 86). Furthermore, HMB has been shown in cultured myoblasts to increase IGF-1 expression compared to control (181). Proteolysis generally occurs via the ubiquitin-proteosome pathway which has been shown to be increase catabolism (270) resulting in increased protein degradation. b-HMB has been shown repeatedly to suppress both ubiquitin-proteosome expression 25 (283) and activity (137, 182, 283, 284). Moreover, during catabolic states, caspases are upregulated and are responsible triggering myonuclear apoptosis (171). Apoptosis, programmed cell death, will cause shrinkage of cells, and is highly dependent upon caspases (cysteine-dependent-aspartyl-specific proteases). Moreover, caspases are involved in the cellular differentiation and proliferation (192). The potential for TNF-α and ANGII to trigger cellular apoptosis can occur via activation of caspase 8, which is responsible for apoptosis initiation; subsequent to activation, caspase 8 will cleave caspase 3 an apoptotic effector (149, 228). The activation of caspases 8 and caspase 3 will activate PKR via the formation of reactive oxygen species (ROS). ROS activation would then activate nuclear factor κB (NF-κB) resulting in increased transcription of key genes of the ubiquitin pathway and degradation of myofibrillar proteins. Eley et al. (88) has shown that murine myotubes incubated with 50 µM of b-HMB possessed attenuated caspase 8 and caspase-3 activation, thus reducing the signaling pathway resulting in protein degradation. Whether in unloading skeletal muscle (126) or culture myotubes exposed to

TNF-α or ANG II (87), b-HMB has been shown to suppress the caspases activation. With age and states of cachexia, there is an impaired capacity for muscle regenesis (280). Using cultured myotubes, Kornasio et al. (181) induced myonuclear apoptosis by serum starving their samples then exposed cultures to varying HMB concentrations (25 - 100µg/mL), Kornasio et al. (181) demonstrated that HMB cultures possessed an increased DNA synthesis 2.5 greater compared to control. These same researchers also revealed an increased dose response of MyoD mRNA expression (an indicator for cell proliferation), myogenin and MEF2 (indicators of cell differentiation), which are activated in satellite cells, suggesting b-HMB has a direct action upon myoblast proliferation and differentiation. Moreover, Kornasio et al. (181) also showed cultures exposed to HMB had a near 2 fold increase in IGF-1 compared to control. 26 In a separate study conducted by Aversa et al. (17) using rodent myotubes treated with HMB a reduction in mRNA and protein levels of muscle atrophy-related ubiquitin ligases atrogin-1 and muscle RING-finger protein-1 (MuRF-1) was observed. Wilson et al. (327) suggests that the findings reported by Kornasio et al. (181) were due in actuality to increased Akt phosphorylation by b-HMB, thus suppressing FoxO3A translocation in turn decreasing atrogin-1 ability to forestall degradation of MyoD. Therefore, b-HMB attenuation of FoxO3A activity will directly reduce activation of proteolysis thereby permitting increases in protein accretion. Furthermore, b-HMB has also been shown to attenuate apoptosis of myoblasts in elderly persons and those suffering from cachexia, suggesting b-HMB as a capable treatment to prevent decreased satellite cell numbers (198, 310). Collectively, in the absence of b-HMB, myonuclear apoptosis remains elevated, a condition which also occurs in catabolic states such as post RE and during remote post prandial periods. Thus, b-HMB has the potential to inhibit myonuclear apoptosis likely via inhibition of caspase 8, reducing skeletal muscle loss as well as inhibiting FoxO3A activation permitting an environment for protein accretion stemming for more than one protein degradation pathway.

MUSCLE ADAPTATION TO RESISTANCE EXERCISE

Muscle response to acute resistance exercise

It is understood that chronic resistance training combined with adequate nutrition will result in protein accretion via elevated muscle protein synthesis and an overall positive nitrogen balance. Fluckey et al. (99) demonstrated the effects of resistance exercise on muscle protein synthesis. They examined both gastrocnemius and soleus muscles of rats that had performed 4 sessions of squat movements, and found that 16 hrs post training, 27 exercising rats possessed elevated rates of muscle protein synthesis compared to non- exercising animals (99). Separately investigating the time course of muscle protein synthesis, Hernandez et al. (135) found in the gastrocnemius of Sprague-Dawley rats, that muscle protein synthesis rates were not elevated until 12 hrs post exercise and remained elevated 24hrs post exercise. This time course has been observed in humans when Chesley et al. examined biceps muscles following high-intensity elbow flexion finding ~2 fold increase in muscle protein synthesis rates up to 24 hrs post exercise compared to control

(56). It was MacDougall et al. that showed muscle protein synthesis rates return to ± 14% of baseline values by 36 hrs post resistance exercise (204). In contrast to muscle protein synthesis rates, Phillips et al. presented evidence that the fractional synthesis rate of mixed muscle peaks as fast as 3hrs remains elevated up to 48hrs post resistance exercise resulting in a greater positive net protein balance (239). Methodological differences in measuring are likely the cause for the observed differences in muscle protein synthesis and fractional synthesis rates. Both Chesley et al. (56) and MacDougall et al. (204) constantly infused L-[13C]-leucine for 6 hrs to detect muscle protein synthesis compared to Phillips (239) who infused of [2H]-phenylalanine. Furthermore, both Chesley et al. and MacDougall et al. supplied food to maintain the rate of appearance compared to Phillips et al. which had their subject fast overnight before the infusion. Complicating these observations further are the findings of Tang et al. (293) , who detected reduced fractional synthesis rates in trained subjects compared to untrained subjects, suggesting that sensitivity to stimulus may be another variable to consider. At its most conservative estimates, muscle protein synthesis will remain elevated 24 hrs post resistance exercise, but the question remains which proteins are being increased. The composition of skeletal muscle is made of several protein types, such as myofibullar, sarcoplasmic, or mitochondrial. Thus, it is with reason to suspect that while one type of 28 protein increase in response to resistance exercise, it is just as possible that another decreases in response to the same stimuli. Skeletal muscle is comprised of ~70% of the myofibrillar proteins actin and myosin responsible for cross-link formations and subsequent force generation (237). Hence, it is reasonable to assume that more myofibrillar proteins found in skeletal muscle, which occurs as a consequence of hypertrophy, the greater the force generating potential of that muscle. Thus, observing changes in myosin and actin concentrations directly would provide information about the efficacy of resistance exercise. Cuthbertson et al. (66) has assessed in the post prandial state, 8 men that performed step-up exercise while carrying 25% of their body weight, and observed a fluctuation in muscle protein synthesis rates with peaks at 6 hr and 24 hrs post exercise. Furthermore, they observed coinciding increases in Akt and p70S6K protein phosphorylation. Therefore, it can be stated that a single bout of resistance exercise can not only directly increase muscle protein synthesis, but specifically the myofibrillar proteins actin and myosin. As previously stated, acute resistance exercise can induce elevated rates in both muscle protein synthesis and muscle protein breakdown (239). Furthermore, while synthesis rates increase, the net nitrogen balance post resistance exercise remains negative in the post absorptive state (31, 239). Determination of muscle protein breakdown is via estimated using the quantification of urine 3- methylhistidine (3-MH). The myofibrillar proteins actin and myosin both contain 3-MH and during proteolysis the protein sequence release the 3- MH, which cannot be re-incorporated once released, thus permitting an indirect marker of protein degradation (249, 250). While 3-MH is still used today as a method for determining protein degradation, its use is limited to estimates of protein breakdown, since 3-MH can be released from both skin and the intestine.

29 Muscle adaptations to chronic resistance exercise

The rapidity in shift from a state of catabolism to one of anabolism post-acute resistance exercise, it is the chronic effects which are sought by a resistance training program. Chronic resistance training has been shown to increase hypertrophy (5, 10, 63, 91, 151) which leads to increased strength (68, 350), resulting in a greater quality of life. Thus, the relative importance of muscle hypertrophy as a result of chronic resistance exercise is of considerable interest. In an effort to design a model to induce hypertrophy Hornberger and Farrar (145) modified a climbing ladder design using weights attached to Sprague- Dawley rat tails to resistance train animals. After completing 8-weeks of progressive resistance training, their rats had increased flexor hallucis longus mass by ~23% which represented at 0.3% per diem accretion of muscle mass. Furthermore, they observed a coinciding increase in FHL cross sectional area with an ~24% increase in myofibrillar proteins in exercising rats compared to non-exercising rats. It is known that muscle fiber types possess varying concentrations in enzymes, myosin heavy chain phenotypes, capillary density, and metabolic preferences. There exists two distinct forms of skeletal muscle fiber types with further subdivisions therein, type I – containing slow twitch motor units, type IIa – containing fast twitch fatigue resistant motor units, and type IIb/x – fast twitch fatigue-able motor units (rat/human, respectively).

Whereas the gastrocnemius and plantaris both contain type IIa fibers, Hornberger and Farrar (145) point out that the extensor digitorum longus and FHL both contain mainly type IIb/x fibers. Differences in adaptability, specifically hypertrophy, do exist between the fiber types. The investigators McCall et al. (211) and Staron et al. (285) both reported that type II fibers are more responsive to hypertrophic signaling compared to type I fibers following progressive resistance training programs. The mechanisms by which the observations McCall et al. (211) and Staron et al. (285) occurred were identified by Tannerstedt, Apró, 30 and Blomstrand (294). Tannerstedt et al. took muscle biopsies from six male subject vastus lateralis muscles either immediately post, at 1hr, and at 2 hrs post resistance exercise. The researchers observed an increase in p70S6K and a 3-4 fold increase in p38 MAPK phosphorylation up to 1 hr post exercise in both type I and type II fiber types, but a significantly greater amount of phosphorylation was observed in the type II fibers. Protein accretion and observable hypertrophy are the result of prolonged periods of positive nitrogen balance stemming from prolonged periods of elevated muscle protein synthesis and suppression of muscle protein breakdown (144). Skeletal muscle responsiveness and adaptation to a bout of resistance exercise necessitates an overloading of the muscle. Several investigators reported attenuated or a lack of response in muscle protein synthesis rates in trained legs compared to untrained legs following an acute bout of resistance exercise in either rat or human muscle (177, 238, 240). It stands to reason that if the trained limb experienced a weight insufficient to overload the muscle, an adaption response would likely not occur. In the study conducted by Phillips et al. (238), they found a reduced muscle protein synthesis rate in the trained compared to untrained subjects using the same absolute workload. Their finds suggest insufficient overload placed on the trained limb being the likely cause of reduced signaling in the trained leg. However, as Burd et al. (47) reports, exercise training, either as aerobic or resistance, may in fact alter both the duration and extent of muscle protein synthesis compared to untrained subjects. When accounting for the relative intensity of resistance exercise, Tang et al. (293) observed increases at 4 hrs and 28 hrs post resistance exercise of the fractional synthetic rates in both untrained and trained subjects. Moreover, when Tang et al. considered the data from Kim et al (177), who was part of the same research group, Tang et al. (293) reported a greater amplitude in the fractional synthesis rate with observable reductions at 16 and 28 hrs post exercise in their resistance trained subjects compared to control. They hypothesized that 31 their results were due to alterations in the responsive window and thus likely that the peak in muscle protein synthesis occurred, but were missed. Support for the hypothesis of Tang et al. was presented by Gasier et al. (111) who resistance trained Sprague-Dawley rats for 5 weeks and examined plantaris and soleus muscles. Gasier et al. when utilizing the typical technique of a flooding dose over 10-15 seconds of I-[2,3,4,5,6,-3H]phenylalanine they found no difference with acute measurements in muscle protein synthesis at 16 hrs post exercise and also found 4E-BP1 phosphorylated and bound to eIF4E. However, Gasier et al. also measured the cumulative, 36 hrs fractional rates of muscle protein synthesis post

2 resistance exercise, by using H2O and found that anabolic responses to resistance exercise occurred post exercise in the chronically trained animals, contrary to the radio-labeled phenylalanine technique. Thus, it appears that the responsive window to resistance exercise still exists in chronically resistance trained muscle, but that the observable window shifts and can likely be missed, which may explain observed elevated rates of protein degradation. As an indicator of muscle hypertrophy and phenotic changes, evidence exists to suggest p70S6K as a marker of adaptation. Early indication of p70S6K as a marker was its tight positive correlation with muscle mass following 6 weeks of resistance training, as reported by Baar and Esser (18). As previously described, mTOR activates p70S6K and it subsequently stimulates mRNA translation for muscle protein synthesis. Evidence of p70S6K importance for inducing hypertrophy was provided by Bodine et al. who supplied rapamycin (an mTOR suppressor) to rats and reported resistance training hypertrophy effects were blocked up to 95% in the plantaris (33). They also reported a concomitant increase in total Akt concentration and its phosphorylation when CSA was enlarged, demonstrating the importance of the Akt/mTOR signaling pathway for inducing hypertrophy. Furthermore, Léger et al. (197) also reported elevated Akt total 32 concentrations in hypertrophied muscles of rats which had completed an 8-week resistance training protocol. Therefore, the importance of the mTOR signaling pathway is paramount for inducing hypertrophy.

EXERCISE EFFECT ON GLUCOSE UPTAKE & GLUCOSE TOLERANCE

The demand of exercise, specifically resistance exercise places a large demand for energy in skeletal muscle. Therefore, it stands to reason that the development of an energy sensing mechanism would regulate unnecessary energy expenditure during times of high energy demand and permit energy expenditure during periods of low energy demand. As discussed previously, AMPK is an energy sensor within the cell. 5´-AMP-activated protein kinase (AMPK) is a heterotrimeric complex comprised by several isoforms, of which the α1/2 and γ3 are of particular interest. The α complexes have been speculated to be sensitive to glycogen inhibition as a previous investigation revealed equal inhibition by glycogen in rat liver (210) yet a difference in sensitivity was found in human skeletal muscle where the α2 complex appeared more sensitive to glycogen inhibition (286, 334). The γ3 complex has been shown to be selectively expressed in white muscle (24, 206, 348) involved in high intensity exercise, as well as being responsible for increasing glycogen synthesis (24). Moreover, the cystathionine-β-synthase (CBS) domain of the first γ3 complex has been shown to be responsible for the glycogen accumulation within white glycolytic skeletal muscle (11). While not a focus of recent investigations, the β submit contains a carbohydrate binding motif which permits AMPK to associate with glycogen particles (29, 148, 210, 243). Moreover, the degree of AMPK activation was shown to be greater in glycogen depleted muscles compared to glycogen loaded human skeletal muscles (256, 334).

33 Early investigations analyzing the relationship between ATP/AMP and ATP/ADP ratio reductions due to high cellular energy turnover (302) have permitted the conclusion that these reductions are responsible for AMPK activation. The activation of AMPK occurs when AMP and ADP bind to three of the four CBS domains (26, 57). When ADP/AMP bind to AMPK a conformational change in the heterotrimeric structure which will inhibit protein phosphatases from accessing the threonine 172 residue, which is the primary residue for phosphorylation activation of AMPK which is located on the α complex (343). AMPK activation as a direct consequence of exercise was first reported in rodents by Winder and Hardie (329) and later confirmed in humans (52, 102, 286, 336). Investigations in how AMPK activation during exercise occurs revealed exercise of a minimum 60%

VO2max is necessary for activating AMPK (53, 334-336), however AMPK activation does occur with exhaustive exercise of 30-40% VO2max (335). Interestingly when exercise of a moderate duration high intensity exercise occurs, the α2 complex is primarily activated (102, 286, 335). The ability to phosphorylate AMPK suggests regulation by upstream kinases or phosphatases. It has been recently identified that liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ) are likely candidates that may activate αAMPK. LBK1 activation requires MO25 and STRAD binding for activation (19, 41); LBK1 complexes in vitro have been shown to directly phosphorylate αAMPK (130, 140). Similar to LBK1, CaMKKβ is expressed in skeletal muscle and has been shown to be both α1 and α2 AMPK kinases (164, 257, 330). Furthermore, when CaMKKβ expression levels were varied there was a similar covariation in AMPK activation within cultured mammalian cells (340).

34 AMPK involvement in glucose homeostasis within skeletal muscle

Early investigations into whether or not AMPK is involved in glucose transport utilized the chemical 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) an adenosine analogue. Specifically, AICAR is phosphorylated to form ZMP, an AMP mimetic that generally does not alter either AMP or ATP intracellular levels (127). While it has been shown using in vitro models (132, 214) and skeletal muscles of conscious rats (30), increased glucose transport has had mixed observations in humans. However, it has been revealed that AMPK activation via contraction/exercise is additive to insulin stimulated skeletal muscle GLUT4 translocation to the PM (132), thus permitting insight into likely AMPK targets and the existence of separate pools of GLUT4 within skeletal muscle cells. It has subsequently been learned that AMPK inhibits TBC1D4 (AS160) action, which when not affect by AMPK would inhibit GLUT4 translocation (51, 172). The ability to aid in GLUT4 translocation is important for maintaining intracellular energy levels, especially during exercise/contraction mediated stimulation. AMPK has been shown to be increased following exercise as well as increasing insulin sensitivity long after the acute post exercise effects have disappeared (165, 332). Moreover, chronic exercise has been shown to increase insulin sensitivity as well as increased basal AMPK concentration and activity in humans (101, 196, 312). Moreover, it has been observed that increased basal AMPK concentrations were observed with increased basal AS160 phosphorylation in both humans and rats (44, 106, 300, 312). Another mechanism by which AMPK can control ATP consumption is by regulating protein synthesis. By phosphorylating eukaryotic elongation factor 2α (eEF2), AMPK can inhibit the elongation phase of protein synthesis (141). However, recently in unpublished data from Ivy’s laboratory, AMPKα phosphorylation and mTOR signaling proteins were elevated post exercise in animals provided a mixture of HMB, Leucine, and whey protein following 35 endurance exercise. This finding suggests that AMPKα phosphorylation can occur from protein enriched supplementation independent of exercise. In agreement with the recent investigation by Nakai et al. (218) which reported that C2C12 myoblast stretched presented with elevated AMPK phosphorylation, as well as mTOR signaling, protein pathways believed to be antagonistic towards one another. Finally, AMPK has been shown to inhibit glucose storage utilization (glycogen utilization) by phosphorylating glycogen synthase (GS) (49). By inhibiting GS it would be reasonable to conclude that AMPK actually inhibits glycogen synthesis. However, recently it was reported that AMPK increases glycogen storage by increasing a “net-effect” with chronic activation whereby an accumulation of glucose-6-phosphate, by mass action kinetics and an allosteric mechanism, overrides the inhibitory phosphorylation on GS (152).

NEGATIVE REGULATION OF INSULIN SIGNALING

As previously mentioned, blood glucose levels are tightly regulated, with dysregulation resulting in observed insulin resistance and the development of diseases such as diabetes. To prevent uncontrolled activity of glucose metabolism, there exist a number of sites where termination of the insulin signal may occur. Moreover, the intensity and duration of the insulin signal factors into how far the signal is transferred downstream and which mechanism potentially feedback to suppress further signaling activation. It is known that tyrosine phosphorylation is pivotal for IR and IRS-1 and IRS-2 to propagate the insulin signal throughout the insulin signaling cascade. Therefore, when serine/threonine phosphorylation occurs within the receptors there is an attenuation of the insulin signal. The increase in serine/threonine phosphorylation can occur in response to several factors

36 including, hyperglycemia and insulin itself activating several downstream kinases such as mTOR and S6K1 (39). Insulin resistance has been observed in rats and humans that have increased serine phosphorylation with decreased tyrosine phosphorylation in the IR and IRS proteins (81, 173, 277, 351). Historically, phosphorylation of IRS-1 on serine residue 307 has been shown to be elevated in obese and diabetic mice (136, 305, 306). aPKC-ζ has also been shown to cause serine phosphorylation of IRS-1 (251) as well as threonine phosphorylation of Akt, inhibiting Akt PM recruitment (244). Finally, when the downstream protein mTOR is activated it will in turn activate S6K1 and both of these proteins have been shown to have negative feedback upon IRS tyrosine phosphorylation by increasing serine phosphorylation. Evidence for this comes from the use of rapamycin which has shown that there are several rapamycin-sensitive phosphorylation residues upon IRS-1 which mTOR and S6K1 can directly phosphorylate (129, 207, 275, 301, 303). Furthermore, within TSC deficient cells, IRS-1 and IRS-2 have been shown to be unresponsive to insulin without prolonged rapamycin exposure (128, 275, 276). Following mTOR elevation S6K1 will phosphorylate mTORC2 component RICTOR which has resulted in attenuated Akt/PKB activation (71, 169). Collectively, these findings suggests that on a cellular level the body will mitigate the amount of glucose which will enter into the cell and consequently maintain elevated extracellular glucose levels in the event that there is overstimulation of the insulin signaling cascade.

b-HMB interactions with blood glucose homeostasis

Recently, the work of Yonamine et al. (346) and Nunes et al. (226) appears to show that ß-

HMB can elicit impairments to peripheral insulin sensitivity and glucose tolerance, but their

37 experimental designs do not control for confounding variables which may account for their findings. To test the hypothesis that chronic ingestion of ß-HMB (320 mg/kg-1 of BW day-1 for 4 weeks) will adversely affect glycaemic homeostasis and peripheral insulin sensitivity in healthy sedentary Wistar rats, Yonamine et al. conducted a glucose tolerance test (GTT) and found that ß-

HMB treatment resulted in impaired insulin sensitivity in liver and soleus tissues after an insulin overload with reduced total GLUT4 and PM GLUT4 concentrations in the soleus. Therefore,

Yonamine et al. concluded that ß-HMB impairs peripheral insulin sensitivity. The Yonamine et al.

(346) finding that ß-HMB impairs peripheral insulin sensitivity rests upon the questionable assumption that the anesthetic thiopental that they used during their GTT did not affect blood glucose levels or insulin sensitivity. A previous investigation that tested the hypothesis of thiopentone (also known as thiopental) can affect blood glucose levels, Dundee (82) used 8 subjects given a GTT with or without thiopentone anesthesia and found thiopentone significantly depressed the ability to cope with an extra load of glucose. Therefore, Dundee (82) concluded that the depressed glucose handling was a result of the hepatotoxic effect caused by a large dose of thiopentone. Furthermore, it is known that one of the most complicated and arduous components during anesthesia is the regulation of blood glucose (167, 288), with most anesthetics blocking insulin secretion from the beta cells (167). Therefore, the use of anesthesia during a GTT of any kind will only server to complicate the findings and provide controversial evidence for hyperglycemia stemming from ß-HMB supplementation.

The investigation by Nunes et al. (226) sought to test the hypothesis that ß-HMB ingestion

(320 mg/kg-1 of BW day-1 for 5 days) could be detrimental to glucose and lipid homeostasis when co-administered with glucocorticoids (dexamethasone) in Wistar rats, so they conducted both an intraperoteneal (ip) insulin tolerance test and a ipGTT, finding that ß-HMB did not attenuate effects of dexamethasone on food intake, BW loss and that ß-HMB rats presented within increased fasting glucose levels and an exacerbated glucose intolerance. Therefore Nunes et al. concluded that ß- 38 HMB did not mitigate the diabetogenic characteristics of glucocorticoid excess and that the possibility exists that ß-HMB can exacerbate the glucocorticoid effects. The results of Nunes et al. contradict the findings of Noh et al. (225) who found that ß-HMB had no influence on dexamethasone-induced serum glucose levels that were significantly increased compared to control. Furthermore, the methodology used during the ipITT and ipGTT disregards previous evidence which has shown that hepatic glucose output is moderately suppressed when either glucose or insulin bypass the portal vein (55, 67) and does not account for the variability of ip injection sites between rats nor does it account for the relative variability of the circulatory system within the tissue that receives the injection. Finally, the findings of Nunes et al. (226) call into question the application of anesthetics during GTTs, revealing the importance of considering methodologies that utilize a conscious rodent during GTT or ITT procedures that attempt to elucidate the effect a substance has on glucose homeostasis.

SUMMARY

It is without question that skeletal muscle protein accretion is a multi-faceted and complex system comprised of multiple avenues of redundant activation throughout the signaling pathway. Thus, the areas of overlap, have been observed to behave as regulatory control steps in the signaling pathways. Furthermore, resistance exercise is a potent stimulator of inducing skeletal muscle protein synthesis with chronic effects evident as increased skeletal muscle hypertrophy. It is also quite clear that proteins involved with muscle protein synthesis are both sensitive to the energy state of the cell but can also act as regulators of cellular energy and glucose homeostasis. Moreover, ß-HMB has the potential effect to directly interact with the mTOR signaling pathway and as such, through it, affect glucose homeostasis and skeletal muscle hypertrophy during a chronic resistance

39 training program. Thus, supplementation with ß-HMB may raise new insights into the acceleration of muscle recovery and/or muscle hypertrophy following resistance exercise.

40 ζ GTP - λ/ aPKC GLUT4 Glucose Glucose Transport GLUT4 Rab P 1 GDP - - 3 Rab TBC1D1 TBC1D4 et al. (2013); Boucher et al. (2014) (2014) al. et Boucher (2013); al. et PDK PIP Aoi P P (2007); Akt 1 & AMPK signaling pathways signaling AMPK & 1 - P Cantley GSK3 Glycogen synthesis P 1 - P 2 Amino Acids PDK PRAS40 Insulin, IGF mTORC2 PIP

p110 K 1 - - Adapted from Manning and Manning from Adapted P S6K1 p85 p55 PI3 TSC P P 2 - mTORC1 BP1 - P P 1 2 1R - - - TSC Protein Synthesis Protein 4E IRS IRS P P IR or IGF or IR 1 - 2+ 1 and AMPK signaling 1 pathways - Ca Insulin orIGF Insulin CaMKK P AMPK . Insulin, .Insulin, IGF LKB1 ADP:ATP 2.1 Low energy Low Muscle Contraction Muscle Figure

41 ζ GTP - λ/ aPKC GLUT4 GLUT4 Glucose Glucose Rab Transport 1 signaling 1 - P 1 GDP - - 3 et al. (2013); Boucher et al. (2014) (2014) al. et Boucher (2013); al. et TBC1D1 TBC1D4 Rab PDK PIP Aoi P P et al. (2009); (2009); al. et Akt

mTORC1 P Vodenik (2007); 2 PIP Cantley P P signaling signaling pathways p110 K 1 - - p85 p55 PI3 BP1 Negative modulators of Insulin and IGF and Insulin of modulators Negative - S6K1 Grb14 Grb10 P P 4E and and IGF

1 2 1R - - - IRS IRS P P Adapted from Manning and Manning from Adapted IR or IGF or IR Protein Synthesis 1 - 2+ Ca CaMKK Insulin or IGF or Insulin P Negative modulators insulin of Negative modulators AMPK . . 2.2 LKB1 ADP:ATP Low energy Low Muscle Contraction Muscle Figure

42

ζ t GTP - λ/ aPKC Glucose Glucose GLUT4 Transpor GLUT4 Rab P et al. (2013); Boucher et al. (2014) (2014) al. et Boucher (2013); al. et 1 GDP - - Aoi 3 TBC1D1 TBC1D4 Rab PDK PIP P P et al. (2009); (2009); al. et Akt Vodenik mTORC1 P (2007); HMB - β Cantley 2 PIP P P p110 1 & MuF1) - K - p85 p55 PI3 BP1 - S6K1 Grb14 Grb10 3K signaling pathways pathway and degradation protein 3K signaling P P - 4E 1 2 1R - - - Adapted from Manning and Manning from Adapted IRS IRS P P Gene Atrogin ( Transcription IR or IGF or IR Protein Synthesis FOXO3A 1 - Insulin IGF HMB with HMB the Akt/PI - 2+ b Ca CaMKK Gluconeogensis P AMPK LKB1 Interactions of Interactions of . AMP/ATP Low energy Low Muscle Contraction Muscle 2.3

Figure 43 ζ GTP - λ/ aPKC Glucose Glucose Transport GLUT4 GLUT4 Rab 1 GDP - - 3 Rab TBC1D1 TBC1D4 et al. (2013); Boucher et al. (2014) (2014) al. et Boucher (2013); al. et PDK PIP Aoi P (2007); Akt P Cantley GSK3 Glycogen synthesis P

1 - P 2 Amino Acids PDK PRAS40 Resistance exercise subcellular effects subcellular exercise Resistance mTORC2 PIP p110 K 1 - - Adapted from Manning and Manning from Adapted P S6K1 p85 p55 PI3 TSC P P 2 - mTORC1 BP1 - P P 1 2 1R - - - TSC Protein Synthesis Protein 4E IRS IRS P P / IR or IGF or IR MAFbx atrogin1 P 1 - FOXO3A MuRF1 2+ Insulin or IGF or Insulin Ca CaMKK Proteolysis P AMPK ADP:ATP Resistance exercise effects on exercise subcellular signaling pathways Resistance Low energy Low Muscle Contraction Muscle . LKB1 2.4

Figure 44 CHAPTER III

THE ACUTE EFFECTS OF BETA-HYDROXY-BETA-METHYLBUTYRATE ON GLUCOSE HOMEOSTASIS , INSULIN SENSITIVITY, AND AKT/MTOR PATHWAY PROTEINS

ABSTRACT

The leucine metabolite, β-hydroxy, β-methylbutyrate (HMB) is a dietary supplement used by recreational athletes, with reported benefits of increased strength, lean body mass, and reduced fat mass. However, recent investigations have suggested that HMB adversely affects blood glucose. We aimed to test varying concentrations of HMB and their effects on plasma glucose concentration, insulin sensitivity, and activation of the Akt/mTOR signaling pathway proteins. Forty-eight Sprague-Dawley rats, ~2-3 months in age were randomly placed into one of 4 treatments, control 450 mg/kg (CON, n = 12), 150 mg/kg bodyweight (LOW, n = 12), 300 mg/kg bodyweight (NORM, n = 12), or 450 mg/kg bodyweight (HIGH, n = 12). HMB was gavaged at two times either 15mins or 150 mins prior an oral glucose tolerance test (OGTT) and 150 min prior to an insulin sensitivity test (ITT). Skeletal muscle harvesting of the quadriceps, gastrocnemius, plantaris, and soleus occurred following completion of the insulin sensitivity test. We observed no effect of HMB on plasma glucose concentrations when given at either 15 min or 150 min prior to an OGTT. However, we did observe the HIGH HMB treatment did have lower plasma insulin concentrations at 210, 240, 270 post HMB administration. We did observe a significant increase, in a signal proteins of the Akt/mTOR signaling pathway, in the HIGH treatment group compared to all other treatment groups and CON. We conclude that HMB ingestion does not adversely affect blood glucose handling but at elevated concentrations increases the Akt/mTOR signaling pathway and potentially muscle

45 protein synthesis rates. Therefore, HMB may be a supplement to further increase protein accretion when supplemented chronically.

INTRODUCTION

Increasing skeletal muscle mass and the amino acid leucine are closely intertwined. Their association has been conclusively demonstrated, with leucine being able to directly affect both the mechanistic target of rapamycin complex 1 (mTOR) as well as p70S6k (12, 156). These proteins are responsible for increasing the activation of protein synthesis. However, recently Pimentel et al. presented data that beta-hydroxy-beta-methylbutyrate

(b-HMB), a metabolite of leucine metabolism, was also capable of directly affecting both mTOR and p70S6k (241).

Initial interest in b-HMB stemmed from an early investigation by Van Koevering and Nissen (308) who established the importance of b-HMB formation as part of leucine metabolism. Since this study Nissen et al., demonstrated growth and muscle metabolism effects during resistance exercise in animals (221, 223, 224) and both Nissen and Gallagher et al., through a series of studies, investigated the effects of b-HMB supplementation on parameters such as hematology, hepatic and renal functioning, strength and fat-free mass when provided both acute and chronically (22, 109, 110, 222). b-HMB has become an exciting potential supplement for reducing muscle wasting during endurance exercise and prolonged caloric restriction (232), cancer cachexia (86, 283), and increasing strength and lean mass (202, 328).

While b-HMB investigations run the gamete from strength gains to cachexia prevention and are historically without adverse effects, there remains a gap in the literature concerning b-HMB supplementation effects on blood glucose. However, investigations by 46 Gerlinger-Romero et al. (112), Nunes et al. (226), and Yonamine et al. (346) have each presented b-HMB supplementation resulting in elevated serum insulin levels, exacerbated glucose intolerance when co-administered with dexamethasone, and peripheral insulin resistance, respectively. Yet, to date no investigation has specifically examined the direct effects b-HMB may have on blood glucose acutely. Therefore, the aim of the current investigation is the determination of the dose response of β-HMB on plasma glucose and plasma insulin during an oral glucose tolerance test and insulin sensitivity test. Furthermore, we will investigate if varying β-HMB doses result in altered activation in Akt pathway proteins.

METHODS

Animals: A total of 48 male SD rats were obtained at approximately 2 to 3 months of age from Charles River (Wilmington, MA) and were ~275 – 350g. Rats were housed 2 per cage and provided standard laboratory chow (Prolab RMH 1800 5LL2, LabDiet, Brentwood, MO) and water ad libitum. Animals singly housed were randomly placed into one of four treatments, isonitrogenous control 450 mg/kg (CON, n = 12), 150 mg/kg bodyweight (LOW, n = 12), 300 mg/kg bodyweight (NORM, n = 12), or 450 mg/kg bodyweight (HIGH, n = 12). The temperature of the animal room was maintained at 21° C with a non-reversed artificial 12 h dark-light cycle was set with the light phase from 7:00 am to 7:00 pm. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas at Austin and conform to the guidelines for the use of laboratory animals published by the United States Department of Health and Human Resources.

47 Experimental protocol: Two oral glucose tolerance tests (OGTTs) and one insulin tolerance test (ITT) were conducted in this experiment separated by two weeks to permit for a washing out period and ample time for tail healing. Animals performed OGTT 1 and 2 then the ITT in that order. Following an 8hr fast, baseline blood samples were taken and rats were gavaged either CON or one of the b-HMB treatments. During the OGTTs animals were gavaged either CON or one of the b-HMB treatments 15 minutes (OGTT 1) or 150 minutes (OGTT 2) prior to the glucose gavage. The glucose gavage was prepared at 1g/kg bodyweight). During the ITT animals were gavaged either CON or one of the b-HMB treatments 150 minutes prior to the intraperitoneal insulin injection. The insulin injection was prepared at 0.1U/kg bodyweight.

Blood samples were collected prior to b-HMB treatment, prior to glucose gavage or insulin injection, 30, 60, 90, and 120 minutes post gavage or injection using a OneTouch Ultra 2 glucometer (LifeScan Inc, Wayne, PA, USA). Following completion of the ITT the animals were anesthetized via an intraperitoneal injection of ketamine/xylazine (KX: 70 mg/kg, 7 mg/kg body weight, respectively). Once analgesia was confirmed the triceps surea and quadriceps were excised. Muscles were clamped and immediately placed in liquid nitrogen, upon completion of the daily cohort, tissues were placed into a -80 °C freezer for later analysis. Once all tissues were harvested animals were euthanized via a thoracotomy.

Blood Analysis: On the day of testing animal tails were pre-warmed to ~38° C via a heating pad. A total of 0.7 mL whole blood was collected at each time point. All blood samples were collected in micro-centrifuge tubes containing 0.1 ml of 24 mg/ml EDTA (pH 7.4) to prevent clotting. The whole blood was centrifuged at 14,000 x g resulting in ~250 – 300 µl of plasma. The plasma was then transferred to a clean micro-centrifuge tube

48 and stored immediately in a -80° C freezer for later analysis of plasma glucose and insulin concentrations.

Insulin: Plasma insulin concentrations (Millipore Corporation, Billerica, MA) were determined using a competitive (Goetz 1963) 125I radioimmunoassay in which the serum hormone competes with its 125I-labeled counterpart for a sub saturating concentration of antibody. Plasma concentration is related with the amount of 125I-labled hormone displaced from the antibody. Following an incubation step, antigen-antibody complexes were isolated by centrifugation and radioactivity counted in a Beckman 5500 gamma counter (Beckman Bioanalytical Systems Group, Fullerton, CA).

Akt/mTOR Pathway Protein Quantification: Plantaris muscle homogenates were prepared from each animal according to the manufacturer’s instructions. The concentration of Akt/mTOR pathway proteins (Akt, Akt-p, GSK3a, GSK3a-p, GSK3ß, GSK3ß-p, IGF1R, IGF1R-p, IR, IR-p, IRS1, IRS1-p, mTOR, mTOR-p, p70S6k, p70S6K-p, PTEN,

PTEN-p, RPS6, RPS6-p, TSC2, TSC2-p) was measured using MILLIPLEXÒ MAP Kits (11-Plex Akt/mTOR Total Protein Magnetic Bead Kit, Cat # 48-612MAG and 11-Plex Akt/mTOR Phosphoprotein Magnetic Bead Kit, Cat # 48-611MAG; Merck KGaA EMDMillipore, Billerica, MA 01821) following the manufacturer’s instructions.

Statistical approach: To account for potential differences in values, data obtained during the OGTT1, OGTT2, and ITT from Low, Norm, or High b-HMB treatments were analyzed as a % change relative to the CON treatment. All other data was analyzed as an absolute value. A two-way analysis of variance (ANOVA) (time x treat) was performed on a between-within mixed model design for the measurement in data obtained 49 from the OGTT1, OGTT2, and ITT glucose and insulin data. When the interactive effect (time x treat) is statistically significant, differences among means was determined using Fisher’s LSD post hoc analysis. A one-way ANOVA was used for analyzing areas under the curve (AUC) and Fisher’s LSD post hoc test was performed to compare mean differences among treatments. Differences with p < 0.05 were considered statistically significant. All statistical analyses was performed using IBM SPSS Statistics v24.0 software (IBM Corporation, Armonk), and all data was expressed as mean ± standard error of the mean (SEM).

RESULTS

Figure 3.1. The blood glucose concentrations (A) and plasma insulin concentrations (B) during an OGTT when b-HMB was provided 15 minutes prior to the glucose bolus. A

190 Glucose Con LD 170 Gavaged ND HD 150 130 110 90

Blood Glucose (mg/dL) 70 50 D0 D15 D45 D75 D105 D135 Sampling Time Points (min)

50 B 350.0

Con Low 300.0 Glucose Norm High Gavaged 250.0

200.0

150.0

Plasma Insulin (pM) 100.0

50.0

0.0 D0 D15 D45 D75 D105 D135 Sampling Time Points (min)

Note: CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed. Values are expressed as mean ± SD.

51 Figure 3.2. The blood glucose concentrations (A) and plasma insulin concentrations (B) during an OGTT when b-HMB was provided 150 minutes prior to the glucose bolus.

A Con LD 200 ND HD 180 HMB Gavaged 160 140 120 100 80 60

Blood Glucose (mg/dL) 40 20 Glucose Gavaged 0 D0 D150 D180 D210 D240 D270 Sampling Time Points (min) B 450.0 Con Low 400.0 Norm High HMB Gavaged *,† 350.0 †† *,† *,† 300.0 †† †† 250.0

200.0

150.0 Plasma Insulin (pM) 100.0

50.0 Glucose Gavaged 0.0 D0 D150 D180 D210 D240 D270 Sampling Time Point (min)

Note: CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in blood glucose. * High treatment significantly different vs CON, p ≤ 0.05, † High treatment

52 significantly different vs. Low p ≤ 0.05, †† High treatment significantly different vs. Norm, p ≤ 0.05.Values are expressed as mean ± SD.

Figure 3.3. The blood glucose concentrations (A) and plasma insulin concentrations (B) during an OGTT when b-HMB was provided 150 minutes prior to the glucose bolus.

A Con LD ND HD 160 HMB 140 Gavaged 120 100 80 60 40 20 Blood Glucose mg/dL 0 Insulin Injected D0 D150 D180 D210 D240 D270 Sampling Time Points (min)

B700.0 Con Low 600.0 Norm High 500.0 HMB Gavaged

400.0

300.0

200.0 Plasma Insulin (pM)

100.0 Insulin Injected 0.0 D0 D150 D180 D210 D240 D270 Sampling Time Points (min)

53 Note: CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed. Values are expressed as mean ± SD.

Figure 3.4 – Glycogen Synthase Kinase 3b (GSK3b) total protein (A) and total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

6000.0

5000.0

4000.0

3000.0 MFI

2000.0

1000.0

.0

Con Low Norm High

B *,† 1000.000 ††

750.000

500.000 MFI

250.000

.000

Con Low Norm High

54 Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total GSK3b protein. * High treatment significantly different vs CON, p ≤ 0.05, † High treatment significantly different vs. Low p ≤ 0.05, †† High treatment significantly different vs. Norm, p ≤ 0.05.Values are expressed as mean ± SEM.

Figure 3.5 – Insulin Like Growth Factor 1 (IGF1R) total protein (A) & total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

200.00 180.00 160.00 140.00 120.00 100.00 MFI 80.00 60.00 40.00 20.00 .00

Con Low Norm High

55 B

20.00

15.00

10.00 MFI

5.00

.00

Con Low Norm High

Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total IGF1R or total phosphorylated IGF1R protein.

56 Figure 3.6 – Insulin receptor subunit-1 total protein (A) and total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

20000.00

15000.00

10000.00 MFI

5000.00

.00

Con Low Norm High B *,† 40.00 ††

30.00

20.00 MFI

10.00

.00

Con Low Norm High

Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total IRS-1 protein. * High treatment significantly different vs CON, p ≤ 0.05, † High treatment significantly

57 different vs. Low p ≤ 0.05, †† High treatment significantly different vs. Norm, p ≤

0.05.Values are expressed as mean ± SEM.

Figure 3.7 – Akt total protein (A) and total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

500.00 450.00 400.00 350.00 300.00 250.00 MFI 200.00 150.00 100.00 50.00 .00

Con Low Norm High

B

100.00 *,†

75.00

50.00 MFI

25.00

.00

Con Low Norm High

Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and 58 High = 450 mg/kg BW b-HMB. No significant differences observed in total Akt protein. * High treatment significantly different vs CON, p ≤ 0.05, † High treatment significantly different vs. Low p ≤ 0.05. Values are expressed as mean ± SEM.

Figure 3.8 – Mammalian target of rapamycin (mTOR) total protein (A) and total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

400.00

350.00

300.00 250.00

200.00 MFI 150.00

100.00

50.00 .00

Con Low Norm High

B

250.00 *, †

200.00

150.00 MFI 100.00

50.00

.00

Con Low Norm High

59 Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total mTOR protein. * High treatment significantly different vs CON, p ≤ 0.05, † High treatment significantly different vs. Low p ≤ 0.05, Values are expressed as mean ± SEM.

Figure 3.9 – p70S6K total protein (A) and total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

3000.00 §, ‡ ‡ ‡

2000.00 MFI

1000.00

.00

Con Low Norm High

60 B

40.0000

30.0000

20.0000 MFI

10.0000

.0000

Con Low Norm High

Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total phosphorylated p70S6K protein. § Norm treatment significantly different vs CON, p ≤ 0.05, ‡ Norm treatment significantly different vs. Low p ≤ 0.05, ‡‡ Norm treatment significantly different vs. High, p ≤ 0.05.Values are expressed as mean ± SEM.

61 Figure 3.10 – Insulin receptor (IR) total protein (A) & total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

2000.00

1500.00

1000.00 MFI

500.00

.00

Con Low Norm High B

40.00

30.00

20.00 MFI

10.00

.00

Con Low Norm High

Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total IR or total phosphorylated IR protein.

62 Figure 3.11 – Phosphatase and Tensin Homolog (PTEN) total protein (A) and total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

1500.00

1000.00 MFI

500.00

.00

Con Low Norm High

B

1500.00 *

1000.00 MFI

500.00

.00

Con Low Norm High

Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total PTEN protein. * High treatment significantly different vs CON, p ≤ 0.05. Values are expressed as mean ± SEM. 63

Figure 3.12 – Glycogen Synthase Kinase 3 a (GSK3a) total protein (A) & total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

200.00

150.00

100.00 MFI

50.00

.00

Con Low Norm High

B

25.00

20.00

15.00 MFI 10.00

5.00

.00

Con Low Norm High

Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and

64 High = 450 mg/kg BW b-HMB. No significant differences observed in total GSK3a or total phosphorylated GSK3a protein.

Figure 3.13 – Tuberous sclerosis complex 2 (TSC2) total protein (A) and total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

2000.00

1500.00

1000.00 MFI

500.00

.00

Con Low Norm High B *,† 300.0000 ††

250.0000

200.0000

150.0000 MFI

100.0000

50.0000

.0000

Con Low Norm High

65 Note: Sampling derived from muscle collected 270 Min after b-HMB was gavaged. CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total TSC2 protein. * High treatment significantly different vs CON, p ≤ 0.05, † High treatment significantly different vs. Low p ≤ 0.05, †† High treatment significantly different vs. Norm, p ≤

0.05.Values are expressed as mean ± SEM.

Figure 3.14 – Ribosomal protein S6 (rpS6) total protein (A) & total phosphorylated protein (B) quantity as determined by median fluorescence intensity. A

125.00

100.00

75.00 MFI 50.00

25.00

.00

Con Low Norm High

66 B

20.000

15.000

10.000 MFI

5.000

.000

Con Low Norm High

Sampling derived from muscle collected 270 Min after b-HMB was gavaged. Note: CON = control rats, Low = 150 mg/kg BW b-HMB, Norm = 300 mg/kg BW b-HMB, and High = 450 mg/kg BW b-HMB. No significant differences observed in total rpS6 or total phosphorylated rps6 protein.

Blood glucose: Average blood glucose was not significantly different at any time point (p

> 0.05) for any treatment dose of b-HMB was provided 15 minutes prior to 150 minutes prior to an OGTT. No statistical difference was observed at any time point during the ITT when varying doses of b-HMB were provided 150 minutes prior to insulin administration.

Plasma insulin: No statistical difference was observed at any time point for plasma insulin concentrations when b-HMB was provided 15 minutes prior to an OGTT. When b-HMB was provided 150 minutes prior to an OGTT there were three time points

D210, D240, and, D270 where the b-HMB high dose treatment were significantly lower in plasma insulin concentrations compared to control animals, low dose, and norm dose treatments. The differences in insulin measurements between the results of when b-HMB

67 was given 150 minutes prior to a OGTT and the similar administration of b-HMB during the ITT is explained by the ITT having insulin injected whereas the OGTT did not.

Akt/mTOR signaling pathway - total and phosphorylation total protein concentrations

GSK3b: Average total protein concentrations (Figure 4A) were not significantly different between (p 0.05) for low dose (124.47 ± 5.17, MFI), norm dose (115.16 ± 2.54 MFI), or high dose (118.16 ± 2.84 MFI) compared to control (121.78 ± 5.50). The total phosphorylated protein concentrations (Figure 4B) in the high dose (711.910 ± 66.215 MFI) b-HMB treatment group presented with concentrations significantly greater (p < 0.05) than low dose (450.190 ± 59.627 MFI), norm dose (494.250 ± 100.673 MFI), or control (444.720 ± 54.785) treatment groups using post hoc Tukey HSD analysis.. IGF-1 receptor: Average total protein concentrations (Figure 5A) in the low dose

(177.06 ± 4.98 MFI), norm dose (175.03 ± 6.13 MFI), high dose (174.88 ± 5.85 MFI), and control (177.69 ± 7.14 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 5B) in the low dose (15.72 ± 1.293 MFI), norm dose (15.59 ± 0.616 MFI), high dose (15.88 ± 0.891 MFI), and control (16.06 ± 1.067 MFI) treatments were not significantly different. IRS-1: Average total protein concentrations (Figure 6A) in the low dose (14,141.88

± 1033.08 MFI), norm dose (13,829.88 ± 1122.41 MFI), high dose (15,831.63 ± 597.03 MFI), and control (15,695.13 ± 787.95 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 6B) in the high dose (30.00 ± 3.60 MFI) b-HMB treatment group presented with concentrations significantly greater than low dose (20.97 ± 2.69 MFI), norm dose (22.31 ± 3.53 MFI) and control (21.25 ± 0.81 MFI) treatment groups using post hoc Tukey HSD analysis.

68 Akt: Average total protein concentrations (Figure 7A) in the low dose (395.81 ± 23.88 MFI), norm dose (431.28 ± 29.34 MFI), high dose (389.94 ± 39.46 MFI), and control (434.06 ± 41.00 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 7B) in the high dose (75.63 ± 5.65 MFI) b- HMB treatment group presented with concentrations significantly greater than low (62.16

± 4.81 MFI) and control (62.56 ± 4.10 MFI) treatment groups using post hoc Tukey HSD analysis. mTOR: Average total protein concentrations (Figure 8A) in the low dose (349.13

± 15.193 MFI), norm dose (333.72 ± 16.249 MFI), high dose (327.72 ± 14.144 MFI), and control (330.28 ± 12.137 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 8B) in the high dose (196.63

± 17.690) b-HMB treatment group presented with concentrations significantly greater than low dose (142.78 ± 16.121 MFI) and control (139.53 ± 5.829 MFI) treatment groups using post hoc Tukey HSD analysis. p70S6K: Average total protein concentrations (Figure 9A) in the norm dose

(2070.69 ± 228.22) b-HMB treatment group presented with concentrations significantly lower than low dose (2620.50 ± 179.33 MFI) and high dose (2546.38 ± 123.63 MFI) but not control (2333.00 ± 104.54 MFI) treatment groups using post hoc Tukey HSD analysis. Average total phosphorylated protein concentrations (Figure 9B) in the low dose (30.41 ± 1.40 MFI), norm dose (31.13 ± 1.364 MFI), high dose (31.25 ± 1.091 MFI) and control (30.22 ± 1.269 MFI) were not significantly different between treatments. Insulin receptor: Average total protein concentrations (Figure 10A) for low dose

(1662 ± 95.575 MFI), norm dose (1472.59 ± 140.540 MFI), high dose (1572.25 ± 82.682 MFI), and control (1492.38 ± 115.379 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 10B) for low dose 69 (23.13 ± 0.957 MFI), norm dose (25.53 ± 1.501 MFI), high dose (25.38 ± 1.741 MFI), and control (23.72 ± 1.290 MFI) were not significantly different between treatments. PTEN: Average total protein concentrations (Figure 11A) in low dose (1,151.31 ± 109.668 MFI), norm dose (921.47 ± 132.969 MFI), high dose (1038.78 ± 93.078 MFI), and control (1018.84 ± 88.722 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 11B) in the high dose

(1,105.44 ± 113.00 MFI) b-HMB treatment group presented with concentrations significantly greater than control (644.56 ± 125.778 MFI) using post hoc Tukey HSD analysis. Average total phosphorylated protein concentrations in the low dose (862.28 ± 156.679 MFI) and norm dose (842.16 ± 194.799 MFI) were not significantly different from control or high dose treatment groups.

GSK3a: Average total protein concentration (Figure 12A) in the low dose (169.88 ± 4.947 MFI), norm dose (162.72 ± 5.881 MFI), high dose (165.56 ± 5.691 MFI), and control (173.47 ± 7.559 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 12B) in the low dose (15.50 ± 1.093 MFI), norm dose (15.25 ± 0.786 MFI), high dose (15.16 ± 0.944 MFI), and control (15.63 ± 0.504 MFI) were not significantly different between treatments. TSC2: Average total protein concentrations (Figure 13A) in low dose (1,516.34 ± 94.04 MFI), norm dose (1,522.28 ± 121.33 MFI), high dose (1605.38 ± 92.42), and control (1,573.63 ± 84.58) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 13B) in the high dose (247.09 ± 28.059 MFI) b-HMB treatment group presented with concentrations significantly greater than low dose (160.53 ± 22.375 MFI), norm dose (165.00 ± 35.102 MFI), and control (176.50 ± 18.254 MFI) treatment groups using post hoc Tukey HSD analysis.

70 rpS6: Average total protein concentrations (Figure 14A) in low dose (124.47 ± 5.17 MFI), norm dose (117.76 ± 2.54), high dose (118.16 ± 2.84 MFI), and control (121.78 ± 5.50 MFI) were not significantly different between treatments. Average total phosphorylated protein concentrations (Figure 14B) in low dose (14.56 ± 0.924 MFI), norm dose (15.00 ± 1.713 MFI), high dose (16.28 ± 0.990 MFI), and control (14.81 ± 0.666 MFI) were not significantly different between treatments.

DISCUSSION

We provided the leucine metabolite, b-HMB, in three doses to Sprague-Dawley rats to determine its effects on blood glucose, plasma insulin and concentrations of the

Akt/mTOR signaling pathway proteins. We observed no effect of b-HMB on blood glucose concentrations when provided either 15 or 150 minutes prior to a glucose tolerance test. We did observe a significant difference at three time points in plasma insulin concentrations when b-HMB was provided 150 minutes prior to a glucose tolerance test (Fig. 3.2B). We collected tissue samples immediately following an insulin tolerance test which was given 150 minutes following the administration of b-HMB. Thus, plantaris samples were collected after a total of 270 minutes had elapsed from b-HMB administration. We observed several effects of b-HMB on phosphorylation of proteins in the Akt/mTOR pathway. Specifically, we observed increases in total phosphorylation concentrations of

IRS-1, Akt, mTOR, TSC2, PTEN, and GSK3b proteins when b-HMB was provided at a dose of 450 mg/kg BW. No dose below 450 mg/kg BW elicited an increasing effect to phosphorylation of Akt/mTOR signaling pathway proteins.

71 Literature on HMB has shown it to be capable of increasing lean body mass (168, 202), reducing fat mass (109, 188), preventing cachexia (236, 283, 284), and increasing strength output (231), attributed by HMB interaction with regulatory proteins involved in protein synthesis (8, 86, 179, 241, 322) and proteolysis (179). HMB literature has shown no adverse effects at any dose (22, 110, 222) until recent investigations have reported dysregulation of blood glucose and decreased insulin sensitivity (112, 226, 346).

Experiments seeking to determine the effects of leucine and its metabolite b-HMB on Akt/mTOR signaling pathway proteins using in vitro and in vivo models, (179, 241) have found it capable of directly interacting with specific proteins. These proteins, specifically, mTOR and p70S6K, have demonstrated their potential for impairing-insulin stimulated glucose uptake (156, 303). The mechanism of impairment, is thought to occur via AMPK stimulated mTOR resulting in p70S6K phosphorylation, whereby once activated they will phosphorylate insulin receptor substrate (IRS)-1 and IRS-2 on serine residues (IRS-1/2 pS) (12, 15, 156, 233, 264) thus suppressing the insulin signal (27, 203) resulting in hyperglycemia. b-HMB supplementation was recently suggested, by Nunes et al. (226) to exacerbate insulin-glucose intolerance when co-administered with dexamethasone and Yonamine et al. (346) concluded that b-HMB possessed the capability to induce hyperglycemia. Our data contradict the findings of Yonamine et al. (346) and Nunes et al. (226), as we did not observe an increased blood glucose concentration at any time point during a glucose tolerance test when b-HMB was administered either at 15 or 150 minutes prior to an oral glucose tolerance test. However, our study design examined the acute administration of b-HMB effects, while Nunes et al. (226) and Yonamine et al. (346) both supplied b-HMB chronically. Interestingly, our results of elevated phosphorylation of Akt and mTOR only occurred in the high dose b-HMB treatment (450 mg/kg BW). Our results are suggestive of a supraphysiological dosage amount of b-HMB 72 needed to elicit the serine phosphorylation of IRS-1 by mTOR; as we did not observe an increase in p70S6K phosphorylation there is the possibility that we missed the ideal time point for its activity consequently limiting our ability to speculate if it can truly serine phosphorylate IRS1/2. Moreover, while we did observe an increased phosphorylation of IRS-1 in the high dose treatment compared to all other treatments, the phosphorylation of Akt provides evidence that the phosphorylation was on tyrosine sites and not serine.

Finally, our observation of b-HMB reducing plasma insulin concentrations when b-HMB was administered 150 minutes prior to an oral glucose tolerance test (Figure 3.2B) is indicative of increasing insulin sensitivity as during the same time points we observed no differences in blood glucose levels between treatments (Figure 3.2A). Interestingly, we did not observe an effect of b-HMB when administered 15 minutes prior to an oral glucose tolerance testing. We believe this discrepancy is due to uptake time for the calcium form of b-HMB to be absorbed and incorporated into insulin sensitive tissues at a concentration sufficient to induce an effect. Fuller et al. (105) examined in humans the differences between two forms of b-HMB the calcium -salt and free-acid forms. They observed that while both forms reached similar endpoint concentrations, the free-acid form of b-HMB was more bioavailable due to its rapidity in absorption. For our investigation, we utilized the calcium salt form of b-HMB as Fuller et al. (104) describes there is limited investigations into the safety of chronic usage of the free acid form of b-HMB. It is known that increasing insulin will result in subsequent increases of phosphatidylinositol 3-kinase (PI-3K) activity and further propagation of the insulin signal. Both eukaryotic initiation factor (eIF2B), a key regulator, which is responsible for catalyzing a step in mRNA translation and protein synthesis are increased following a rise in insulin concentrations (163, 178, 318, 320). Previous studies have established phosphorylation of Akt is generally responsible for the upstream inactivation of GSK3 (64, 73 65, 122, 278). GSK3b phosphorylation inactivates its inhibition of glycogen synthase permitting storage of glucose while also permitting de-phosphorylation of eIF2B on its

Ser540 site (318-320) thereby permitting mRNA translation. High dose b-HMB treatment resulted in significantly greater phosphorylation of GSK3b compared to all other treatments, which supports the purported findings that b-HMB supplementation could potentially increase lean body mass (168, 202, 230). Furthermore, we observed increased mTOR phosphorylation in the high dose treatment compared to all other groups. Our mTOR observation is in agreement with Pimentel et al. (241) and Eley et al. (86) both of whom reported a direct effect of b-HMB on mTOR phosphorylation and demonstrates two independent means by which b-HMB is affecting protein synthesis. While we did not observe an increase in p70S6K, this could be due to missing its activation window. It is established that mTOR phosphorylation is required for p70S6K activation (95-97) and that the action of p70S6K and eIF2B are responsible for activating ribosomal protein S6 (rpS6) (162), while we did not observe a significant increase in rpS6 activity in our study we did observe an increasing trend of rpS6 activity, suggestive of missing the ideal sampling time point. Moreover, the increased GSK3b and mTOR, would confirm the hypothesis that b- HMB possesses the capability of directly influencing muscle protein synthesis rates at doses of 450 mg/kg BW in rodents. Additional studies in humans would be needed to confirm these results. Phosphatase and tensin homolog (PTEN) is found on chromosome 10q23 and is responsible for converting PtdIns (3,4,5) P3 to PtdIns (4,5) P2 (205), via inhibition of PDK1 (3-phosphoinositide-dependent kinase). It can therefore inhibit the activation of Akt. Moreover, PTEN can be regulated at its post-translational level when phosphorylated. When PTEN is phosphorylated on sites Ser380, Ser385, Thr382, and Thr383 its phosphatase activity is reduced by removing PTEN from its intracellular membrane 74 domain to the cytosol (199, 309). Once PTEN is remanded to the cytosol, its ability to interact with PtdIns(3,4,5) P3 is removed since PtdIns(3,4,5) P3 is located on the inter side of the plasma membrane (7). Finally, PTEN activity can be reduced by GSK3b phosphorylation on residues Ser362 and Thr366 (161). Our observations of elevated phosphorylation of PTEN in the high dose treatment of b-HMB compared to control and elevated concentrations (not significantly different) in the remaining b-HMB treatments suggests b-HMB can more thoroughly activate the Akt/mTOR signaling pathway than just insulin alone. Interestingly, TSC2 phosphorylation was significantly elevated in only the high dose b-HMB treatment. However, our TSC2 observation is reasonable in light of our GSK3b observations which presented to significantly elevated concentrations overall, since GSKb has been shown to directly be capable of phosphorylating TSC2 (134) among other Akt/mTOR signaling pathway proteins.

In summary, we tested three doses of b-HMB and observed no adverse effects on blood glucose, an increased insulin sensitivity, and interactions of b-HMB on the Akt/mTOR signaling pathway. The increased insulin sensitivity we observed occurred only when b-HMB was provided 150 minutes prior to an oral glucose tolerance test and during the final 90 minutes. Overall, b-HMB appears to not adversely affect blood glucose as previously reported and may contribute to increases in protein synthesis rates.

75 CHAPTER IV

HYDROXY-BETA-METHYBUTYRATE CHRONIC SUPPLEMENTATION ON BLOOD GLUCOSE, STRENGTH, AND HYPERTROPHY IN A NOVEL RESISTANCE METHOD FOR INDUCING MUSCULAR STRENGTH

ABSTRACT

Resistance exercise is known to be a potent stimulus for protein synthesis. Beta- hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite shown to increase muscle protein synthesis. Furthermore, limited information suggests HMB may adversely affect blood glucose chronically. We designed a two condition investigation to determine the effects of HMB on sedentary and resistance trained animals to determine its effect on blood glucose, maximal force production, Akt/mTOR signaling pathway activation, and skeletal muscle mass. We constructed a novel resistance training ladder to diffuse the load pulled across a greater amount of muscle mass, thereby more closely resembling how humans perform resistance exercise. Fischer 344 BN rats were randomly assigned to either sedentary control (CON, n=5; 355.2g ± 23.74g), sedentary control HMB (CON-HMB, n=5; 359.6g ± 17.72g), resistance exercise (EX, n=5; 350.6g ± 15.6g) or resistance exercise with HMB (EX-HMB, n=5; 360.2g ± 6.1g) treatment groups. Animals completed three separate oral glucose tolerance tests, force assessments after 16 weeks supplementation, and muscles of the hindlimb were harvested for wet weight and biochemical analyses. We observed no effect of HMB in any condition for any measurement made, we therefore collapsed treatments and compared sedentary animals to resistance trained animals. We observed a significant difference for body weight, muscle mass, increased maximal force output/muscle mass, and an effect on MAFbx in gastrocnemius medial head and quadriceps muscles following the 16 week resistance exercise regimen. We observed no difference in 76 skeletal muscle hypertrophy using the novel method and conclude that this observation was due to a diffusing of the weight across multiple muscles.

INTRODUCTION

The United States Census Bureau in 2017 reported 15.6% of the total population was greater than 65 years of age (2). Sarcopenia, the loss of strength due to age, often is associated with reductions in quality of life. Strength, the ability to produce force in opposition to external resistance, is the result of integration of neurological and morphological adaptations in skeletal muscle. Therefore, designing training regimens to induce alterations in one or both of the factors affecting strength production should be carefully considered in light of the shift in population demographics. Moreover, supplementations which have been previously shown to increase protein synthesis rates should be considered as a means of induce protein accretion and subsequent increases in strength. Inducing greater rates of protein synthesis would benefit a number of at risk populations. Individuals who are suffering from either cancer induced cachexia, sarcopenia, or injury induced muscle atrophy may be limited in their mobility and struggle to build muscle mass via resistance exercise. Therefore, a potential supplement with the capabilities of inducing increased rates of protein synthesis or reducing degradation of muscle should be explored. One such supplement is HMB which previous investigations have revealed increases rates of protein synthesis and/or decreasing rates of protein degradation when it is supplemented at ~3g/day (70, 85, 87, 88, 181). For example,

Wilkinson et al. (322) provided 3.42 g of free acid HMB to young men 22 yrs ± 1.0 yr and found a 70% increase in muscle protein synthesis coinciding with a 57% decrease in muscle

77 protein breakdown. Recently, the findings of Wilkinson et al. were shown to hold true in an older population. Din et al. (72) using older men 68.5 yrs ±1.0 yrs provided free acid- HMB in addition to a resistance exercise program, found an increase in maximum voluntary contractions and an increase in muscle protein synthesis rates during the initial weeks of their regimen, but no increase after 2 weeks. Therefore, the applicability of HMB to either young or old individuals has been shown to effect two separate mechanisms responsible for protein accretion/maintenance. Finally, whereas leucine has shown the ability to increase tumor size (200), HMB has not thus making it a more suitable supplement to prevent cancer induced cachexia without exacerbating the disease state. Increased strength and skeletal muscle hypertrophy are adaptations to chronic resistance exercise. They are the result of repeatedly overloading skeletal muscle which forced it to continually adapt. The hallmarks of adaptation, as they are understood today, are comprised of duration, intensity, frequency of training, and specificity of training; when applied chronically they will result in increased strength and cross sectional area (CSA) of the exercised muscles (58, 150, 269). Exploring this phenomena, Häkkinen and Keskinen (123) presented a strong correlation (r = 0.70) between CSA and larger force production potential in elite strength and endurance trained athletes. The findings of Häkkinen and Keskinen support the previous observations of Henneman et al. (133) who previously demonstrated that normal patterns of motor unit activation and the subsequent activation of muscle fiber types are from small to large soma size (e.g., type 1a à type IIa à type IIb/x). Therefore, the greater a resistance placed on a muscle the larger the CSA needed to generate the opposing force. Moreover, as Henneman et al. (133) previously demonstrated, the recruitment of muscle fibers is sequential neurological and consequentially also sequential by their CSA. Type IIb/x fibers possess the greatest force generating potential (84, 314). Finally, as Yarasheski et al. demonstrated in rodent skeletal muscle, type IIb/x 78 fibers are responsible for the largest increases in observable CSA when sufficiently recruited through repeated use during a progressive resistance training regimen (345). In an effort to elucidate physical activity and its’ benefits to health, the National Institute of Health Common Fund’s Molecular Transducers of Physical Activity in Humans program has been formed with three aims: 1) catalogue exercise effects on biological molecules, 2) generate a comprehensive map of molecule changes in response to movement, and 3) when possible relate the alterations to physical activity benefits. Animal models of hypertrophy will be utilized as a compliment to the human exercise study and will permit analysis of tissues and organs otherwise inaccessible in their human counterparts. Therefore, we have designed a novel multi-joint resistance training method in animals to more closely mimic whole body resistance exercise typical of human whole body workouts. We hypothesize chronic administration of HMB will elicit an increase in muscle mass and subsequent force generating measurements by itself. We further hypothesize that our novel resistance training model will induce greater muscle mass which will be enhanced further with chronic HMB supplementation.

METHODS

Experimental Design and Dietary Treatment All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin. Twenty male Fischer 344x Brown Norway (F344xBN) rats (180 days of age on arrival) were obtained from the National Institute of Ageing (NIA). All animals were randomly assigned to either sedentary control (CON, n=5; 355.2g ± 23.74g), sedentary control HMB (CON-HMB, n=5; 359.6g ± 17.72g), resistance exercise (EX, n=5; 350.6g ± 15.6g) or resistance exercise with HMB (EX-HMB, n=5; 360.2g ± 6.1g) groups. All

79 animals were housed two per cage with their cage mate in the same treatment group and weighed daily. A 12:12-hr reverse dark (10:00 – 20:00hrs) / light cycle was used to allow training to occur during the animals’ naturally active stage. Animals were allowed water and food ad libitum. Animals were trained in darkness using a 60W equivalent red spiral CFL light source dimmed using a rheostat to lowest possible light level.

Resistance exercise harness construction During early piloting of our resistance exercising training regimen, we believed the best course of action for attaching the weight to animals for equal distribution of weight across all muscles, was an attachment between the shoulders (Figure 4.1 A). The harnesses were each constructed from Kevlar (to prevent rats from biting through the harness). The weight attachment was through a looped segment of Kevlar with a small swivel hook sewn between the looped segment. As a result of the line of pull from the attached weight, the animals were able to hold the weight while stationary. However, when the animal was prodded to climb we Figure 4.1: Weight attachment observed the animals struggle to climb. We points. Note: A – initial harness, B – surmised that the animals struggle to climb was second harness attempt due to two factors. One, the weight remained constant and therefore required all of the muscles of the animal to prevent the animal being 80 pulled backward. Second, the ambulatory nature of a quadruped, is such, that the weight would shift between the left and right sides of the animal and thus overload the muscle on one half of the animal, thus preventing the animal from climbing successfully. We therefore re-designed the Kevlar harness so that the line of pull was lower on the animals and would therefore reduce the magnitude of weight placed over the upper torso (Figure 4.1 B). We observed that even with the weight lowered to over the hips, the animal was overloaded and be pulled to one side if the animal did not have a completely straight line of pull. Since the animals rarely pulled completely straight the animals were continuously pulled perpendicular to the weight and would struggle to maintain their grasp on the ladder and halt their backward movement. We would have to continuously re- position the animals and intercede immediately to prevent injury to the animals. Once again we re-designed the Kevlar vest so that the line of pull was attached on a piece of Kevlar descending from the torso to the base of the tail. The animals were able to climb somewhat better with this placement but the weight would shift as Figure 4.2: Training apparatus. A rat is shown climbing a 1.52-m, the Kevlar attachment was loose to permit 20° incline ladder with weight threading of the tail through it. We therefore attached to tail. At the top of the ladder a blacked-out housing concluded that the tail attachment as seen in chamber is where animals rested for 2-min between repetitions. Figure 4.2 was ideal. Weight attachment 81 placement on the tail, resulted in animals immediately ascending the ladder with little difficulty with minimal disruption to their natural movement pattern. Therefore, weight attachment at the tail was used for the duration of the exercising protocol.

Exercise Protocol

Resistance trained rats were familiarized with climbing a ladder (1.52 ´ 0.125 m, 2-cm grid, 20° incline) with a load secured to a cable system secured to the tail. The apparatus has individual lanes with blacked out plexi-glass lane dividers. The length of the ladder required the animals to make 10-16 dynamic movements (reps) per climb. The load apparatus consisted of double looped ended flexible galvanized gauge wire (3.657 m) run through a pulley system. The pulley wheels were secured directly over one another; the lowest wheel was secured at the bottom of the climbing ladder and off set (.152 m) to permit direct vertical pull and the highest wheel was secured 2.1336 m above ground. The galvanized gauge wire possessed a stainless-steel screw and pin anchor shackle through a crown bolt (3/8 in. ´ 4 in.) stainless eye bolt with nut to hold the weights attached at one end and a small swivel hook at the other end. Weights were in the form of ½ in. zinc plated flat washers (~40g/each) or 3/8 in. zinc plated cut washers (~10g/each). The load apparatus was secured to the tail by wrapping the proximal portion of the tail with athletic cohesive adherent tape cut to a 1.5cm width (2 in. ´ 7.5 yds./roll, Powerflex, Salisbury, MA) which was run through a nickel-plated D-ring. With the load secured to the tail, rats were placed at the bottom of the ladder and familiarized with climbing. Initially, rats were motivated to climb with firm tail taps. At the top of the ladder, rats reached a housing chamber (20 ´ 25 ´ 40cm) that was blacked out, where they were allowed to rest for 120 seconds. Refer to Figures 1 for detailed images of the training apparatus. This procedure was repeated until

82 the rats would voluntarily climb the ladder, three consecutive times without encouragement. Our resistance training regimen was consistent with the training protocol described by Hornberger and Farrar (146) with slight modifications. Briefly, three days following familiarization with the ladder climbing, RT rats began a high-intensity progressive resistance exercise regimen. At the start of the resistance training regimen, animals were given weighted resistance equal to 75%, 100%, 150%, and 200% of their body weight over successive training days (e.g. day 1 = 75%, day 2 = 100%, etc.). After reaching 200% bodyweight, the subsequent training session consisted of 4 to 10 ladder climbs. During the first four ladder climbs, the rats carried 50%, 75%, 90%, and 100% of their previous maximal carrying capacity, respectively. Upon successful completion of the previous 100% load, an additional 40-g weight was added to the load apparatus. This procedure was successively repeated until a load was reached where the rat could not climb the entire length of the ladder. Failure to complete a climb was defined as the inability to make progress up the ladder even after the application of 5 firm tail taps. It was observed that once rats were pulling 3´ their body weight, the incidence of failure increased dramatically resulting in a lack of progression. Therefore, we amended the distance pulled to reflect 3- 7 dynamic movements, a distance of 0.75m. Once we reduced the distance we observed an increase in pulling weight by the animals. This adjustment is similar to human repetition recommendations (193). Resistance training occurred every three days for 16 weeks, a total of 37 training sessions.

In-Situ Contractile Properties Following 16 weeks of resistance training, in situ evaluations of triceps surea and quadriceps muscle force production was performed on the left leg of the animal. Contractile 83 properties of RT animals were assessed 72 ± 5hrs after their final training session. Our technique is a modification of previous work conducted by Hammers et al. (125). Briefly, animals were anesthetized using 2.0-2.5% isoflurane gas before and during the in situ contractile properties assessments. Once the animal was anesthetized, the skin of the hindlimb was removed to expose the quadriceps, hamstrings, and lower leg with particular care to stay subcutaneous and avoid major arteries or veins. The muscles were kept moist using isotonic saline (0.9 % NaCl), and any bleeding that occurred during the following procedure was stopped with cauterization. The initial step was to expose the patellar tendon and remove any connective Figure 4.3: In situ setup of rodent hind limb for quadricep testing. tissue around it. Once the patellar tendon was Note: cross bar and anchoring of clean the animal hindlimb was bent at a 90° condylar pin as well as stimulator placement angle to allow exposure to the femoral condyles. A 1/16” (1.6mm) split point titanium drill bit (DeWalt, DW1370) was drilled through the femoral condyles. After the bit was placed, it was removed from the drill and the area was examined for excess bleeding, any bleeding found was immediately staunched via cauterization. Once the bit was placed, scissors were used to carefully remove the connective tissue on the medial side of the femur permitting the reflection of the medial 84 musculature touching the quadriceps. After the medial side of the leg was prepared, the animal hindlimb was bent at a 90° angle which clearly shows a separating line between the lateral aspect of the quadriceps and the biceps femoris. Using the lateral landmark, scissors were gently used to remove any connective tissue between the quadriceps and the biceps femoris from the patella to just below the greater trochanter. A small slip knot of 2/0 black braided silk was tied around the patellar tendon after the quadriceps were cleaned of connective tissue. A small piece of tibia was then cut using bone cutters to provide a secure piece of bone for the future knot to slip against. After the piece of tibia was cut, the braided silk was used to gently reflect upwards the patella. Small scissors were then used to cut along the bone free the quadriceps from the femur. The length of the cut did not surpass 1/3 the length of the quadriceps, so that the vastus intermedius remained attached to the femur. A braided fishing line was then knotted around the tibial bone piece and pulled tight. Following the quadriceps preparation, the Achilles tendon was exposed and a small fishing wire was secured around the tendon via a slide knot. The calcaneus was then cut with bone cutters to provide a secure piece of bone for the knot to slip against. The triceps surea was then freed from lateral and medial connective tissue with care to avoid damaging the muscle. The animal being fully prepared for the in situ measurements was then transferred to the assessment area. The drill bit was then used as the superior anchor and stabilizer of the hindlimb during contractile measurements. Animals were placed into a prone position for triceps surea assessment and switched to a supine position for quadriceps assessments. The superior anchor was stabilized in place using plexi-glass, and slack was removed from the fishing wire connected to the lever arm of a dual mode servomotor (Aurora Scientific Model 310B Inc.; Aurora, ON, Canada). Following anchor stabilization, a metal rod was placed across the pelvis of the animal and secured in place (Figure 4.3). This procedure was critical for preventing 85 slippage and torque at the hip during contractile measurements. The sciatic nerve was isolated and approximately 2 cm proximal to the femoral condyles. The sciatic nerve was gently attached to an electrode for stimulation. Muscle temperature was regulated with the use of a radiant heat lamp and a mineral oil bath. The mineral oil temperature was maintained between 36.5 and 37.5° C. The input source to the electrode was a Grass 88 stimulator. The dual mode servo output was interfaced with a computer (Dell 8250) and Labview software (Version 3.0, National Instruments, Austin, TX). The dual mode servo was integrated with the Labview software and DA board (National Instruments) for data presentation and analysis. To determine the twitch contractile characteristics, we applied a single squarewave pulse of 5 volts, 0.5 msec duration. With the use of a micrometer, the muscle length was gradually increased until a plateau in twitch tension was observed; this was designated as optimal length (Lo). The triceps surea and quadriceps were then maximally activated by stimulating the sciatic nerve with a 330-ms, 100–150 Hz, 0.5-ms duration, and 7–14 volt pulse. The force frequency response of the muscle was determined using one train pulse of 330 ms duration and 14 volts at frequencies of 100, 125, and 150 Hz. After each high frequency stimulation, the muscle was allowed to rest for 120 sec. The highest tension produced during these stimulations was recorded as peak tetanic tension (Po).

Tissue harvesting Immediately following completion of the force measurements, the gastrocnemius, plantaris, and soleus, biceps femoris, semitendinosus, semimembranosus, quadriceps, gluteus medius, and wrist flexors were quickly harvested from both the resistance measured and non-resistance measured legs, and muscle wet weights were recorded. The gastrocnemius was then divided into medial and lateral heads. Sections of each muscle 86 were then embedded in OCT compound and all muscle segments were then frozen in liquid nitrogen-cooled isopentane and stored at −80°C for later protein content and histological analyses. Animal hearts, epididymal fat, and adrenal glands were also excised and weighed. Rats were euthanized via a thoracotomy.

Protein Concentration Protein content quantification was determined in a manner previously described by Hammers et al. (125). Briefly, approximately 100 mg of muscle tissue was removed from the frozen gastrocnemius medial and lateral heads, plantaris, soleus, biceps femoris, semitendinosus, semimembranosus, quadriceps, gluteus medius, and wrist flexors muscles of both the resistance measured and non-resistance measured legs, trimmed of all visible connective tissue, and homogenized in a buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 1% Triton X-100, 20 mM β-glycerol phosphate, 10 mM NaF, 1 mM Na3VO4, 10 ng/ml each of leupeptin and aprotinin, 1 mM PMSF, and 1:100 dilutions of phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). The resulting homogenate was centrifuged at 12,000 g for 30 min, and the supernatant was kept for analysis. Protein concentrations of all samples were determined as described by Bradford (42) and run in triplicate to verify results.

Western blotting Gastrocnemius medial head and quadriceps muscle protein concentrations determined by the Bradford Assay described above, were utilized in determining loading values of 100ug of protein in western blots. Samples were boiled in 2× Laemmli's sample buffer (Bio-Rad, Cat #at a ratio of 1:1, and equal amounts of total protein were loaded into each well of a 5% stacking-15% separating polyacrylamide gel. SDS-PAGE was run for 87 80v (constant) for 110 mins. Following SDS-PAGE, proteins were wet-transferred to a

0.2µ nitrocellulose membrane (Bio-Rad, Cat# 1620112) using a Towbin buffer without SDS. Following transfer, membranes were stained with Ponceau S (Sigma Aldrich, Cat # P7170) to verify staining and then de-stained with 3 5-min washes of 25 mL 0.1% Tween- 20 in TBS (TBST, pH 7.4). Once de-stained, membranes were blocked with 7% milk in 0.1% Tween-20 in TBS (TBST,) for 20 min. Membranes were incubated in 1:250 dilutions of either anti-phospho- FoxO3a (Ser 253; Cell Signaling Technology, Cat #9466S), anti-phospho-Akt (Ser 473; Cell Signaling Technology, Cat# 4060S), 1:500 for MuRF 1 (Santa Cruz Biotechnology, Cat# sc-398608), atrogin-1 (Santa Cruz Biotechnology, Cat# sc-166806) , anti-FoxO3a (Cell Signaling Techology, Cat# 12829S), anti-Akt primary (Cell Signaling Technology, Cat#9272S), and/or 1:1000 with GAPDH (Cell Signaling Technology, Cat #5174S) primary antibodies in 5% BSA-TBST for 18.5hrs at 4°C, then in 1:2,000 dilutions of either anti-rabbit horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Cat #7074S) or anti-mouse horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Cat# 7076S) in 5% milk-TBST for 1-2 hrs. Blots were imaged with the Chemidoc XRS system (Bio-Rad, Cat#1708265). Band volumetric analysis was performed using Quantity One software and is expressed as arbitrary units of volume [intensity (INT) × mm2]. Phosphorylation status of each protein species was calculated by dividing the abundance of the phosphorylated form by the total protein abundance. All experiments were repeated in duplicate to verify results.

Determination of tissue dry weight Portions of the gastrocnemius medial and lateral heads, plantaris, soleus, biceps femoris, semitendinosus, semimembranosus, quadriceps, gluteus medius, and wrist flexors 88 muscles were weighed and then dried at 95°C in an oven. The muscles were then weighed on consecutive days until a stable weight was obtained (i.e., dry weight). Ratios of wet to dry weight were then determined (Table 4.7).

Oral glucose tolerance test The effects of the resistance training regimen on glucose handling was tested three times during this investigation. Testing occurred prior to resistance exercise familiarization, 5 weeks into the resistance exercise regimen, and 11 weeks into the resistance exercise regimen. Animal tails were cut with a sterilized razor and 0.5 mL whole blood was extracted into micro-centrifuge tubes containing 0.1 mL of 24 mg/mL EDTA (pH 7.4) to prevent clotting a total of 6 times. Blood sampling occurred prior to glucose administration (time point 0) to determine baseline plasma glucose measurements and at 15, 30, 60, 90 and 120 minutes post glucose administration. Plasma glucose measurements were taken using a Freestyle glucometer (Therasense, Alameda, CA, USA) at each of the sampling time points. The glucose bolus was gavaged at a concentration of 1.0g/kg bodyweight of glucose via a feeding bulb. Following the final blood collection animal tails were cauterized and returned to their home cages. Animal health was assessed 1 hr. after being returned to their home cages.

RESULTS – FOUR TREATMENT ANALYSES

Blood glucose concentrations. Three separate OGTTs were performed on CON and CON-HMB treatments groups. No statically significant difference was observed at any time point between treatments (Figure 4.4 A-C). Three separate OGTTs were performed on EX and EX-HMB treatments groups. No statically significant difference was observed

89 at any time point between treatments (Figure 4.5 A-C). Area under the curve (AUC) was calculated for CON and CON-HMB blood glucose during the OGTTs, no significant difference between treatments was observed (Figure 4.6). AUC was calculated for EX an EX-HMB blood glucose during the OGTTs, no significant difference between treatments was observed (Figure 4.7) In-situ Contractile Measurements. Following completion of 16 weeks supplementing with HMB, sedentary animals (CON & CON-HMB) under went in situ force measurements of their quadriceps and triceps surae muscle groups. No significant differences were observed between treatments for total force in the triceps surae (Figure 4.8A) or quadriceps muscle groups (Figure 4.8B) or force/muscle mass calculations (data not shown). Following completion of 16 weeks of resistance training, resistance trained animals had under went in situ force measurements of their quadriceps and triceps surae muscle groups. No significant differences were observed between treatments for total force in the triceps surae (Figure 4.9A) or quadriceps muscle groups (Figure 4.9B) or force/muscle mass calculations (data not shown). Upon completion of force measurements, CON & CON-HMB animal hindlimb muscles were harvested and their wet weight was acquired. No significant differences were observed between treatments for any muscle in either the force measured or non-force measured hindlimbs (Table 4.1). Upon completion of force measurements, EX & EX-HMB animal hindlimb muscles were harvested and their wet weight was acquired. No significant differences were observed between treatments for any muscle in either the force measured or non-force measured hindlimbs (Table 4.2).

90 Figure 4.4 Blood glucose during oral glucose tolerance tests which occurred prior to HMB administration (A), 5 weeks (B), and 11 weeks (C) with chronic HMB supplementation. A

140 120 100 80 60 40 20 0 0 D15 D30 D60 D90 D120 Blood Glucose (mg/dL) Sampling Time Points (min)

CON CON !-HMB B

140 120 100 80 60 40 20 0 0 D15 D30 D60 D90 D120 Blood Glucose (mg/dL) Sampling Time Points (min)

CON CON !-HMB C

140 120 100 80 60 40 20 0

Blood Glucose (mg/dL) 0 D15 D30 D60 D90 D120 Sampling Time Points (min)

CON CON !-HMB

91 Note: CON = sedentary control rats, CON-HMB = sedentary control with HMB. No significant differences observed. Values are expressed as mean ± SD.

Figure 4.5 Blood glucose during oral glucose tolerance tests which occurred prior to training (A), 5 weeks (B), and 11 weeks (C) into resistance training. A

140 120 100 80 60 40 20 0 0 D15 D30 D60 D90 D120 Blood Glucose (mg/dL) Sampling Time Points (min)

EX EX !-HMB B

140 120 100 80 60 40 20 0 0 D15 D30 D60 D90 D120 Blood Glucose (mg/dL) Sampling Time Points (min)

EX EX !-HMB

92 C

140 120 100 80 60 40 20 0 0 D15 D30 D60 D90 D120 Blood Glucose (mg/dL) Sampling Time Points (min)

EX EX !-HMB

Note: EX = resistance trained rats, EX-HMB = resistance trained + HMB. No significant differences observed. Values are expressed as mean ± SD.

Figure 4.6 Area under the curve (AUC) for blood glucose during tolerance tests which occurred prior to HMB administration (A), 5 weeks (B), and 11 weeks (C) with chronic HMB supplementation.

CON 15000 14500 14000 13500 13000 12500 12000

(mg/dL x 120 min) x 120 (mg/dL 11500 Blood Glucose AUC 11000 OGTT 1 OGTT 2 OGTT 3 Oral Glucose Tolerance Tests

Note: CON = sedentary control rats, CON-HMB = sedentary control with HMB. No significant differences observed. Values are expressed as mean ± SEM.

93 Figure 4.7 Area under the curve (AUC) for blood glucose during tolerance tests which occurred prior to resistance exercise (A), 5 weeks (B), and 11 weeks (C) with into resistance exercise.

EX EX !-HMB 14500 14000 13500 13000 12500 12000 (mg/dL x 120 min) x 120 (mg/dL

Blood Glucose AUC OGTT 1 OGTT 2 OGTT 3 Oral Glucose Tolerance Tests

Note: EX = resistance trained rats, EX-HMB = resistance trained + HMB. No significant differences observed. Values are expressed as mean ± SEM

Figure 4.8 Total Force (N) measured in Triceps Surae (A) and Quadriceps (B) muscle groups by treatment.

A B

CON CON !-HMB CON CON !-HMB 45 50 40 45 35 40 30 35 30 25 25 20 20 Force (N) 15 Force (N) 15 10 10 5 5 0 0 CON CON !-HMB CON CON !-HMB Treatments Treatments

Note: CON = control rats, CON-HMB = rats fed 450mg/kg/BW daily for 16 weeks. Values are expressed as mean ± SEM

94 Figure 4.9: Total Force (N) measured in Triceps Surae (A) and Quadriceps (B) muscle groups by treatment. A B

EX EX !-HMB EX EX !-HMB 45 50 40 45 35 40 30 35 30 25 25 20 20 Force (N) Force (N) 15 15 10 10 5 5 0 0 EX EX !-HMB EX EX !-HMB Treatments Treatments

Note: EX = resistance trained rats, EX-HMB = resistance trained rats fed 450mg/kg/BW daily for 16 weeks. Values are expressed as mean ± SEM

Table 4.1 Wet weights of muscles harvested from sedentary animals.

95 STATISTICAL CONSIDERATIONS Our rationale for analyzing HMB and our dosage, were based on observations found in Study 1, wherein we observed several proteins of the Akt/mTOR signaling pathway phosphorylated to a greater extent compared to a no HMB treatment and HMB treatments at lower dosages. The findings from Study 1 were similar to both Din et al. (72) and Wilkinson et al. (322) who both observed an increase in protein synthesis rates when HMB was administered. Therefore, it was reasonable to expect that chronic HMB supplementation would promote greater protein accretion when chronically supplemented. We also were interested to observe the chronic effects of resistance training and HMB supplementation on blood glucose as our acute findings in Study 1 demonstrated no effect but which there is limited literature of chronic HMB supplementation effects on blood glucose. Consequently, we analyzed two separate conditions. The first condition, sedentary rats compared to sedentary rats fed 450 mg/kg/BW, was to determine the efficacy of chronic administration of HMB on developing muscle mass sans additional stimuli. We observed no effect of HMB on blood glucose, maximal force generation of either the triceps surae or quadriceps muscle groups, nor did we find any differences in muscle wet weights. In the second condition, resistance trained rats compared to resistance trained rats fed 450 mg/kg BW, was performed to assess if HMB could enhance protein accretion and force generation of the triceps surae and quadriceps muscle groups to a greater degree than resistance training alone. We observed no significant difference between treatments of the resistance exercising animals in either blood glucose effects, maximal force generation of the triceps surae or quadriceps muscle groups, or muscle wet weights. Therefore, we conclude that HMB does not possess any significant effect on protein accretion when supplemented chronically alone, nor does it adversely affect blood glucose. Our results are 96 similar to Jakubowski et al. (160) and Teixeira et al. (297) who each observed no effect of HMB when incorporated into a resistance training regimen. As a consequence of the lack of HMB effect we collapsed treatment groups, into sedentary and resistance trained animals. We re-analyzed the data with respect to the new treatment parameters and present our results below.

RESULTS – TWO TREATMENTS ANALYSES

All of the EX animals successfully complete the 16 weeks of training. The training protocol promoted an increase in performance as assessed by the ability to carry progressively heavier loads (Figure 4.10). After 16 weeks of training, EX animals increased their maximal carrying capacity by 198.2% (871.6 g ± 14.0 g to 1727.6 ± 31.6g), t £ 0.0001. The maximal carrying capacity by the final training week represents 427 ± 11.4% of the animal’s body weight.

PROPERTIES OF THE QUADRICEPS AND TRICEPS SURAE

In-situ Contractile Properties. Measurements of the in-situ contractile properties of the quadriceps are reported in Table 3.5 and triceps surae are reported in Table 3.6. PSCA and fiber length (Flo) measurements were calculated using data procured by Eng et al., (2008) who thoroughly investigated Sprague-Dawley rat hindlimb musculature characteristics (89). No difference was observed between treatments for Pt or Po, however when Po was calculated with respect to muscle mass (N/g) a significant difference was observed between EX and CON groups in the triceps surae (12.839 N/g ± 0.43 N/g to

97 11.548 N/g ± 0.46 N/g, respectively), t £ 0.05, and quadriceps muscle groups (10.3839 N/g ± 0.27 N/g to 8.855 N/g ± 0.36 N/g, respectively), t £ 0.01, data presented in Figure 4.11.

Table 4.2 Wet weights of muscles harvested from resistance trained animals.

Figure 4.10: Maximal carrying load per training week.

2000

1600

1200

800

400

Maximal Carrying Capacity (g) Capacity Carrying Maximal 0 0 1 2 3 4 5 6 7 8 9 10 11 Training Week Note: Over the course of 11 weeks the animal’s maximal carrying capacity increased 198.2%. Values are expressed as mean ± SD.

98 Figure 4.11: Force (N) / Muscle Weight (g) in both the Triceps Surae and Quadriceps muscle groups by treatment.

Triceps Surae Quadriceps 14 * 12 †

12 10 10 8 8 6 6 4 4 2 2 0 0 Force (N) / Muscle Weight (g) Force (N) / Muscle Weight (g) CON EX CON EX

Note: CON = control rats, EX = resistance trained rats * Significant difference vs. CON, t ≤ 0.05. † Significant difference vs. CON, t ≤ 0.01Values are expressed as mean ± SEM

Body weight, tissue weights, and wet/dry weight ratios. Resistance exercising animal body weights were significantly reduced compared to sedentary animals, EX = 404g

± 17g vs CON = 431g ± 17g, t £ 0.0001. Training did not increase the wet weight of any muscle in the EX group, however, compared to CON animals, every muscle except the plantaris and semitendinosus were significantly smaller, t ≤ 0.0001, the weights are presented in Table 4.3. Epididymal fat wet weight and adrenal gland mass was significant reduced in the EX animals compared to CON, t ≤ 0.001, whereas the heart mass remained unchanged, the weights are presented in Table 4.4. Significant differences were observed in the force measured leg semitendinosus of EX (0.19 ± 0.01 mg) animals compared to CON (0.24 ± 0.01 mg), t ≤ 0.01. A significant difference was found in the force measured leg semimembranosus of EX (0.21 ± 0.01 mg) animals compared to CON (0.23 ± 0.01 mg), t ≤ 0.001. Significant differences were observed in the semitendinosus (EX: 0.26 ± 0.01 mg compared to CON: 0.27 ± 0.01 mg, t ≤ 0.01) and gluteus medius (EX: 0.23 ± 0.01 99 mg compared to CON: 0.25 ± 0.01 mg, t ≤ 0.05) of the non-forced measured leg. No significant differences were observed in any other muscle measured (Table 4.7)

Signaling Protein Properties. Western blotting of Akt, pAkt, forkhead box 3A (FoxO3A), p-FoxO3A, atrogin-1/MAFbx, MuRF1, and GAPDH were taken in both the gastrocnemius medial head and quadriceps muscles in non-force measured legs. Relative protein abundance was determined by dividing phosphorylated protein concentration by total protein concentrations. No difference was observed in pAkt, pFoxO3A, or MuRF1 proteins. Significant differences were found for atrogin-1/MAFbx in both the GMB and quadriceps muscles. In the GMB, the EX treatment resulted in significantly greater protein concentration compared to CON animals, t ≤ 0.001, data presented in Figure 4.12A. In the quadriceps muscles, the EX treatment resulted in significantly less protein concentration to CON animals, , t ≤ 0.05, data presented in Figure 4.12B.

Blood glucose concentrations. Three separate OGTTs were performed on CON and EX treatment groups. No statically significant difference was observed at any time point between treatments (Figure 4.13 A-C). AUC was calculated for CON and EX blood glucose during the OGTTs, no significant difference between treatments was observed (Figure 4.14)

100 DISCUSSION

This study was conducted to determine the efficacy of chronic HMB supplementation. Specifically, examined the effects of HMB on blood glucose, skeletal muscle mass and force generation in muscles of sedentary rats and rats which completed 16 weeks of resistance exercise using on a novel resistance training model. The importance of resistance exercise as part of an exercise regimen is without question (166, 252, 339), our model provides an additional means of testing resistance exercise effects. Additionally, our model demonstrates an efficacy in developing strength which is beneficial to populations seeking strength gains over hypertrophy. To investigate any impact on glucose homeostasis, we performed three oral glucose tolerance tests which occurred before training, five weeks, and eleven weeks into training. Our resistance exercise (RE) model did not result in differences in glucose handling overall (Figures 4.6 A-C).

101 Figure 4.12: Western blotting was performed on sedentary (CON) and resistance trained (EX) animal gastrocnemius medial heads (A) and quadricep muscles (B) of the non-force measured hindlimb. A

CON EX 1.8 † 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 Relative Protein Abundance (AU) 0 pAkt pFoxO3A MuRF1 MAFbx GAPDH

B

CON EX 1.2 1 * 0.8

0.6

0.4

0.2

Relative Protein Abundance (AU) 0 pAkt pFoxO3A MuRF1 MAFbx GAPDH

Note: CON = control rats, EX = resistance trained rats. Proteins assessed included the phosphorylated and total protein forms of Akt, FoxO3A, atrogin-1/MAFbx and MuRF1. GAPDH was measured as a loading control. * Significant difference vs. CON, t ≤ 0.05, † Significant difference vs. CON, t ≤ 0.001. Values represented are the M ± SEM.

102 Table 4.3 Body and Muscle Weights

CON (n=9) EX (n=10) (M ± SD) (M ± SD) Initial Body Weight (g) 431 ± 17 416 ± 19 Final Body Weight (g) 478 ± 21 404 ± 17 † Soleus (mg) 218 ± 0.007 204 ± 0.005 † Plantaris (mg) 464 ± 0.3 445 ± 0.02 Gastrocnemius (g) 2.3 ± 0.09 2.1 ± 0.04 † Quadriceps (g) 4.43 ± 0.2 4.0 ± 0.1 † Biceps Femoris (g) 3.9 ± 0.2 3.4 ± 0.1 † Semitendinosus (g) 1.4 ± 0.04 1.4 ± 0.04 Semimembranosus (g) 2.2 ± 0.1 1.9 ± 0.04 † Gluteus Medius (g) 3.4 ± 0.05 2.9 ± 0.05 † Note: CON = control rats, EX = resistance trained rats. † Significant difference vs. CON, t ≤ 0.0001. Values are expressed as mean ± SD.

Table 4.4 Ancillary Tissue Weights

CON (n=9) EX (n=10) (M ± SD) (M ± SD) Adrenal Glands (mg) 65 ± 0.001 55 ± 0.001 † Epididymal Fat (g) 11.7 ± 0.48 7.3 ± 0.46 † Heart (g) 1.3 ± 0.2 1.3 ± 0.2 Note: CON = control rats, EX = resistance trained rats. † Significant difference vs. CON, t ≤ 0.001. Values are expressed as mean ± SD.

Table 4.5 Quadriceps Contractile Properties

CON (n=9) EX (n=10) (M ± SD) (M ± SD)

Lo (cm) 4.88 ± 0.11 4.77 ± 0.14 Flo 1.49 ± 0.085 1.49 ± 0.085 ‡ CSA (cm2) 0.4225 ± 0.085 0.4225 ± 0.085 ‡ Pt (N) 4.98 ± 0.07 4.95 ± 0.09 Po (N) 39.36 ± 2.96 41.44 ± 1.87 SPt (N/cm2) 11.79 ± 0.18 11.73 ± 0.21 SPo (N/cm2) 93.16 ± 6.98 96.98 ± 3.40 SPt/Spo 0.12 ± 0.02 0.12 ± 0.01 103 Note: CON = control rats, EX = resistance trained rats. ‡ Data calculated using results from Eng, CM et al. (2008) (89). Values are expressed as mean ± SD.

Table 4.6 Triceps Surae Contractile Properties

CON (n=9) EX (n=10) (M ± SD) (M ± SD) Lo (cm) 4.12 ± 0.07 4.12 ± 0.12 Flo 1.49 ± 0.085 1.49 ± 0.085 ‡ CSA (cm2) 0.36 ± 0.133 0.36 ± 0.133 ‡ Pt (N) 4.89 ± 0.19 4.78 ± 0.14 Po (N) 35.03 ± 2.75 34.01 ± 2.62 SPt (N/cm2) 13.57 ± 0.54 13.29 ± 0.38 SPo (N/cm2) 95.76 ± 7.05 92.70 ± 5.71 SPt/Spo 0.147 ± 0.01 0.140 ± 0.01 Note: CON = control rats, EX = resistance trained rats. ‡ Data calculated using results from Eng, CM et al. (2008) (89). Values are expressed as mean ± SD.

While a plethora of resistance exercise studies have demonstrated an increase in insulin sensitivity indicated by increased glucose clearance, these studies all show an increase in lean body mass in their supplementation groups (183, 187, 191). Furthermore, there is limited evidence that resistance exercise alone is capable of increasing glucose clearance (185, 189) in the absence of an increase in insulin sensitive tissues, some studies even report finding no effect (342). Our EX animals did not increase in body mass and instead show a significantly reduced body mass when compared to CON animals at the end of the training regimen (Table 4.3). The body mass difference we observed is likely the cause of why we observed no changes in glucose handling as the amount of insulin sensitive tissue was not significantly different from the amount of tissue in control animals.

104 It is known that skeletal muscle possess two distinct glucose transporter 4 (GLUT4) pools, one which is sensitive to insulin while the other activates in response to mechanical signaling (138, 139). Moreover, both pools of GLUT4 are reliant on Akt substrate of 160 kDa (AS160) phosphorylation (50, 106, 265). While we did not harvest skeletal muscle immediately post glucose tolerance testing, Deshmukh et al (69) demonstrated that AS160 phosphorylation was sensitive to the mode of exercise as they reported no change in AS160 concentration in resistance exercise groups but an observable increase following a single bout of aerobic exercise. Therefore, it is within reason that our lack of an observable difference in glucose handling across a resistance exercise regimen may in part be due to a combination of insufficient stimulus of AS160 in conjunction with no changes in the amount of skeletal muscle mass. Finally, our resistance training model is novel and designed as a whole body exercise aimed at diffusing the weight so no single muscle is overloaded, as such, the typical signals of inducing adaptation are likely reduced and thus the muscle adaptation is stunted. Models of resistance training necessitate fundamental parameters to inducing morphological adaptations. The first is known to be repeated muscular overload which will induce hypertrophy (60, 338). The second is progressive overloading on a muscle is necessary for continual stimulation that results in observable/measurable hypertrophy (1, 185). Finally, adaptations manifest in two temporal segments, the initial adaptations being neurological while the latter are morphological (2, 80, 108, 145, 155, 266, 269). Currently, the applicability of these tenets are derived, in large part, from animal studies (58, 145, 155, 189, 201, 291, 298). A substantial benefit to using animal models of resistance exercise is the measuring of training effects on extractable muscles such as the quadriceps (189, 291), flexor hallucis longus (145, 315) , and plantaris (155, 291). However, animal studies are atypical in their applicability to the generalized public. Currently, one goal of 105 the Molecular Transducers of Physical Activity Consortium (MoTrPAC) is to understand how “physical activity improves and preserves health”. In an effort to measure physical activity effects, MoTrPAC has sought an animal resistance exercise model that can closely mimic human resistance exercise responses. To investigate the efficacy of our novel resistance exercise model we measured both the quadriceps and triceps surae muscle groups. We observed no significant difference between either group for Pt or Po, however, when we controlled for the lack of differences in Po force, per unit muscle mass, we found a significant difference in the triceps surae and quadriceps groups (Figure 3.11). Furthermore, the result of finding a significant difference in both muscle groups supports the conclusion that the training apparatus is different from Hornberger and Farrar (145). While Hornberger utilized a ladder apparatus as well, his ladder was set at 80° whereas our ladder was set at 20°, as a consequence of his ladder, Hornberger’s found a 23% increase in the flexor hallucius longus (FHL) weight and no differences in muscles of the triceps surae or quadriceps muscle groups. The lack of triceps surae and quadriceps development was due to the action by the animals to ascend the ladder, an action which was primarily reliant on flexion of the FHL as well as an eccentric component, hence its’ responsiveness to the training while the other muscles failed to respond. Using an apparatus at a reduced angle permitted a greater amount of weight overall compared to Hornberger and Farrar, which consequently diffused the weight being pulled over more muscle. Our resistance exercising animals were significantly smaller than our sedentary animals and yet absolute force production was similar between treatments. When controlling for force production per unit mass, we observed a significant difference in the EX groups compared to control. The question then, is how did animals with smaller muscle mass pull so much weight?

106 Our training regimen elicited an increased weight pulled of 198.2% from start to finished, which represented ~427% of the animal body weight. Additionally, our training ladder was designed to diffuse the weight across a greater amount of muscle mass. Similar movements requiring large muscle mass to move heavy weights relative to the body weight of the lifter is found in Olympic lifting. The movements of Olympic lifts are known to be multi phasic and requiring a synchronization of multiple joints and muscle groups to achieve maximum strength. Gradations in lifting potential has been previously assessed in Olympic lifting to permit relatively equal competition between lifters. Since 1979 the International Weightlifting Federation method by which they assess the effects of body mass on performance, is via the application of a quadratic equation designed to account for a lifter’s handicap (175), which the Sinclair formula (282) is the most commonly utilized. The formula calculates the previous four years Olympic lifting world records and develops a Sinclair coefficient which is multiplied against a lifters performance to generate a Sinclair score, independent of body mass. However, the Sinclair calculation reveals two outcomes, an upper limit wherein heavier lifters are penalized and an “ideal” body mass for maximal lifting performance exists. A separate equation for calculating the effects of body mass on lifting performance was the Siff equation (281). When Cleathers (59) applied the Siff equation to the squat, bench, and deadlifts of men, it represented a tight correlation for body mass and strength for both the squat and bench. However, the deadlift was shown to rely heavily on body mass for initial force production but as the subjects got heavier there was less of a reliance on body mass. Cleather concluded that the limiting factor representing the rapid decline in body mass importance was the limiting muscular mass of the forearm causing reduced grip strength and the inability to hold onto heavier weights. In observations of our animals climbing action, the animals displayed two methods of approaching the task. One way was 107 to firmly grasp the leading bar with their forearm grip and rapidly jump with their the hindlimb forwards while not moving their forearms. This action placed the forearms in a momentary period of overloaded. At upper weights when the animal force production was near maximum, they were unable to produce sufficient force for the jumping action and would stumble. In the second approach to climbing, the animals would ascend the ladder similar to humans, one rung at a time. In this action the burden of maintaining a firm grasp oscillated back and forth between each forearm individually. We observed when animals employed this technique, it occurred only during the lighter warmup loads. Thus, a factor impacting further progression in weight was the limited CSA of the forearm to produce the necessary force to hold weight when the hindlimbs were in motion. Wolfe (337) and Stump et al. (287) both presented evidence of the importance in maintaining muscular strength in preventing chronic disease. Specifically, loss of strength has been shown to increase all-cause cardiovascular death risk factors (14, 103, 174, 247), decrease glucose homeostasis (20), and increase adipose tissue stores (157, 208). Moreover, decreases in strength are associated with the loss of functional independence of older adults, increasing risk of early death. For example, using computer tomography and dual-energy x-ray absorptiometry measurements of legs, Newman et al. (220) analyzed 2292 men and women between 70-79 yrs old and reports that strength is more determinate than muscle size in estimating mortality risk. Interestingly, the capacity to gain strength is not regulated to only the young. Fiatarone et al. (94), demonstrated in nonagenarians performing a high intensity weight training program an average of 174% ± 31% in total strength. Moreover, the subjects of the study had led a sedentary lifestyle, had more than one adverse health diagnosis, and all were at risk of cardiovascular complications. Yet, as they discovered, the maintenance of an evenly paced resistance exercise training program did not increase cardiovascular complications. Instead, they observed a 48% increase in 108 tandem gait speed, a task dependent on both strength and balance. Fiatarone et al. observed an small increase in hypertrophy in some but not all subjects but noted increases in strength occurring within 2 weeks of training. Therefore, they concluded that neurological development was the primary factor influencing strength gains. Funato et al. compared elite senior and college Olympic lifters (107) and reported the highest mass lifted in both the snatch and clean and jerk lifts were in the elite senior lifters even when measured per unit of fat free mass. Interestingly, Funato et al. did not find any difference between groups for fat-free mass, muscle CSA or force values except in knee flexion where elite senior lifters produced more force compared to college lifters. Finally, Funato et al., reported increased ratios of force to muscle CSA in both elbow concentric and eccentric contractions. These findings are revelatory in that they demonstrated that force production in multi-joint movements is more reliant on coordination of muscle groups than raw force production of any one muscle group. Furthermore, several researchers have found that training type significantly impacts recruitment and adaptation characteristics of motor units during contractile activation (78- 80, 266, 307), suggesting a strong neurological malleability dependent upon the task. It has been established in the literature, that early neurological adaptation facilitates initial gains in strength. While the subject of neurological adaptation to strength exercise is beyond the scope of this investigation, Enoka (90) previously articulated that the maximum force generated by a motor-unit is influenced by numerous factors (e.g., innervation ratio, CSA, and specific force). The various influencing factors account for the force of a single motor unit possessing the potential to increase 300 – 1500% when the discharge rate is increased from the minimum to maximum above baseline in motor unit activation. Furthermore, Enoka states that muscle force is further stratified by concurrent recruitment and discharge rate variations (90). Yet, while the motor unit force outputs range 109 is large, the recruitment pattern of the motor neurons maintains Henneman’s size principle (133). It has been shown that resistance training can induce neurological adaptation, specifically, synchronicity in motor unit firing rates. Milner-Brown, Stein, and Lee (215) demonstrated increased motor unit synchronicity in the first dorsal interosseous muscle of the hand after just 6 weeks of resistance training. Furthermore, Semmler and Nordstrom compared weightlifters, musicians, and untrained subjects, they found increased motor unit synchronicity in both the dominate and non-dominate hands of weightlifters but not musicians (272). Combined these findings suggest the repeated application of weight and not skill resulted in the observed neurological adaptations. Neuromuscular inhibition, the reduction in neural activation of muscles that activate from myotendinous feedback involuntarily in opposing action to voluntarily active muscle contractions, appears to be damped over repetitions of the same action (108). Aagaard et al. had 15 males perform 14 weeks of heavy resistance training and found significant declines in neural inhibition with concurrent increases in force production in quadriceps muscles. In particular, they found reductions of neuromuscular inhibition in vastus lateralis and vastus medialis with complete removal in the subject’s rectus femoris muscles. The researchers also reported increased electromyography of 21-52, 22-29, and 16-32% for the vastus lateralis, vastus medialis, and rectus femoris, respectively. Aagaard et al. concluded that down regulation of Ib afferent feedback resulted in the reduced neural inhibition and their observable increases in force production (3). We observed significantly increased MAFbx concentrations in the non-force measured gastrocnemius medial head with concomitant significant declines in concentrations in the quadriceps muscle group of the non-force measured leg. Regulation of E3 ligases occurs via transcription factors, such as, forkhead box O (FoxO) 3A found in the nucleus. FoxO3A has been shown to directly affect both muscle atrophy F-box 110 (MAFbx/atrogin-1) which is both enriched in skeletal, cardiac, and smooth muscle and has been shown to be elevated under atrophy conditions (34). Reduced expression of MAFbx/atrogin-1 occurs when its regulator FoxO3A is phosphorylated causing it to be trapped in the cytosol. In opposition to one another, Akt and AMPK have been shown to interact with FoxO3A. Specifically, Akt possesses the capability of phosphorylating FoxO3A (46) thereby inhibiting its activation while AMPK directly stimulates FoxO3A to translocate into the nucleus thus increasing the expression of MAFbx/atrogin-1 transcription (219). Our observations are likely due to the time course of MAFbx activity and where in its activity we harvested our sample. Since we homogenized tissue from the non-force measured leg the rise in MAFbx concentrations is not due to the simulation during force measurements. Instead, the concentrations of MAFbx suggest the differences in muscle group activations during the final bout of resistance exercise. Specifically, the gastrocnemius medial head had a greater concentration of MAFbx compared to control animals even though they had completed a resistance bout 72 hours prior, and which FoxO3A levels were no different from control. This observation is suggestive of the fact that the gastrocnemius medial head was not overly damaged during the final resistance training bout. Moreover, if this circumstance had persisted throughout the duration of training regimen it would have resulted in a marked decline in mass compared to control, which was absent. Therefore, resistance training induced sufficient protein synthesis rates to offset the degradation induced by MAFbx as observed by the lack of difference in muscle wet weights between treatments. Finally, the difference in its expression in gastrocnemius medial head and quadriceps muscles is likely due to the relative activity of the muscle during the resistance exercise. As the quadriceps possess a considerably greater CSA than the gastrocnemius medial head, it is within reason that the quadriceps activation was not

111 activated to same extent as that of the gastrocnemius medial head. Thus, a greater amount of MAFbx was released in the gastrocnemius compared to the quadriceps. In summary we compared sedentary rats and resistance trained rats with or without chronic HMB supplementation at 450 mg/kg/BW and found no effect of HMB in any parameter measured. We then collapsed our treatments and compared the results between sedentary and resistance treatments. We observed a significant difference in the force/per unit mass in both the triceps surae and quadriceps muscle groups representing an increase of strength without an increase in hypertrophy. Additionally, the amount of force generated by animals which exercised was significant compared to body weight. Thus, we conclude that our novel resistance exercise model was successful in diffusing the amount of weight placed on each muscle thereby limiting the stimulus for hypertrophy. Consequently, our observations in force generation are likely the result of a potent neurological adaptation. We conducted subcellular signaling protein investigations in muscles harvested 72 hrs after the final resistance training bout. We designed our study to examine the chronic effects of HMB and resistance training, therefore we timed our harvesting of muscle to reflect the chronic environment as opposed to the immediately post resistance exercise environment. Finally, our observations regarding protein signaling may alter with respect to both the time course of harvesting muscle post resistance exercise and the alterations in the timing window which occurs in response to chronic training.

112 Figure 4.13: Oral glucose tolerance test which occurred prior to training (A) , 5 weeks (B), and 11 weeks (C) into training.

A

140 120 100 80 60 40 20

Plasma Glucose (mg/dL) 0 0 D 15 D 30 D 60 D 90 D 120 Sampling Timpoints (Min)

CON EX

B

140 120 100 80 60 40 20 Plasma Glucose (mg/dL) 0 0 D 15 D 30 D 60 D 90 D 120

Sampling Timepoints (Min) CON EX

113 C

140 120 100 80 60 40 20 0

Plasma Glucose (mg/dL) 0 D 15 D 30 D 60 D 90 D 120 Sampling Time Points (Min)

CON EX

Note: CON = control rats, EX = resistance trained rats. No significant differences observed. Values are expressed as mean ± SD.

Figure 4.14: AUC calculations from oral glucose tolerance test which occurred prior to training (A) , 5 weeks (B), and 11 weeks (C) into training.

CON EX 14000

13500

13000

12500 (mg/dL x 120 min) x 120 (mg/dL

Plasma Glucose AUC 12000 OGTT 1 OGTT 2 OGTT 3 Oral Glucose Tolerance Tests

Note: CON = control rats, EX = resistance trained rats. No significant differences observed. Values are expressed as mean ± SEM.

114

Table 4.7 Effect of Resistance Exercise on Tissue Dry Weight/Wet Weight Ratio

CON EX CON EX Force Measured Leg Non-Force Measured Leg (M ± SEM) (M ± SEM) (M ± SEM) (M ± SEM) Soleus 0.24 ± 0.01 0.24 ± 0.01 n.s. Soleus 0.24 ± 0.01 0.25 ± 0.01 n.s. Plantaris 0.24 ± 0.01 0.24 ± 0.01 n.s. Plantaris 0.23 ± 0.02 0.21 ± 0.02 n.s. Gastroc. Medial Head 0.23 ± 0.01 0.21 ± 0.02 n.s. Gastroc.Medial Head 0.22 ± 0.01 0.21 ± 0.01 n.s. Gastroc. Lateral Head 0.21 ± 0.01 0.22 ± 0.01 n.s. Gastroc. Lateral Head 0.25 ± 0.04 0.25 ± 0.04 n.s. Quadriceps Muscle Group 0.23 ± 0.01 0.24 ± 0.01 n.s. Quadriceps Muscle Group 0.27 ± 0.01 0.26 ± 0.01 n.s. Biceps Femoris 0.22 ± 0.002 0.23 ± 0.002 n.s. Biceps Femoris 0.23 ± 0.01 0.23 ± 0.01 n.s. Semitendinosus 0.24 ± 0.01 0.19 ± 0.01 † Semitendinosus 0.27 ± 0.01 0.26 ± 0.01 † Semimembranosus 0.23 ± 0.01 0.21 ± 0.01 †† Semimembranosus 0.23 ± 0.01 0.23 ± 0.01 n.s. Gluteus Medius 0.22 ± 0.02 0.21 ± 0.01 n.s. Gluteus Medius 0.25 ± 0.01 0.23 ± 0.01 * Wrist Flexors 0.24 ± 0.01 0.25 ± 0.01 n.s. Wrist Flexors 0.24 ± 0.10 0.25 ± 0.10 n.s. Note: CON = control rats, EX = resistance trained rats. Values are expressed as the dry weight (mg) ÷ wet weight (mg); n = 6-10 per treatment. n.s. = nonsignificant (t ≤ 0.05). * Significant difference vs CON, t ≤ 0.05, † Significant difference vs. CON t ≤ 0.01, †† Significant difference vs. CON, t ≤ 0.001.

115 CHAPTER V

GENERAL DISCUSSION

Summary of Results

1) HMB failed to induce any significant effect on blood glucose, plasma insulin, or proteins of the Akt/mTOR signaling pathway, when gavaged at 150 or 300

mg/kg/BW in Sprague Dawley rats. However, at a dose of 450 mg/kg/BW, HMB, was capable of acutely increasing the phosphorylation of proteins in the Akt/mTOR signaling pathway indicative of inducing an increase in protein synthesis, but still had no effect on blood glucose. 2) Two different conditions of HMB were tested chronically. Using Fisher 344 Brown Norwegian rats given a dosage of 450 mg/kg/BW, they were either sedentary or resistance exercised. HMB failed to induce any effect on blood glucose during three separate oral glucose tolerance tests which were given prior to treatment, 5-weeks, and 11-weeks after daily dosing in either condition. 3) Finding no effect of HMB, in either sedentary or exercising conditions, for glucose tolerance testing, maximal force production, and muscle wet weights, we collapsed

our treatment groups to permit comparisons between sedentary and exercising treatment effects. 4) The novel resistance model which we designed to elicit a more diffuse force application to the body was successful in producing a 198% increase in lifted

weighted representing 427 ± 11.4% of their body weight. However, the resistance model did not elicit a hypertrophic response. Instead we observed the exercising animals completed the protocol with a significantly reduced body weight compared to sedentary animals.

116 5) Maximal force between sedentary and exercising animal triceps surae and quadriceps muscle groups were no different. However, when we controlled for the muscle weight of the animal performing the task we observed a significant increase in force per unit mass of the exercising animals in both the triceps surae and quadriceps muscle groups compared to sedentary animals. In addition, the force generated by the animals performing exercise was significant compared to their body weights relative to sedentary animals.

Conclusions

Our data clearly demonstrate that acute beta-hydroxy-beta-methylbutyrate supplementation was capable of increasing phosphorylation of proteins of the Akt/mTOR signaling pathway while not effecting blood glucose. However, chronic supplementation of beta- hydroxy-beta-methylbutyrate failed to elicit an effect on either blood glucose, muscle strength, phosphorylation of proteins within the Akt/mTOR signaling pathway, or increases in muscle wet weights. Furthermore, our novel resistance model was capable of increasing force per unit mass in both the triceps surae and quadriceps muscle groups. Therefore, we believe that a progressive resistance training program targeted at increasing the strength instead of hypertrophy of the individual would be of benefit with respect to the caloric demands needed to maintain an increase in hypertrophy.

Future Directions

Animal resistance training models which evoke a response from a greater amount of muscle mass will permit a wider applicability of their findings to the general population. Moreover, we believe that using our resistance training model and varying the angle of incline to maximize the hypertrophic response while ensuring the greatest amount of activated and contributing muscle

117 mass will be more beneficial to the scientific community than models which historically induce a response in single muscles or atypically trained muscles in humans. The effects of HMB have historically been associated with or in tandem with supplementation of various other nutrients. Our observations indicated that HMB alone under the conditions we have designed did not produce an effect. However, we did not calculate the daily caloric intake of our exercising rats with respect to the sedentary rats. Therefore, it would be of interest to determine if our resistance model produces similar HMB results in animals known to be consuming the same amount of calories per day.

118 APPENDICES

APPENDIX A: EXPANDED METHODS

Resistance Exercise and Gavage

Solutions:

1mg/mL stock solution of b-hydroxy b-methylbutyrate H2O

Procedure:

Once animals cleared quarantine, they were handled daily. After two days of handling, animals received daily oral gavages of 1mL H2O to familiarize the animals with the oral gavage technique and reduce stress response. Rats were trained in the dark with the light source for investigators being red lights to minimize circadian rhythm perturbations. Resistance trained rats were familiarized with a fabricated climbing ladder in addition to receiving the daily oral gavage. The climbing ladder had the following dimensions and construction design:

1.52m ´ 0.125m ´ 2.1336m 2-cm climbing bars distance

20° incline Pulley system using galvanized gauge wire with a stainless-steel pin&anchor shackle through a crown bolt to hold the weights at one end with a swivel hook to secure to animal at the other end Pulley was off-set by 0.152m to permit direct vertical pull of weight

119 Animals ran up the incline towards a housing chamber (20 ´ 25 ´ 40cm) that was blacked out Pre-measured ½” (~40g) and 3/8” (~10g) zinc plated washers were used as weights. The crown-bolt weight was ~150g. Animals were removed from their cages then weighed daily and returned to cages until their turn exercising. The volume of stock solution was calculated based off their weight and syringes were filled with a 1mg/kg dose of the appropriate fluid. To get familiar with the desired movement, animals were placed on the climbing ladder with no weights attached. Animals received encouragement to ascend the ladder quickly with their pauses in movement being rewarded with firm tail taps. After successfully ascending the ladder, the animals had a respite of 2 minutes before being placed back on the lowest part of the ladder to repeat the ascension process. This process was performed for a total of 10 completed ascensions on a single familiarization day, with 3 days of familiarization being used prior to weight attachment. Cohesive adherent tape was measured into 15cm lengths and cut in half ~1.5cm width. Exercising animals were removed from their cages gently and placed on a workstation. Their tails were wrapped in the cohesive adherent tape and a small swivel hook was threaded through the tape for a firm hold. The tape was labeled with the animals number and animals were placed into the housing chambers at the top of the ladder. The initial resistance weight was calculated as 75% of the animal’s bodyweight (BW) with subsequent weights at 100%, 150%, and 200% of the BW. The weight was placed on the crown bolt and the cable was secured using the swivel hooks. During familiarization, rats performed a single ascension at 75% BW followed by a 2 minute respite. This process occurred for 4 familiarization days with added weight (e.g., day 1 = 75%, day 2 = 100%, etc.). On the fifth training day with weight, once the animal reached 200% of their BW as a resistance weight, subsequent weights of ~40grams were attached to the crown bolt for the animal

120 to pull (i.e. 200% +40g, 200% +80g, etc.). This process continued until failure, which was defined as refusal to perform the movement following repeated encouragement with no progression in ascension or an inability to hold onto the bars with continual slippage down the ladder. Failure typically occurred between 4-10 full length ladder repetitions. The next training bout (6th à n) used the maximum weight of the previous training bout to re-calculate 50%, 75%, 90%, and 100%. Once animals attained a pull weight equal to 3x BW, it was observed that animals failure rates increased dramatically. We amended the distanced pulled to be 0.75m with 3-7 dynamic pulls. Once animal had completed their repetitions for the day, they were quickly removed from the ladder, the cohesive adhesive tape was removed, and they were gavaged with the treatment solution. After gavage the animals were returned to their respective housing cages and monitored for 30 minutes. Resistance training occurred for 16 weeks with a total of 37 training sessions.

Muscle and Tissue Removal and Storage

Solutions: 1X Dulbecco’s Phosphate Buffered Saline (DPBS)

Procedure: Prepare workstations for tissue harvest. Pre-cut foil into ~2.5 inch squares with triple folds at edges to ensure a strong cavity. Label all cassettes for histohemistry. Label freezer bags for each muscle and muscle type. Label must include: Date, Muscle Name, Basal (non-force measured) or Force (measured limb), Animal Number, and Section (to be used for histochemistry).

121 Suspend ~50mL of isopentane in a hanging dish over a pool of liquid nitrogen. The isopentane can at times freeze solid, this can be reverse by removing it from liquid nitrogen for a minute, then return it back to liquid nitrogen to prevent it get too warm. Get ice and place a glass plate on the surface. Quickly excise muscles from the animal and transfer to glass plate and using DPBS wipe off any blood from muscle. Using a freshly tared weigh-boat, remove the muscle from the glass plate using forceps, blot off excess liquid using Kim wipes, and weigh muscle. Record the weight into the prepared Excel spreadsheet or printed spreadsheets. Remove muscle from the balance and return to glass plate. Locate midline of muscle and make a single fluid cut with scissors to transect the muscle into two parts. Cut a piece of muscle ~1-2 mm in thickness to be then transferred into a cassette, then submerge it in liquid nitrogen- cooled isopentane. Continue this process for remaining muscles. Pay special attention to balance and ensure all weigh-boats are tared for accurate weight measurements. After collecting all muscles, the heart, adrenal glands, and epididymal fat was removed, weighed, and the data recorded for later analysis. Once all samples are finished being processed, remove them from liquid nitrogen and place them into properly labeled freezer bags. Place freezer bags into a -80º C storage box for later processing. Dispose of weigh-boats, turn off balance, pour remaining isopentane back into original container (can be reused many times), pour remaining liquid nitrogen back into original container, and prepare animal for proper disposal. Update surgical records and at end of experiment notify EHS for pickup and disposal of carcasses.

122 Muscle Homogenization

Solutions: 1 M HEPES (pH 7.6; NaOH) 5 M NaCl 10% Triton X-100 1 M β-glycerol phosphate 1 M NaF 100 mM PMSF (Store these at 4º C; may be made on prior date; check viability of chemical storage prior to use)

Procedure: This homogenization buffer is specific for determining phosphorylated states of proteins other goals may require alternative buffers. Before homogenization procedure, calculate an estimated volume of buffer to be required. If more than 50 mL is required, prepare only 50 mL at a time. Keep buffer on ice for duration of homogenization procedure. 1.) Prepare all stock solutions and store at 4º C. Gather all materials needed; label all microcentrifuge tubes (you will need at least 5 for each sample) properly. Begin icing dH2O, 50 mL conical tube, microcentrifuge tubes, homogenation tubes, Teflon pestals (or polytron rotor). It is essential that all materials are kept as cold as possible throughout entire homogenization process. 2.) Mix the pre-lysis buffer as indicated below:

Pre-Lysis Buffer [Final] [Stock] For 50 mL 50 mM HEPES (pH 7.6; NaOH) 1 M 2.5 mL 150 mM NaCl 5 M 1.5 mL 1% Triton X-100 10% 5.0 mL 20 mM β-glycerol phosphate 0.5 M 1 mL 10 mM NaF 1 M 0.5 mL

123 dH20 32.5 mL dH20

*this solution can be mixed prior to day of homogenization

Immediately before beginning homogenization, add the following ingredients:

Protease Inhibitors (add fresh) [Stock] For 50 mL 1 mM Na3VO4 10 mM 5 mL 10 ng/mL Leupeptin 1 mg/mL 500 uL 10 ng/mL Aprotinin 2 mg/mL 250 uL 1:100 Phosphatase Inhibitor Cocktail 1 500 uL 1:100 Phosphatase Inhibitor Cocktail 2 500 uL 1 mM PMSF 100 mM 500 uL

*Leupeptin must be mixed from powder form prior to addition

3.) Install either Teflon pestal in drill press or rotor to polytron; surround with ice. Make sure all equipment is working.

4.) Remove 3-4 muscle samples at a time from -80º C freezer and place in ice.

5.) Remove 1 sample from ice, carefully remove foil, and excise ~200 mg of connective tissue- free muscle tissue sample. Rewrap remaining muscle sample in foil and refreeze in liquid nitrogen.

6.) Record weight of excised muscle in laboratory notebook, mince muscle with scissors, and transfer to cold homogenization tube. Add homogenization buffer to muscle in a 15:1 volume to mass ratio (i.e. 0.2 g x 15 mL/g = 3 mL of buffer).

7.) Remove ice surrounding Teflon pestal or polytron rotor. Keep homogenization tube surrounded by ice and carry to drill press or polytron.

8.) For Teflon pestal: Turn-on drill press, slowly raise homogenization tube until pestal reaches bottom of tube, then pull back down until suction is breached. After 5 passes, place tube back in ice to cool, then perform 5 more passes. Repeat this until all major portions of muscle are absent from homogenate. For polytron: Raise homogenization tube until rotor is in contact with bottom of tube.

Turn-on polytron and set at desired speed for 10 seconds. After 10 seconds, turn-off polytron and place tube in ice. Repeat if necessary.

124 9.) Allow homogenate to sit in ice to let foam settle. During this time wipe Teflon pestal with

Kim wipe, or manually remove connective tissue from polytron rotor and run at high speed in clean, cold dH2O, then replace ice until next run.

10.) Distribute homogenate evenly between 2 (more may be necessary) pre-chilled, properly- labeled microcentrifuge tubes. Aliquot 100 uL into a 3rd tube. This portion will be used to determine total protein content of muscle, therefore can be stored at -80º C at this point. Place all other tubes on ice.

11.) Repeat starting at step 5 until all samples are homogenized, then repeat starting at step 4 until no more than 32 microcentrifuge tubes are prepared.

12.) Make sure centrifuge is working properly, place samples into F-20/MICRO centrifuge rotor head in a manner that keeps balance across the head, and secure the lid. Set the rotor speed to

9650 rpm (12000g; RCF = 0.00001118r N^2) and run for 30 minutes. Make sure the temperature is at

4º C.

13.) Remove centrifuged samples from head and return to ice. Remove lipid layer with micropipette and dispose. Carefully transfer supernatant into a clean, chilled, properly-labeled microcentrifuge tube, without disturbing the pellet.

14.) Store homogenates in a properly labeled container in -80º C freezer until further needed.

Protein Concentration Determination

Solutions:

20% solution of Bio-Rad Protein Assay dye reagent (can be stored at 4º C for 2 weeks)

10 mg/mL BSA

Procedure:

Remove samples from -80º C freezer and place in ice to allow a slow thaw. Properly label all tissue culture tubes that will be needed for entire procedure (one for each sample + 4 for BSA standards

+1 for blank). Turn on spectrophotometer and set wavelength to 595nm.

125 Make serial dilutions of 10mg/mL BSA such that you end with dilutions of 1.25, 2.5, 5, and 10 mg/mL. Aliquot 2.5 mL of diluted protein assay solution into 5 tissue culture tubes (one will be your blank the other 4 will be for your BSA standards). Add 5 uL of each BSA standard into its respective tissue culture tube; add nothing to the tube designated as the blank. Vortex each tube after protein addition; solution should turn from dark red to blue; intensity of blue should increase with increasing protein concentration. Let sit for 5 minutes before reading absorbance. Signal will deteriorate after 30 minutes.

Vortex blank briefly and carefully transfer to an empty, clean quartz cuvette. Wipe the sides of the cuvette carefully with a Kim wipe, place blank into the spectrophotometer in the correct orientation, and “Blank” the system. Repeat to take sequential measurements of BSA standards and record the absorbance. Dispose of BSA standards and retain blank in cuvette, as it will be used for the duration of this procedure. Dispose of used cuvettes and tissue culture tubes properly when finished.

Open an Excel spreadsheet and plot your BSA standard data as absorbance vs. [BSA]. Generate a scatter plot and run a linear regression analysis to obtain the equation and R2 value. If your R2 is above 0.98, continue to next step; if not, repeat, starting at step 3. BSA standard solutions may be stored in freezer for up to 1 year.

Check homogenized samples on ice. If fully thawed, prepare tissue culture tubes with 2.5 mL protein assay solution. If not fully thawed, DO NOT agitate samples, this will cause proteins to fractionate. Take thawed sample from ice, vortex briefly , and aliquot 5 uL into proper tube containing protein assay solution. Quickly return sample to ice and vortex tube. Repeat for each sample. Repeat for each sample. Prepare not more than 10-15 samples or the number of samples that can be run in the spectrophotometer at one time to prevent possible deterioration of signal. Return homogenates to -80º

C as soon as possible.

“Blank” spectrophotometer with blank and begin taking absorbance measurements of homogenate samples. Record the measurement for each sample in your lab notebook or excel spreadsheet. Dispose of all waste properly.

126 With the recorded absorbance data, return to the Excel spreadsheet with the BSA standard curve to calculate protein concentration of each homogenate. The equation for the standard curve will be in the form of y=mx+b, where y is the absorbance (Abs;AU) and x is the protein concentration ([P]; mg/mL). Rearrange equation to read [P] = (Abs-b)/m. Plug in absorbance values to determine respective protein concentrations. Record in lab notebook and create a table detaining this data.

Manipulate [P] data to determine proper lading amounts of sample for 100ug protein content using 2X Laemmli’s Buffer.

Sample Preparation

Obtain ice, properly label microcentrifuge tubes, and begin to heat beaker of H2O. Remove protein samples from -80 ºC freezer and place on ice to slow thaw.

Transfer appropriate amount of 4X Laemmli’s sample buffer to each tube. When protein samples are completely thawed, vortex protein sample, and transfer sample to tube in a 3:1 ratio (i.e.

25 uL of 4X sample buffer + 75 uL of protein sample = 100 uL of prepared sample). Quickly return original samples to freezer.

Make sure H2O bath is boiling (95-100 ºC) . Place samples in sample floater and put in boiling water for 5 minutes. Ensure caps are secured with a weight to prevent popping and potential intrusion by water bath.

Remove from H2O after 5 minutes. Sample can be loaded into a gel when cooled to room temperature, or can be frozen for future use.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Solutions

10X Running Buffer (2L)

127 Reagent Amount (g)

Trisbase 60.56

Glycine 288.4

SDS 20

Add reagents into 1.5L ddH2O stirring on a magnetic plate and bring to the volume with ddH2O Procedure:

Obtain ice, remove protein samples from -80 ºC freezer and place on ice to slow thaw.

Assemble the Mini-Protean® apparatus. Open Bio-Rad Mini-Protean® TGX gels and place appropriate side against rubber gasket. Ensure that the bottom of the gels are flush with the plastic bottom of the Mini-Protean® apparatus to prevent leakage of inner chamber fluid.

Add 1L 1X running buffer to inner chamber so that all wells are filled and flushed with the fresh 1X running buffer. Keep pouring the remaining buffer into the inner chamber to remove as many bubbles as possible.

Place 7uL of Precision Plus ProteinTM Dual Color Standards and 5uL Precision Plus ProteinTM

WesternCTM Standards to lanes 1 or 10. Both standards can be added to a single lane.

Load 100ug prepared standard and samples into correct lanes. Record this arrangement in lab notebook.

Once all lanes are loaded place cover securely over the banana plugs of the Mini-Protean® apparatus aligning black to black and red to red.

Turn ON power supply and set to constant current, 80V and run for 147 minutes. This was the ideal voltage and run time using the power supply, however, the voltage and time can vary and should be determined for best protein band spread (using the standards as an indicator) and preventing

“smiling” bands which are the result of a voltage set to high.

128 Transferring to Nitrocellulose Membrane

Solutions:

Wet-transfer Buffer (2L for 2 tanks, Modified Towbin Buffer) Reagent Amount (g) Glycine 28.8 TrisBase 6.04 Methanol 300 mL SDS 0.75

Add reagents in order into 1400mL ddH2O while stirring on a magnetic plate. Bring the volume into 2L with ddH2O.

Procedure: Thirty minutes prior to the electrophoresis completion, prepare for wet-transfer. Begin by preparing fresh transfer buffer as described above. Cut 9x6 cm rectangular Nitrocellulose Membranes, label them appropriately, and place membranes into ~50 mL of the fresh transfer buffer on a rocker for a minimum of 5 minutes to equilibrate. Get ice or use the previous ice, transfers produce heat and so the ice will mitigate the heat produced and allow for a more efficient transfer. Place Mini-ProteinTM tanks into the ice and stabilize them so they do not slide around. Fill tanks 60% with fresh buffer and place transfer carriage into the tanks. Fill a small basin with fresh transfer buffer to be used to assemble transfer sandwich. Carefully separate casing plates using appropriate tool, cut off stacking gel and place resolving gel in fresh wet-transfer buffer for 5 minutes to permit equilibration. Soak four of the spongy pads and 4 pieces of extra thick blot paper (2 for each gel) until saturated. Assemble the transfer sandwich:

129 Place cassette in the basin filled with transfer buffer, black side down. From the bottom to the top the sandwich is as follows: Spongy pad Extra thick blot paper Gel Nitrocellulose membrane Extra thick blot paper Spongy pad Ensure that all air bubbles are pressed out using either a roller or a fleaker on its side. Close cassette Keeping the cassette submerged, move to where you have placed the tank and rapidly transfer the cassette to the transfer carriage with the clear side facing towards the red and black side facing the black back. Place the cover over the corresponding colored banana plugs (black to black, red to red). Surround the transfer tank with ice. The running voltage and time vary depending on the protein kD and are as follows:

Protein( kD) Voltage (V) Duration (min) 40-45 90 42 60 90 or 100 75 or 60 85-100 90 or 100 90 or 65

Immunoblotting

10X TBST (2L, pH 7.4) Reagent Amount (g) TrisBase 24.2 NaCl 175. 36 Tween 20 12 mL

130 Add the reagents into 1.5L ddH2O while stirring on magnetic plate and bring to the volume. Bring pH down to 7.4 using 12M HCL.

Blocking – 5% BSA, 30mL per membrane Reagent or Solution Amount (mL or g) TBST 30 BSA 1.5

*To be used for phosphoproteins

Blocking – 5% Non-Fat Dry Milk (NFDM), 30mL per membrane Reagent or Solution Amount (mL or g) TBST 30 NFDM 1.5

*To be used for non-phosphoproteins

Procedure: During transfer prepare 1X TBST

Add 100mL 10X TBST into 900 mL ddH2O while stirring on a magnetic plate. Prepare blocking solution as described above ensuring you prepare appropriate blocking for phosphorylated protein probing. It is acceptable to go as high as 7% blocking concentration. Use pilot blots to indicate if blocking should exceed 5%. Once transfer is complete, remove sandwich and place gel/nitrocellulose membrane (likely adhered to one another) into a small amount of transfer buffer. This will permit a complete separation of the adhering items. The gel MUST be complete removed from the membrane to prevent antibody trapping. After separated place membrane into ~50mL Ponceau S solution for 15-30 seconds to stain proteins and ensure proper transfer.

131 Remove membrane from Ponceau S solution and place into a clean container and add ddH2O until the membrane is covered. Agitate with vigorous shaking for 10 seconds and remove ddH2O, now stained red. Photograph membrane and trim membrane to remove excess. Place trimmed membrane into container with appropriate blocking solution and place on rocker for 1 hr with gentle agitation. Blocking can be left over night if needed. During blocking, prepare primary antibody solution. This solution can be used more than once, no more than 5 times, and must be kept at 4ºC for periods less than a week or -20ºC if the period of use exceeds 1 week. The primary solution should be made in accordance with manufacturer’s recommendations, typically in a TBST solution containing 5% BSA at concentration of 1:1000. This dissertation utilized a concentration of 1:500 for non-phosphorylated proteins and a 1:250 concentration for phosphorylated proteins. The concentration needed varies by lot and manufacturer and should be piloted to determine the best concentration. The volume of working primary antibody for this dissertation was 10 mL per membrane per antibody.

After blocking is complete, decant blocking and add 25mL of TBST to membrane and place back on rocker with medium agitation for 5 min. After 5 min, decant TBST wash, place container on side to remove as much TBST as possible. Place 10mL of primary antibody onto membrane and return to rocker with a gentle agitation for 18.5 hours (variable and dependent on image quality) and place at 4ºC. After primary antibody incubation, collect primary to be re-used, and wash membrane 3 x 5min in 15 mL TBST with medium agitation on a rocker. During washes prepare secondary antibody at a concentration of 1:2000 in TBST. Pay close attention to reacting conjugant species. Prepare 10mL per membrane. The solution contains only the antibody (5uL) and 10mL TBST.

132 After final wash, add 10mL secondary antibody solution to membrane and incubate for 1.25 hrs or longer depending on image quality (up to 2 hrs can be done successfully) at room temperature with gentle agitation on a rocker. After incubation, wash membranes 3 x 5 min with 15 mL TBST. Visualize protein bands using an ECL detection kit and Bio-Rad ChemiDoc detection system according to manufacturer’s instructions. Quantify the density of the bands using provided software from Bio-Rad. This dissertation utilized Quantity One Analysis software, current software is Lab View both are Bio-Rad specific software.

In situ Force Measurements

Make an incision around the ankle of the anesthetized animal being careful not to damage the

Achille’s tendon. Begin to separate the skin and connective tissue from the musculature as you cut up from the ankle to the midline of the animal.

Once the hindlimb has been skinned, begin to remove the connective tissue around the patella.

Upon completion of the cleaning, position the knee on a cork board bent to a 90° angle to allow exposure to the femoral condyles.

After positioning the knee, using a 1/16” drill bit, drill through the condyles of the femur. You must ensure that the drill line is perpendicular to the shank. After the drill is placed, remove it from the drill and leave the bit in the condyles for the duration of the in situ measurements. Any bleeding must be stemmed and cauterized.

Carefully expose the medial musculature of the upper leg by removing the connective tissue covering the quadriceps and hamstrings. Then bend the knee and using small scissors make an incision along the lateral aspect of the femur to free the vastus lateralis from the lateral aspect of the hamstrings.

Perform a similar incision on the medial aspect of the femur freeing the vastus medialis from the adductors.

133 Isolate the femoral nerve, paying special attention to avoid stretching or damaging the nerve in any way. The nerve is encased is several layers of connective tissue and each layer needs to be pulled back and removed to free the nerve and create a pocket underneath for the stimulator to slide into and thus be able to stimulate only the nerve.

Once the quadriceps are freed medially and laterally, using a hemostat pinch a small length of

2/0 black braided silk under the patellar ligament followed by bone cutters cutting a shallow piece of the tibia to be used as an anchor. Holding the braided silk, lift the patella to expose the vastus intermedius and cut it free using small scissors. The cut should be approximately 1/3 the length of the femur (if you cut to long, you risk completely cutting the vastus intermedius free and rendering its contribution of force production as null).

After freeing the quadriceps from the femur, place a braided fishing line with a looped knot around the piece of tibial bone and cinch it down tight.

Carefully make an incision along the medial and lateral aspects of the shank to free the gastrocnemius from the tibia.

Using a hemostat, pinch a piece of 2/0 braided black silk behind the Achille’s tendon and secure with a knot. Cut the calcaneus. Transfer the animal from the surgical area to the force measurement area and place a cross bar across the hips of the animal as it lays in the supine position. The placement of the bar correctly will influence force of the rectus femoris (bi-articulate) and ensure force is not lost through hip movement.

Using the tibial bone piece as an anchor, attach the quadriceps to the lever arm of the dual- mode servomotor (Cambridge Technologies, model 301 LR) via the braided fishing line.

Stimulate the muscle to contract using the stimulator (A-M Systems, model 2100) by placing electrodes under the femoral nerve such that it is held in suspension by the stimulator hooks.

Keep the muscle warm and moist using 37.5 C mineral oil and a radiant heat lamp.

Determine optimal length by finding maximum twitch force using a stimulation of 0.5 Hz.

Stimulate muscle at 150 Hz to obtain maximum peak tetanic contraction. Allow muscle to rest for 2 minutes following each contraction. Record data.

134 Once the quadriceps data is recorded, place the animal in a prone position and attach the triceps surea muscles via the calcaneus piece and braided fishing line.

Stimulate the muscle to contract using the stimulator (A-M Systems, model 2100) by placing electrodes on the tibial nerve.

Keep the muscle warm and moist using 37.5 C mineral oil and a radiant heat lamp.

Determine optimal length by finding maximum twitch force using a stimulation of 0.5 Hz.

Stimulate muscle at 150 Hz to obtain maximum peak tetanic contraction. Allow muscle to rest for 2 minutes following each contraction. Record data.

Tissue Sectioning

Set the cryostat specimen head temperature at -20 degrees Celsius.

Frozen tissue was transported from the -80°C freezer placed within the cryostat chamber.

Using single-edge razor blades, the portion of the tissue that is important for analysis is cut off before mounting onto specimen disks for sectioning.

Mount tissue onto specimen disks using optimal cutting temperature compound (OCT).

Insert specimen disk into specimen head and orient specimen head if necessary.

Initially adjust base of the blade holder to bring blade close to tissue using coarse feed settings and handwheel.

Begin sectioning tissue; ensuring sections are sliding under the anti-roll plate.

Gently place sectioned tissue from blade holder using a microscope slide; two tissue sections are placed on each microscope slide.

Appropriately label microscope slide and place in slide holder for future use.

Tissue Dry Weight Prepare a spreadsheet with the current weight of the frozen sample, the size of muscle to be removed, and the new total for the remaining muscle. Collect liquid nitrogen in two dours with equal volume.

135 Fill one dour with all the muscles of a single muscle type to be weighed. The foils need to be submerged in the liquid nitrogen. Label small pieces of foil with the animal number and muscle. Taking forceps, select a single foil and remove the sample from the liquid nitrogen. Place the foil containing the sample on a piece of foil laid out on the workbench and remove the muscle inside. Tare a weigh-boat on the balance. Quickly, using a fresh razor blade, slice ~50-80mg of tissue. Then return the muscle to its storage foil and place in liquid nitrogen. Using the back end of a Sharpie marker, fold the foil around it creating a small vessel to hold the muscle sample. Place the folded foil in a microcentrifuge tube block. Continue this process until all samples of the muscle group are processed.

Place the microcentrifuge block into the oven which is at 95°C. Continually check on the samples until they appear dehydrated. Then remove from oven and weigh. This typically takes a minimum of 3 days but can take as long as 5 days. Ratios of wet to dry weight were then determined.

Glucose Tolerance Test Food was removed from the animals 12 hours prior to the glucose tolerance test. A 1mg/mL glucose solution was prepared the day before and keep in the refrigerator. The animals to be assessed were removed from their cages the day of and placed into a separate area and their bodyweight was obtained. The amount of necessary glucose solution was calculated and loaded into the gavage syringes. The glucose bolus was gavaged at a concentration of 1.0g/kg bodyweight of glucose via a feeding bulb. Six microcentrifuge tubes were labeled appropriately and placed on ice and filled with 0.1 mL of 24 mg/mL EDTA (pH 7.4). Animals were placed on a heating pad 2 minutes prior to blood collection.

136 Animal tails were cut with a sterilized razor and 0.5 mL whole blood was extracted into micro-centrifuge tubes. Blood sampling occurred prior to glucose administration (time point 0) to determine baseline plasma glucose measurements using a Freestyle glucometer. The baseline had two measurements, if a 10% difference between samples was observed this required a third measurement. Sampling timepoints following the glucose bolus occurred at 15, 30, 60, 90 and 120 minutes post glucose administration. All plasma glucose measurements were taken using a Freestyle glucometer. Following the final blood collection animal tails were cauterized and returned to their home cages. Animal health was assessed 1 hr. after being returned to their home cages.

Insulin Tolerance Test Food was removed from the animals 12 hours prior to the glucose tolerance test. The animals to be assessed were removed from their cages the day of and placed into a separate area and their bodyweight was obtained. A 0.1 U/kg Humulin insulin dose was loaded into syringes calculated from the animals bodyweight. Six microcentrifuge tubes were labeled appropriately and placed on ice and filled with 0.1 mL of 24 mg/mL EDTA (pH 7.4). Animals were placed on a heating pad 2 minutes prior to blood collection. Animal tails were cut with a sterilized razor and 0.5 mL whole blood was extracted into micro-centrifuge tubes. Blood sampling occurred prior to Humulin insulin administration (time point 0) to determine baseline plasma glucose measurements using a OneTouch glucometer. The baseline had two measurements, if a 10% difference between samples was observed this required a third measurement.

137 After baseline was determined, the animal received the appropriate dose of Humulin insulin via intraperitoneal injection. Sampling timepoints following the IP injection occurred at 15, 30, 45, 60, 90 and 120 minutes post glucose administration. Following the final blood collection animal tails were cauterized and returned to their home cages and provided food. Animal health was assessed 1 hr. after being returned to their home cages.

Akt/mTOR Phosphoprotein & Total Protein 11-Plex Magnetic Bead Kits 1) Generate a 96-well plate map, include standards, quality control, and muscle sample orientations.

2) Add 50µL Assay Buffer per well. 3) Shake 10 minutes at room temperature 4) Decant buffer

5) Add 25µL 1X beads to wells. Add 25µL Assay Buffer to the blank well. Add 25µL control and sample lysates (in duplicate) to appropriate wells

6) Incubate overnight (16-20 hours) at 4°C with shaking in the dark. 7) Wash 2x with 100µL Assay Buffer. Then add 25µL 1X detection antibody. 8) Incubate 1hr at room temperature with shaking in the dark.

9) Remove Detection Antibody and add 25µL 1X Streptavidin-PE (SAPE) 10) Incubate 15 minutes at room temperature with shaking in the dark.

11) No removing SAPE, ADD 25µL Amplification Buffer 12) Incubate 15 minutes at room temperature in the dark.

13) Remove SAPE/Amplification buffer and resuspend beads in 150µL Assay Buffer. 14) Read results using appropriate Luminex® instrument. 15) Remember to use the magnet during every wash step to ensure that the beads are not lost during the decanting process!

138

Rat Insulin RadioimmunoAssay (RIA)

Procedure: Rat plasma insulin was determined using an RIA method (#RI-13K, Millipore, Millipore

Corporation, MA). 125I-labeled insulin was added to glass tubes (12 x 75 mm) containing standards, controls, or plasma samples. Rat insulin antibody was added to the tubes containing standards or samples. All tubes were incubated at 4°C overnight (20-24 hours). During that time, 125I-labeled and unlabeled insulin competed for binding sites on the antibody. Thus, the amount of tracer bound to antibody was decreased as the concentration of unlabeled antigen increased. The next day, the antigen-antibody complex was precipitated by adding precipitating solution, incubating the tubes, and centrifugation. Once the supernatant was poured off, the tubes were counted for 2 min in a gamma counter (Perkin Elmer life sciences, Turku, Finland). A standard curve was generated by the counter and used to calculate sample concentration. In study 1, the insulin in plasma samples was measured in duplicate. If the difference between duplicates was greater than 10% CV, the sample was reanalyzed. The fasting level of insulin in the rat is generally in a range of 0.5-2.0 ng/ml.

139 APPENDIX B: RAW DATA CHAPTER III Plasma Glucose During OGTT 1 (15 min HMB delay) (mg/dL) CON D0 D15 D45 D75 D105 D135 1 121 142 136 135 141 131 5 108 117 121 128 123 123 9 92 110 181 136 113 131 13 100 133 118 109 120 116 17 128 136 130 129 127 136 21 118 148 137 127 130 142 25 116 146 139 130 112 118 29 75 9 156 105 103 104 LOW 150 mg/kg HMB 2 124 163 116 128 125 124 6 99 116 152 128 122 112 10 101 119 164 156 124 115 14 96 119 129 106 114 99 18 118 151 119 117 115 113 22 85 107 159 143 117 117 26 99 134 213 140 132 125 30 99 109 158 106 101 91 NORM 300 mg/kg HMB 3 122 137 144 122 109 108 7 96 115 114 121 126 142 11 120 129 139 113 113 107 15 85 111 173 125 113 105 19 97 118 177 141 130 126 23 124 157 141 149 136 129 27 117 127 165 111 102 111 31 89 111 146 114 103 108 HIGH 450 mg/kg HMB 4 111 118 136 104 109 105 8 105 113 176 136 137 129 12 107 126 130 119 122 101 16 80 103 173 110 104 99 20 90 107 175 153 139 122 24 132 143 123 147 131 135 28 78 103 223 116 121 120 32 105 132 141 133 115 115

140 Plasma Glucose During OGTT 2 (150 Min Delay) (mg/dL) CON D0 D150 D180 D210 D240 D270 1 104 96 145 118 118 124 5 97 88 187 134 138 124 9 ------13 80 91 138 117 102 108 17 101 90 143 113 110 126 21 154 112 170 144 143 139 25 96 104 179 134 126 131 29 90 95 192 131 131 136 LOW 150 mg/kg HMB 2 117 96 134 117 117 113 6 90 82 174 132 110 111 10 111 74 168 145 107 103 14 91 88 141 113 97 104 18 72 78 183 124 126 126 22 122 108 123 124 124 120 26 89 83 157 129 121 113 30 99 108 188 149 149 136 NORM 300 mg/kg HMB 3 108 95 140 115 115 109 7 101 91 137 138 127 135 11 97 87 193 113 104 102 15 83 75 138 130 103 103 19 120 90 147 123 124 117 23 110 96 173 143 125 127 27 110 99 127 131 124 117 31 89 11 160 143 132 133 HIGH 450 mg/kg HMB 4 103 90 182 123 102 103 8 77 78 185 121 112 103 12 88 68 124 116 104 97 16 79 96 164 163 140 133 20 ------24 134 109 142 153 124 125 28 81 78 167 150 137 147 32 117 94 153 135 124 128

141 Plasma Glucose During ITT - (150 Min Delay) (mg/dL) CON D0 D150 D180 D210 D240 D270

1 5 105 114 123 114 163 161 9 135 100 152 147 130 137 13 118 133 159 114 130 133 17 102 88 117 134 124 123 21 120 101 117 133 141 151 25 91 85 108 97 106 83 29 72 80 84 60 65 66 LOW 150 mg/kg HMB 2 6 87 101 111 86 91 107 10 132 163 190 168 200 254 14 79 89 97 95 82 74 18 87 89 99 119 98 131 22 90 83 88 43 37 43 26 88 75 108 93 83 65 30 82 76 100 109 116 120 NORM 300 mg/kg HMB 3 7 93 90 96 96 97 106 11 97 89 113 81 67 61 15 88 87 78 70 75 69 19 112 97 134 123 108 107 23 89 84 95 88 75 69 27 95 88 92 68 69 81 31 90 83 97 115 130 122 HIGH 450 mg/kg HMB 4 8 100 86 95 72 91 117 12 98 92 99 77 69 80 16 76 92 110 99 116 106 20 127 133 129 128 138 165 24 112 83 118 83 85 77 28 80 92 105 89 69 67 32 109 92 120 125 128 132

142 Plasma Insulin Concentration (pM) During - OGTT1 (15 Min Delay) Animal D0 D15 D45 D75 D105 D135 Number Treatment 1 CON 112.259 95.730 153.581 92.975 29.270 40.634 5 CON 42.700 279.614 140.840 57.507 58.540 88.154 9 CON 12.397 40.634 35.468 62.328 47.521 22.383 13 CON 139.463 202.824 329.890 80.234 172.865 75.069 17 CON 391.185 277.893 241.391 115.702 15.840 28.926 21 CON 54.408 356.749 240.358 67.837 80.234 42.011 25 CON 26.171 99.518 207.300 18.595 16.873 14.118 29 CON 0.000 3.444 176.309 4.821 0.000 13.774 2 150 mg/kg BW HMB 5.510 0.000 3.099 5.854 15.152 2.755 6 150 mg/kg BW HMB 20.317 115.358 99.174 26.171 90.565 35.813 10 150 mg/kg BW HMB 206.956 140.840 270.317 228.650 77.135 79.201 14 150 mg/kg BW HMB 146.350 165.978 212.810 104.683 73.347 34.435 18 150 mg/kg BW HMB 15.840 25.138 10.331 21.006 16.873 0.000 22 150 mg/kg BW HMB 13.085 2.066 18.595 12.397 0.000 8.264 26 150 mg/kg BW HMB 0.000 4.132 120.523 0.000 12.052 4.821 30 150 mg/kg BW HMB 108.471 10.331 99.174 15.152 12.397 14.807 3 300 mg/kg BW HMB 68.526 29.959 137.741 37.879 64.394 116.736 7 300 mg/kg BW HMB 172.865 80.234 123.278 62.672 68.871 151.515 11 300 mg/kg BW HMB 0.000 407.369 624.311 500.000 234.160 122.590 15 300 mg/kg BW HMB 168.733 120.179 342.975 140.152 59.917 152.893 19 300 mg/kg BW HMB 0.000 22.039 34.780 11.708 7.576 4.821 23 300 mg/kg BW HMB 201.102 130.854 119.146 82.300 112.603 48.209 27 300 mg/kg BW HMB 66.116 3.444 254.132 13.774 6.887 58.540 31 300 mg/kg BW HMB 86.088 161.501 283.058 124.656 276.171 269.972 4 450 mg/kg BW HMB 10.331 47.176 46.143 22.383 9.986 0.000 8 450 mg/kg BW HMB 46.488 83.678 146.006 56.129 59.229 57.163 12 450 mg/kg BW HMB 130.165 272.039 272.727 410.468 219.697 105.372 16 450 mg/kg BW HMB 126.377 178.719 569.215 85.055 76.791 71.970 20 450 mg/kg BW HMB 12.052 0.000 60.606 27.204 0.000 0.689 24 450 mg/kg BW HMB 175.620 183.884 60.262 69.215 60.606 29.959 28 450 mg/kg BW HMB 0.000 33.058 81.956 1.377 0.000 #DIV/0! 32 450 mg/kg BW HMB 402.548 451.102 453.512 386.708 311.295 372.590

143

Plasma Insulin Concentration (pM) During- OGTT2 (150 Min Delay) Animal D0 D15 D45 D75 D105 D135 Number Treatment 1 CON 266.873 288.912 391.529 370.868 344.697 305.785 5 CON 185.606 301.653 336.777 212.810 250.344 210.399 9 CON 89.532 213.154 259.986 163.223 105.372 #DIV/0! 13 CON 150.482 135.331 217.975 188.705 132.576 146.694 17 CON 127.410 180.785 310.606 197.314 101.240 129.477 21 CON 688.705 697.658 764.463 543.733 419.077 404.614 25 CON 183.884 201.102 329.890 263.430 185.262 221.074 29 CON #DIV/0! 74.725 189.050 112.948 120.868 97.107 2 150 mg/kg BW HMB 196.970 210.399 358.127 264.463 196.970 262.741 6 150 mg/kg BW HMB 127.755 90.220 193.526 138.085 373.623 147.383 10 150 mg/kg BW HMB 234.504 107.094 433.196 264.807 151.859 186.295 14 150 mg/kg BW HMB 124.656 137.741 166.322 184.917 155.647 78.168 18 150 mg/kg BW HMB 105.716 68.871 377.410 161.157 198.691 197.658 22 150 mg/kg BW HMB 341.942 224.862 274.449 258.609 200.758 184.917 26 150 mg/kg BW HMB 150.826 139.463 346.074 155.647 130.165 128.444 30 150 mg/kg BW HMB 140.840 234.848 212.466 214.187 121.212 133.953 3 300 mg/kg BW HMB 373.967 263.774 595.386 365.358 319.559 287.879 7 300 mg/kg BW HMB 262.052 174.242 263.085 284.435 205.579 125.689 11 300 mg/kg BW HMB 118.802 104.683 291.322 122.590 136.364 106.061 15 300 mg/kg BW HMB 150.138 164.945 144.284 193.526 155.992 127.066 19 300 mg/kg BW HMB 170.799 170.110 338.499 152.548 179.752 139.118 23 300 mg/kg BW HMB 288.912 167.011 387.397 363.292 209.366 239.325 27 300 mg/kg BW HMB 140.152 212.810 212.466 209.366 174.587 220.730 31 300 mg/kg BW HMB 69.559 101.928 206.612 104.683 130.165 127.755 4 450 mg/kg BW HMB 253.444 204.545 741.391 240.358 196.281 154.614 8 450 mg/kg BW HMB #DIV/0! 80.923 227.961 223.485 92.975 149.105 12 450 mg/kg BW HMB 164.256 107.782 190.427 142.218 131.887 114.325 16 450 mg/kg BW HMB 90.909 144.628 189.050 158.402 204.545 168.733 20 450 mg/kg BW HMB 79.545 174.587 189.738 196.625 159.091 141.529 24 450 mg/kg BW HMB 460.055 365.014 411.501 367.769 356.061 277.893 28 450 mg/kg BW HMB 129.132 50.275 233.471 148.416 92.631 141.529 32 450 mg/kg BW HMB 200.069 161.501 356.061 259.986 219.697 197.658

144 Plasma Insulin Concentration (pM) During - ITT (150 Min Delay) Animal D0 D15 D45 D75 D105 D135 Number Treatment

1 CON 5 CON 250.344 207.989 379.132 135.675 137.052 126.033 9 CON 715.909 555.785 740.702 710.055 130.854 270.317 13 CON 251.722 299.587 590.909 664.945 383.264 371.901 17 CON 109.160 75.069 127.066 90.909 318.871 314.394 21 CON 357.438 320.937 70.937 87.810 65.771 113.292 25 CON 275.517 160.813 399.793 495.523 413.912 425.275 29 CON 34.780 14.807 537.879 586.088 441.116 426.653 2 150 mg/kg BW HMB 6 150 mg/kg BW HMB 125.344 116.047 529.270 231.061 83.333 26.171 10 150 mg/kg BW HMB 600.551 320.248 728.994 573.347 508.264 456.956 14 150 mg/kg BW HMB 724.174 559.573 580.578 384.298 262.741 277.548 18 150 mg/kg BW HMB 323.347 326.446 775.482 349.518 316.116 299.931 22 150 mg/kg BW HMB 249.656 307.163 275.826 231.749 197.658 312.672 26 150 mg/kg BW HMB 182.851 906.336 540.634 517.562 204.201 193.182 30 150 mg/kg BW HMB 201.102 259.642 176.653 201.102 231.061 279.614 3 300 mg/kg BW HMB 7 300 mg/kg BW HMB 287.190 256.887 569.904 1508.953 320.248 357.438 11 300 mg/kg BW HMB 140.152 65.427 262.741 380.854 242.769 234.504 15 300 mg/kg BW HMB 271.350 161.157 365.358 289.945 257.920 307.507 19 300 mg/kg BW HMB 367.080 489.325 456.612 609.160 376.722 484.504 23 300 mg/kg BW HMB 37.534 #DIV/0! 773.416 554.063 381.887 257.920 27 300 mg/kg BW HMB 64.394 35.813 609.504 235.882 123.623 98.140 31 300 mg/kg BW HMB 175.964 277.548 125.344 206.956 213.843 183.884 4 450 mg/kg BW HMB 8 450 mg/kg BW HMB 362.948 370.179 1117.080 690.083 616.736 409.091 12 450 mg/kg BW HMB 203.857 106.749 584.366 416.322 448.003 416.322 16 450 mg/kg BW HMB 125.000 139.118 335.055 289.945 220.730 187.328 20 450 mg/kg BW HMB 357.438 320.937 70.937 87.810 65.771 113.292 24 450 mg/kg BW HMB 316.804 322.314 492.080 481.061 676.653 356.405 28 450 mg/kg BW HMB 9.642 48.209 273.416 305.096 335.399 282.369 32 450 mg/kg BW HMB 358.815 257.576 324.380 284.435 416.667 346.763

145 Total Protein Concentration Using Median Fluorescence Intensity (MFI) Animal Treatment GSK3beta IGF1R IRS1 Akt mTOR p70S6K IR PTEN GSK3alpha TSC2 RPS6 Number 1 CON 3987 207.25 15568 462.5 366 2509 1406 689 211 1302 144.5 5 CON 4139 206 20062 578.5 358 2125 1313 906 197 1603 144.5 9 CON 3569 178 13676 326 288.5 2442 1214 1379 172 1397 113.75 13 CON 3483 166 13658 630.25 333.75 1741 1975 745.5 171.75 1120 109 17 CON 4593 165.5 15625 400.25 344.5 2247 1967 917.25 171.25 1648 122.75 21 CON 4515 183.5 17276 409.5 366.5 2362 1601 1030 162.5 1714 125 25 CON 4031 163 16174 335 286.25 2577 1126 1291 143.75 1244 105.5 29 CON 4370 152.25 13522 330.5 298.75 2661 1337 1193 158.5 1761 109.25 2 150 mg/kg BW HMB 4211 201 14109 443.25 409 2866 1777 1752 193 1572 152.5 6 150 mg/kg BW HMB 4353 184 16278 453.5 343.5 2850 1715 1120 174.25 1572 134.75 10 150 mg/kg BW HMB 4769 173 9898 376.75 364 3035 1320 1372 178.25 1764 123.5 14 150 mg/kg BW HMB 4446 183 15925 319 276.25 1649 1946 751.5 173.5 941.75 122 18 150 mg/kg BW HMB 5236 160.5 12372 360.5 389.75 2634 1779 1308 164 1674 117.75 22 150 mg/kg BW HMB 4737 182.25 17225 515.75 351.5 3170 2009 1025 169 1688 123 26 150 mg/kg BW HMB 4326 176 16898 344.25 355.5 2102 1396 1153 162.5 1324 121 30 150 mg/kg BW HMB 4723 156.75 10430 353.5 303.5 2658 1357 1529 144.5 1595 101.25 3 300 mg/kg BW HMB 4065 200.5 17061 375.5 283 1435 1962 495 184.75 1109 126.75 7 300 mg/kg BW HMB 3853 176.5 14357 428.5 338.75 1627 1265 700.75 174.75 1462 119 11 300 mg/kg BW HMB 4871 152.75 15183 384.5 343.5 2760 2185 1311 172 1676 123.75 15 300 mg/kg BW HMB 4491 173.5 13021 314.25 347 2563 1406 1396 167.5 1638 109 19 300 mg/kg BW HMB 3908 151.75 13704 317.75 265.75 2545 1246 842.5 155.5 1505 113 23 300 mg/kg BW HMB 4230 158.75 15588 363.75 285.25 2543 1387 1101 165.75 1312 113 27 300 mg/kg BW HMB 1658 153 6583 152 217 998.5 986.75 379.5 131.5 617.25 108.5 31 300 mg/kg BW HMB 4436 153.5 15142 314 269.5 2094 1343 1146 150 1259 108.25 4 450 mg/kg BW HMB 4660 195 16231 406.75 375 2328 1955 681 186.75 1838 118.75 8 450 mg/kg BW HMB 4686 181.25 16352 637.5 348.75 3231 1494 1006 180.25 1891 127.25 12 450 mg/kg BW HMB 3813 165.5 14837 335.25 322 2362 1482 917.5 171 1438 124.75 16 450 mg/kg BW HMB 4644 199.25 17353 316.75 287 2277 1273 856.25 181 1345 125 20 450 mg/kg BW HMB 4193 156.5 14483 363.75 363.75 2202 1445 1440 154.5 1428 116.25 24 450 mg/kg BW HMB 4658 154 12733 417.25 359.75 2734 1791 1376 154.75 1850 105.25 28 450 mg/kg BW HMB 4709 176 16942 377.5 267.5 2804 1384 1144 152.25 1794 120 32 450 mg/kg BW HMB 3689 171.5 17722 264.75 298 2433 1754 889.5 144 1259 108

146

Total Phosphorylated Protein Concentration Using Median Fluorescence Intensity (MFI) Animal Treatment GSK3beta IGF1R IRS1 Akt mTOR p70S6K IR PTEN GSK3alpha TSC2 RPS6 Number 1 CON 461.25 14.5 23.25 40.25 176.25 32.25 20.25 546.5 14.5 124.75 11.75 5 CON 322 17 24 74.75 141.75 31.75 23.25 333.75 16 142 15 9 CON 300 21.5 18.75 76.25 126.25 31 25.5 1283 18 164.5 18.5 13 CON 655.25 14.25 17.5 65.75 138 29 31.75 729.5 15 196.5 14.5 17 CON 318.5 14.75 23 53 127.75 33.5 23 397.75 14 131 14 21 CON 341 11.75 21.75 62.5 133.5 34.25 20.5 361.5 16.25 156.5 15.75 25 CON 690 16 21.5 62.75 145.75 25.25 23.25 1069 14.25 221.5 14.5 29 CON 469.75 18.75 20.25 65.25 127 24.75 22.25 435.5 17 275.25 14.5 2 150 mg/kg BW HMB 768.5 11.75 24.5 73 248.75 29.5 20.5 1404 12 309.25 15 6 150 mg/kg BW HMB 341.25 16.5 36.5 83.5 145 30.75 23.5 637 15.25 109.75 18.75 10 150 mg/kg BW HMB 479.5 15.5 16.5 71 134.75 37.75 29 1674 21.25 169 17 14 150 mg/kg BW HMB 515 12.5 18.5 62.5 118 32.75 24.25 659 12.5 146.25 12 18 150 mg/kg BW HMB 334.75 21.75 15 60 110 25 22.5 860.5 17.5 155 12.5 22 150 mg/kg BW HMB 345.25 15.5 20 46.5 107.5 26 20.5 392.75 15.5 127.25 12.5 26 150 mg/kg BW HMB 571.5 12.25 24.5 58 151.25 31.25 22.5 694.5 17 149.75 16.5 30 150 mg/kg BW HMB 245.75 20 12.25 42.75 127 30.25 22.25 576.5 13 118 12.25 3 300 mg/kg BW HMB 518.5 17 24.25 54 119 27.5 27 681.5 17.5 103.5 13.5 7 300 mg/kg BW HMB 472.75 14.5 14.5 55.5 177.25 34.5 29.25 891.5 14.5 194.25 14 11 300 mg/kg BW HMB 1172 15.5 45.5 128.75 380 36.25 31.75 2079 19 394 26.25 15 300 mg/kg BW HMB 336.25 19 15.25 68.5 155.25 33 31.5 496.25 15.75 122.25 15.25 19 300 mg/kg BW HMB 414.5 16 20.25 48 107.75 26 22 275.5 15 130.5 10.75 23 300 mg/kg BW HMB 301.75 14.5 22.75 68.75 111.5 34 26 1001 12 114.75 11 27 300 mg/kg BW HMB 430.25 14.75 19.5 62 152.5 27.25 24.75 782.5 13.25 175 14.75 31 300 mg/kg BW HMB 308 13.5 16.5 74.25 108.5 30.5 20 530 15 85.75 14.5 4 450 mg/kg BW HMB 544.5 18.5 35 59.75 171.5 33.75 35.75 746.25 15.5 174.25 18.5 8 450 mg/kg BW HMB 710 15.5 19.75 64.75 140.5 27.75 25.75 1176 17.75 262.75 16.5 12 450 mg/kg BW HMB 888.5 19 31.5 74 216.25 32.25 25.75 1286 18.75 253 18.5 16 450 mg/kg BW HMB 660.5 17.5 40.5 66.25 189 30.75 21.25 880.75 16.25 203.25 17.25 20 450 mg/kg BW HMB 1051 16.5 32.5 93.5 270.25 30 22.25 1741 14.75 403.75 14 24 450 mg/kg BW HMB 514 11.75 12 92 219 28.75 24 910 11.25 196.5 12.5 28 450 mg/kg BW HMB 774.75 15 42 96.5 242.5 37.25 28 893.5 15.5 310.25 20 32 450 mg/kg BW HMB 552 13.25 26.75 58.25 124 29.5 20.25 1210 11.5 173 13

147 CHAPTER IV

Force Measured Leg Untrained Animals Max Tetanic Contraction (N) Animal Name Animal Weight (g) Quadriceps Triceps Surea 1 CON 470 32.85 15.9385 3 CON 555 36.3484 33.0112 5 CON 465 39.2692 38.3969 7 CON 500 38.3765 33.4829 9 CON 456 44.9726 28.152 CON AVERAGE 489.2 38.363 29.796 CON STD. ERROR 18.032 1.987 3.825 2 CON_HMB 496 45.671 37.7385 4 CON_HMB 461 39.227 31.1769 8 CON_HMB 511 47.3532 34.2213 10 CON_HMB 462 37.9638 37.2173 CON HMB AVERAGE 482.5 42.554 35.089 CON HMB STD. ERROR 12.507 2.325 1.517

148 Force Measured Leg Untrained Animals Muscle Weights (g) Animal Biceps Gluteus Wrist Name Soleus Plantaris Gastroc. Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 1 CON 0.187 0.507 2.32 4.298 3.807 1.297 2.202 3.103 0.901 3 CON 0.23 0.481 2.518 4.859 4.306 1.755 2.477 3.684 1.102 5 CON 0.222 0.438 2.255 4.512 3.823 1.405 1.916 3.598 1.047 7 CON 0.22 0.542 2.449 4.634 3.981 1.353 2.225 3.282 1.092 9 CON 0.209 0.419 2.256 4.12 3.725 1.366 2.041 3.224 0.904 CON AVERAGE 0.214 0.477 2.360 4.485 3.928 1.435 2.172 3.378 1.009 CON STD. ERROR 0.007 0.0224 0.053 0.129 0.103 0.082 0.095 0.112 0.0445 2 CON_HMB 0.21 0.497 2.229 4.486 4.015 1.104 2.366 3.563 0.895 4 CON_HMB 0.191 0.432 2.191 4.181 3.383 2.292 2.18 3.459 1.06 8 CON_HMB 0.246 0.477 2.481 4.677 4.113 1.421 2.425 3.704 1.083 10 CON_HMB 0.221 0.403 2.163 4.197 3.67 1.301 1.986 3.297 1.062 CON HMB AVERAGE 0.217 0.452 2.266 4.385 3.795 1.530 2.239 3.506 1.025 CON HMB STD. ERROR 0.011 0.021 0.073 0.120 0.167 0.262 0.099 0.086 0.044

149 Force Measured Leg Untrained Animals Muscle Lengths (cm) Biceps Gluteus Wrist Animal Name Soleus Plantaris Gastroc. Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 1 CON 2.2 3.3 3.6 3.7 4.8 5.3 4.1 3.7 3.1 3 CON 3.1 3.3 3.5 3.6 4.6 5.8 4.2 4 2.5 5 CON 2.5 3.7 3.3 3.6 4.1 4.2 4 3.4 3.3 7 CON 2.5 3.7 3.7 3.9 5.3 5.1 4 3.8 2.5 9 CON 2.5 3.5 3.4 3.7 4.9 5.1 4.7 3.7 2.4 CON Average 2.56 3.5 3.5 3.7 4.74 5.1 4.2 3.72 2.76 CON SEM 0.147 0.089 0.071 0.055 0.196 0.259 0.130 0.097 0.183 2 CON_HMB 2.5 4 3.6 - 5.1 4.7 3.9 3.8 2.3 4 CON_HMB 2.2 3.6 3.6 3.6 4.4 5.6 4.4 3.4 2.4 8 CON_HMB 2.4 3.8 3.7 3.7 4.7 5.4 4.3 3.9 2.9 10 CON_HMB 2.3 3.5 3.1 3.9 5 5.4 - 3.5 2.5 CON_HMB Average 2.35 3.725 3.5 3.7333333 4.8 5.275 4.2 3.65 2.525 CON HMB SEM 0.065 0.111 0.135 0.088 0.158 0.197 0.153 0.119 0.131

150 Force Measured Leg Resistance Trained Animals Max Tetanic Contraction (N) Animal Name Animal Weight (g) Quadriceps Triceps Surea 11 EX 437 40.9284 32.1381 13 EX 411 40.4769 28.2435 15 EX 442 44.7041 32.6768 17 EX 432 46.3697 33.1904 19 EX 400 40.0343 39.5901 EX Average 424.4 42.503 33.168 EX SEM 8.066 1.274 1.828 12 EX_HMB 412 38.8712 34.1106 14 EX_HMB 400 35.2601 31.1339 16 EX_HMB 404 41.5204 32.8042 18 EX_HMB 414 43.4739 38.452 20 EX_HMB 439 41.5185 37.5529 EX_HMB Average 413.8 40.129 34.811 EX HMB SEM 6.8 1.420 1.393

151

Force Measured Leg Resistance Trained Animals Muscle Weights (g) Bicep Gluteus Wrist Animal Name Soleus Plantaris Gastroc. Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 11 EX 0.24 0.474 2.161 4.56 4.032 1.529 2.12 3.153 1.093 13 EX 0.208 0.471 2.012 3.9 3.268 1.263 1.902 2.821 1.072 15 EX 0.196 0.466 2.085 4.15 3.977 1.446 1.975 2.997 1.147 17 EX 0.221 0.425 1.989 3.953 3.62 1.481 1.979 2.976 1.076 19 EX 0.218 0.488 2.095 3.913 3.402 1.243 1.951 2.73 1.071 EX Average 0.217 0.465 2.068 4.095 3.660 1.392 1.985 2.935 1.092 EX SEM 0.007 0.011 0.031 0.125 0.152 0.059 0.036 0.076 0.014 12 EX_HMB 0.182 0.436 2.047 4.103 3.372 1.348 1.865 2.946 0.988 14 EX_HMB 0.22 0.393 2.01 3.98 3.597 1.306 1.855 2.681 0.999 16 EX_HMB 0.175 0.412 1.944 3.888 3.344 1.423 1.917 2.828 1.048 18 EX_HMB 0.189 0.459 2.089 3.952 3.253 1.411 2.008 2.916 1.092 20 EX_HMB 0.174 0.42 2.075 4.065 3.555 1.568 1.972 3.054 1.005 EX_HMB Average 0.188 0.424 2.033 3.9976 3.424 1.411 1.923 2.885 1.026 EX HMB SEM 0.008 0.0112 0.026 0.039 0.065 0.045 0.030 0.062 0.019

152

Force Measured Leg Resistance Trained Animals Muscle Lengths (cm) Bicep Gluteus Wrist Animal Name Soleus Plantaris Gastroc Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 11 EX 2 3.6 3.2 3.7 4.7 5.5 4.1 3.6 2.4 13 EX 2.6 3.3 3.4 3.5 4.3 5.5 4 3.4 2.5 15 EX 2.2 3.4 3.7 3.7 5.4 5.6 4 3.4 2.6 17 EX 2.4 3.4 3.6 3.6 4.8 5.4 3.9 3.6 2.7 19 EX 2.4 3.7 3.4 3.4 4.5 - 4.1 3.3 2.7 EX Average 2.320 3.480 3.460 3.580 4.740 5.500 4.020 3.460 2.580 EX SEM 0.102 0.073 0.087 0.058 0.186 0.041 0.037 0.060 0.058 12 EX_HMB 2.2 3.5 3.4 3.5 3.5 5.4 3.9 3.5 2.4 14 EX_HMB 2.6 3.2 3.3 3.7 5 5.5 4.1 3.2 2.6 16 EX_HMB 1.8 3.4 3.5 3.5 4.2 5 3.8 3.4 2.7 18 EX_HMB 2.2 3.4 3.6 3.6 4.5 5.2 3.8 3.5 3.1 20 EX_HMB 2.1 3.4 3.4 3.5 4.7 5.6 4 3.6 2.3 EX_HMB Average 2.180 3.380 3.440 3.560 4.380 5.340 3.920 3.440 2.620 EX HMB SEM 0.128 0.049 0.051 0.040 0.256 0.108 0.058 0.068 0.139

153

Non-Force Measured Leg Untrained Animals Muscle Weights (g) Animal Biceps Gluteus Wrist Name Soleus Plantaris Gastroc Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 1 CON 0.19 0.438 2.185 4.033 0 0 2.144 0 3 CON 0.185 0.414 1.994 4.088 3.464 1.511 2.201 2.94 5 CON 0.179 0.398 2.091 4.281 4.307 1.046 1.551 3.568 7 CON 0.179 0.445 2.174 4.351 3.823 1.453 2.316 3.052 9 CON 0.174 0.391 2.027 3.804 3.489 1.001 2.021 3.385 CON Average 0.181 0.417 2.094 4.111 3.017 1.002 2.047 2.589 Con SEM 0.003 0.011 0.038 0.097 0.769 0.271 0.133 0.657 2 CON_HMB 1.82 0.427 2.136 4.138 3.575 0.444 2.132 3.424 4 CON_HMB 0.153 0.447 2.238 3.993 3.516 1.532 2.188 2.922 8 CON_HMB 0.213 0.463 2.396 4.482 3.592 1.462 2.29 3.569 10 CON_HMB 0.165 0.434 2.096 3.822 3.685 1.211 1.991 3.14 CON_HMB Average 0.588 0.443 2.217 4.109 3.592 1.162 2.150 3.264 CON HMB SEM 0.411 0.008 0.067 0.140 0.035 0.249 0.062 0.145

154

Non-Force Measured Leg Untrained Animals Muscle Lengths (cm) Animal Biceps Gluteus Wrist Name Soleus Plantaris Gastroc Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 1 CON 2.3 3.1 3.7 3.7 - - 4.2 3.6 3.1 3 CON 2.5 3.8 3.4 3.9 4.3 5.7 3.9 3.8 2.5 5 CON 2.6 3.5 3.2 3.8 5 4 3.7 3.6 3.3 7 CON 2 3.5 3.4 3.8 6.1 4.9 4 3.7 2.5 9 CON 2.2 3.5 3.3 3.6 5.1 4 4.3 3.5 2.4 CON Average 2.56 3.5 3.5 3.7 4.74 5.1 4.2 3.72 2.76 Con SEM 0.147 0.089 0.071 0.055 0.196 0.259 0.130 0.097 0.183 2 CON_HMB 2.5 3.8 3.4 - 4.6 - 3.6 3.7 - 4 CON_HMB 1.8 3.5 3.4 3.5 4.7 5.4 4.6 3.4 2.4 8 CON_HMB 2.2 3.9 3.6 3.8 4.8 5.2 4.2 3.9 2.9 10 CON_HMB 2.4 3.4 3.3 3.8 5.1 5.3 3.4 3.7 CON_HMB Average 2.35 3.725 3.5 3.733 4.8 5.275 4.2 3.65 2.525 CON HMB SEM 0.065 0.111 0.135 0.088 0.158 0.197 0.153 0.119 0.131

155

Non-Force Measured Leg Resistance Trained Animals Muscle Weights (g) Bicep Gluteus Wrist Animal Name Soleus Plantaris Gastroc Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 11 EX 0.21 0.449 2.161 3.991 3.46 1.468 1.9 3.043 - 13 EX 0.155 0.406 1.827 3.712 3.005 1.319 1.772 2.682 - 15 EX 0.187 0.471 1.951 3.997 3.518 1.52 1.867 2.988 - 17 EX 0.191 0.426 1.908 3.781 3.273 1.644 1.868 3.046 1.083 19 EX 1.53 0.349 1.752 3.536 3.632 1.458 1.765 2.588 1.069 EX Average 0.4546 0.4202 1.9198 3.8034 3.3776 1.4818 1.8344 2.8694 1.076 EX SEM 0.269 0.021 0.069 0.087 0.110 0.052 0.028 0.097 0.007 12 EX_HMB 0.149 0.396 1.857 3.683 3.116 1.464 1.798 2.754 - 14 EX_HMB 0.172 0.363 1.788 3.643 3.323 1.383 1.738 2.643 - 16 EX_HMB 0.15 0.416 1.824 3.608 3.26 1.429 1.76 2.852 - 18 EX_HMB 0.209 0.438 1.993 3.866 3.349 1.942 1.928 2.969 1.09 20 EX_HMB 0.212 0.478 2.121 3.978 1.614 2.031 2.883 1.11 EX_HMB Average 0.1784 0.4182 1.9166 3.7556 3.262 1.5664 1.851 2.8202 1.1 EX HMB SEM 0.014 0.019 0.062 0.071 0.052 0.102 0.056 0.056 0.010

156 Non-Force Measured Leg Resistance Trained Animals Muscle Lengths (cm) Animal Biceps Gluteus Wrist Name Soleus Plantaris Gastroc Quadriceps Femoris Semitendinosus Semimembranosus Medius Flexors 11 EX 2.4 3.7 3.6 3.6 4.4 5.3 3.9 3.5 2.4 13 EX 2.2 3.5 3.1 3.4 4.2 - 4.3 3.5 - 15 EX 2.3 3.6 3.4 3.7 4.6 5.4 3.8 3.5 - 17 EX 2.3 3.3 3.5 3.6 4.3 5.2 3.9 3.6 2.8 19 EX 2.3 3.5 3.4 3.5 4.9 - 4.3 3.3 2.4 EX Average 2.3 3.52 3.4 3.56 4.48 5.3 4.04 3.48 2.533 EX SEM 0.032 0.066 0.084 0.051 0.124 0.058 0.108 0.049 0.133 12 EX_HMB 2 3.2 3.6 3.4 4 5.5 4 3.5 2.4 14 EX_HMB 2.2 3.3 3.4 3.6 4.1 5.1 - 3.4 - 16 EX_HMB 2.1 3.5 3.4 3.5 4.9 5.3 3.6 3.1 - 18 EX_HMB 2.3 3.7 3.4 3.7 4.5 5.1 3.9 3.5 2.9 20 EX_HMB 2.1 3.4 3.4 3.6 - 5.4 3.4 3.5 2.7 EX_HMB Average 2.14 3.42 3.44 3.56 4.375 5.28 3.725 3.4 2.667 EX HMB SEM 0.051 0.086 0.040 0.051 0.206 0.080 0.138 0.077 0.145

157

Ancillary Weights (g) Animal Name Epididymal Fat Adrenal Gland Heart 1 CON 11.317 0.06 1.562 3 CON 13.734 0.073 1.603 5 CON 8.56 0.07 1.25 7 CON 14.765 0.064 1.32 9 CON 11.12 0.056 1.361 CON Average 11.899 0.065 1.419 Con SEM 1.088 0.003 0.069 2 CON_HMB 11.882 0.069 1.199 4 CON_HMB 11.08 0.074 1.034 8 CON_HMB 12.556 0.064 1.28 10 CON_HMB 10.301 0.061 1.067 CON_HMB Average 11.455 0.067 1.145 CON HMB SEM 0.489 0.003 0.057 11 EX 7.071 0.061 1.202 13 EX 7.277 0.058 1.603 15 EX 8.388 0.06 1.326 17 EX 7.836 0.058 1.203 19 EX 8.43 0.051 1.02 EX Average 7.800 0.058 1.271 EX SEM 0.278 0.002 0.096 12 EX_HMB 6.231 0.053 1.209 14 EX_HMB 6.167 0.053 1.11 16 EX_HMB 5.856 0.059 1.33 18 EX_HMB 7.329 0.047 1.153 20 EX_HMB 8.767 0.053 1.363 EX_HMB Average 6.870 0.053 1.233 EX HMB SEM 0.536 0.002 0.049

158 Oral Glucose Tolerance Test Prior to Resistance Exercise Regimen Blood Glucose (mg/dL) Blood Glucose (mg/dL) Animal Name Baseline 1 Baseline 2 15 min 30 min 60 min 90 min 120 min 1 CON 30 43 102 118 106 96 107 3 CON 65 63 95 114 85 93 93 5 CON 68 78 125 130 141 119 121 7 CON 66 65 91 123 116 97 94 9 CON 70 62 99 119 104 91 88 CON Average 59.800 62.200 102.400 120.800 110.400 99.200 100.600 CON SEM 7.499 5.598 5.946 2.709 9.147 5.064 5.988 2 CON_HMB 62 72 103 131 95 100 96 4 CON_HMB 67 65 112 128 117 102 6 CON_HMB 58 65 112 118 105 107 115 8 CON_HMB 72 63 104 121 94 86 93 10 CON_HMB 69 65 109 105 98 101 109 CON HMB Average 65.600 66.000 108.000 120.600 101.800 99.200 103.250 CON HMB SEM 2.502 1.549 1.924 4.545 4.259 3.513 5.234 11 EX 64 63 116 118 103 101 102 13 EX 67 61 115 102 101 93 95 15 EX 71 67 114 106 90 97 90 17 EX 61 63 88 115 98 90 100 19 EX 67 67 112 96 90 84 83 EX Average 66.000 64.200 109.000 107.400 96.400 93.000 94.000 EX SEM 1.673 1.200 5.292 4.069 2.731 2.915 3.450 12 EX_HMB 65 64 93 126 131 106 101 14 EX_HMB 66 62 115 113 89 89 93 16 EX_HMB 66 57 93 121 115 120 94 18 EX_HMB 61 62 97 100 116 108 107 20 EX_HMB 70 66 98 104 99 99 98 EX HMBAverage 65.600 62.200 99.200 112.800 110.000 104.400 98.600 EX HMBSEM 1.435 1.497 4.079 4.913 7.294 5.124 2.542

159

Oral Glucose Tolerance Test 5 weeks into Resistance Exercise Regimen Blood Glucose (mg/dL) Blood Glucose (mg/dL) Animal Name Baseline 1 Baseline 2 15 min 30 min 60 min 90 min 120 min 1 CON 70 79 94 97 101 112 102 3 CON 83 64 105 89 118 99 92 5 CON 66 62 110 113 102 106 98 7 CON 59 70 97 10 111 99 102 9 CON 67 70 102 103 97 74 79 CON Average 69.000 69.000 101.600 82.400 105.800 98.000 94.600 CON SEM 3.937 2.966 2.839 18.519 3.813 6.473 4.308 2 CON_HMB 79 73 106 118 98 90 91 4 CON_HMB 68 83 95 119 103 89 83 6 CON_HMB 8 CON_HMB 66 62 109 109 95 97 87 10 CON_HMB 67 70 96 109 82 81 83 CON HMB Average 70.000 72.000 101.500 113.750 94.500 89.250 86.000 CON HMB SEM 3.028 4.340 3.524 2.750 4.481 3.276 1.915 11 EX 78 80 104 105 117 93 98 13 EX 79 73 104 106 103 92 99 15 EX 66 66 128 114 104 111 116 17 EX 73 67 123 108 107 107 105 19 EX 60 60 116 99 95 93 102 EX Average 71.200 69.200 115.000 106.400 105.200 99.200 104.000 EX SEM 3.625 3.397 4.879 2.421 3.555 4.055 3.240 12 EX_HMB 94 92 110 108 99 86 91 14 EX_HMB 75 70 106 107 113 102 109 16 EX_HMB 79 81 122 112 101 94 88 18 EX_HMB 59 60 123 130 100 99 101 20 EX_HMB 83 71 117 100 107 96 107 EX HMBAverage 78.000 74.800 115.600 111.400 104.000 95.400 99.200 EX HMBSEM 5.710 5.435 3.326 5.036 2.646 2.713 4.200

160

Oral Glucose Tolerance Test 11 weeks into Resistance Exercise Regimen Blood Glucose (mg/dL) Blood Glucose (mg/dL) Animal Name Baseline 1 Baseline 2 15 min 30 min 60 min 90 min 120 min 1 CON 81 82 127 107 82 101 95 3 CON 87 89 123 110 104 109 103 5 CON 90 91 130 108 90 95 101 7 CON 81 74 132 113 91 90 102 9 CON 90 82 126 106 92 89 86 CON Average 85.800 83.600 127.600 108.800 91.800 96.800 97.400 CON SEM 2.035 3.010 1.568 1.241 3.527 3.720 3.172 2 CON_HMB 87 80 133 110 103 86 97 4 CON_HMB 70 71 121 113 92 96 87 6 CON_HMB 8 CON_HMB 76 67 117 106 89 86 85 10 CON_HMB 76 69 122 97 86 97 101 CON HMB Average 77.250 71.750 123.250 106.500 92.500 91.250 92.500 CON HMB SEM 3.544 2.869 3.425 3.476 3.708 3.038 3.862 11 EX 70 75 121 104 78 85 91 13 EX 84 64 118 115 89 89 92 15 EX 75 76 128 104 91 100 113 17 EX 86 93 133 119 95 77 97 19 EX 92 93 102 87 94 86 84 EX Average 81.400 80.200 120.400 105.800 89.400 87.400 95.400 EX SEM 3.945 5.634 5.297 5.562 3.043 3.723 4.864 12 EX_HMB 81 76 118 115 89 85 94 14 EX_HMB 76 77 125 120 90 85 88 16 EX_HMB 73 81 145 120 111 118 97 18 EX_HMB 73 110 111 98 100 93 109 20 EX_HMB 81 82 125 124 91 94 85 EX HMBAverage 76.800 85.200 124.800 115.400 96.200 95.000 94.600 EX HMBSEM 1.800 6.304 5.678 4.578 4.188 6.058 4.179

161

Maximal Carrying Capacity per Week Animal Number Treatment 0 1 2 3 4 5 6 7 8 9 10 11 11 EX 910 1110 1190 1405 1409 1450 1452 1505 1585 1635 1635 1680 12 EX 810 890 1170 1243 1403 1405 1526 1526 1605 1645 1645 1685 13 EX 864 985 1125 1224 1315 1315 1355 1437 1682 1682 1682 1682 14 EX 840 1000 1161 1283 1326 1406 1446 1527 1647 1685 1805 1885 15 EX 902 942 1142 1262 1344 1429 1429 1469 1824 1824 1824 1824 16 EX HMB 842 922 1082 1242 1247 1367 1367 1491 1790 1790 1790 1790 17 EX HMB 956 1036 1036 1324 1468 1546 1546 1593 1713 1753 1790 1790 18 EX HMB 860 940 1100 1339 1416 1416 1456 1620 1662 1662 1702 1702 19 EX HMB 832 872 992 1073 1111 1151 1231 1272 1394 1394 1554 1714 20 EX HMB 900 940 1229 1308 1359 1399 1444 1486 1489 1524 1524 1524 Averag 871.60 963.70 1122.70 1270.30 1339.80 1388.40 1425.20 1492.60 1639.10 1659.40 1695.10 1727.60 S.E.M 14.05 22.50 22.77 27.89 32.18 32.31 28.53 30.02 41.03 39.95 33.70 31.68 S.D. 44.42 71.16 72.02 88.20 101.75 102.17 90.22 94.94 129.76 126.34 106.58 100.17

162

Animal Quadriceps Muscle Number Treatment Group Weight CSA Lo Po Pt Spo SPt SPt/Spo 1 CON 4.298 0.4225 4.67 32.85 5 77.7514793 11.8343195 0.152207 2 CON 4.486 0.4225 5.02 45.671 5.1 108.097041 12.0710059 0.11166824 3 CON 4.859 0.4225 4.83 36.3484 4.78 86.031716 11.3136095 0.1315051 4 CON 4.181 0.4225 4.91 39.227 5.21 92.8449704 12.3313609 0.13281668 5 CON 4.512 0.4225 4.78 39.2692 4.98 92.9448521 11.7869822 0.12681695 7 CON HMB 4.634 0.4225 5.04 38.3765 4.87 90.8319527 11.5266272 0.12690058 8 CON HMB 4.677 0.4225 5.11 47.3532 4.76 112.07858 11.2662722 0.10052119 9 CON HMB 4.12 0.4225 4.77 44.9726 4.95 106.444024 11.7159763 0.11006702 10 CON HMB 4.197 0.4225 4.85 37.9638 4.99 89.8551479 11.8106509 0.131441 11 EX 4.56 0.4225 5.04 40.9284 4.96 96.8719527 11.739645 0.12118724 12 EX 4.103 0.4225 4.96 38.8712 5.03 92.0028402 11.9053254 0.12940172 13 EX 3.9 0.4225 4.88 40.4769 5.11 95.8033136 12.0946746 0.12624485 14 EX 3.98 0.4225 4.83 35.2601 4.65 83.455858 11.0059172 0.13187711 15 EX 4.15 0.4225 4.69 44.7041 4.88 105.808521 11.5502959 0.10916225 16 EX HMB 3.888 0.4225 4.72 41.5204 4.93 98.2731361 11.6686391 0.11873681 17 EX HMB 3.953 0.4225 4.52 46.3697 5.01 109.750769 11.8579882 0.10804469 18 EX HMB 3.952 0.4225 4.39 43.4739 4.82 102.896805 11.408284 0.11087112 19 EX HMB 3.913 0.4225 4.41 40.0343 5.07 94.7557396 12 0.1266414 20 EX HMB 4.065 0.4225 4.81 41.5185 4.57 98.2686391 10.816568 0.11007141

163

Animal Triceps Surea Number Treatment Weight CSA Lo Po Pt Spo SPt SPt/Spo 1 CON 3.014 0.36 4.05 15.9385 4.76 44.2736111 13.2222222 0.29864793 2 CON 2.936 0.36 4.13 37.7385 4.67 104.829167 12.9722222 0.12374631 3 CON 3.229 0.36 4.14 33.0112 5.22 91.6977778 14.5 0.15812815 4 CON 2.814 0.36 4.21 31.1769 4.99 86.6025 13.8611111 0.1600544 5 CON HMB 2.915 0.36 4.17 38.3969 5.07 106.658056 14.0833333 0.13204191 7 CON HMB 3.211 0.36 4.51 33.4829 5.16 93.0080556 14.3333333 0.15410852 8 CON HMB 3.204 0.36 3.92 34.2213 5.21 95.0591667 14.4722222 0.15224436 9 CON HMB 2.884 0.36 4.01 28.152 4.88 78.2 13.5555556 0.1733447 10 CON HMB 2.787 0.36 4.1 37.2173 4.67 103.381389 12.9722222 0.12547928 11 EX 2.875 0.36 4.32 32.1381 4.86 89.2725 13.5 0.15122238 12 EX 2.665 0.36 4.16 34.1106 4.72 94.7516667 13.1111111 0.13837341 13 EX 2.691 0.36 4.01 28.2435 4.76 78.4541667 13.2222222 0.16853435 14 EX 2.623 0.36 3.99 31.1339 4.83 86.4830556 13.4166667 0.15513636 15 EX 2.747 0.36 4.04 32.6768 4.62 90.7688889 12.8333333 0.14138471 16 EX HMB 2.531 0.36 3.89 32.8042 4.51 91.1227778 12.5277778 0.1374824 17 EX HMB 2.635 0.36 4.13 33.1904 4.61 92.1955556 12.8055556 0.13889558 18 EX HMB 2.737 0.36 4.07 38.452 4.75 106.811111 13.1944444 0.12353064 19 EX HMB 2.801 0.36 4.44 39.5901 5.04 109.9725 14 0.12730455 20 EX HMB 2.669 0.36 4.25 37.5529 4.89 104.313611 13.5833333 0.13021631

164 Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Gastroc Medial Head Force 0.027 0.005 0.185 2 CON Gastroc Medial Head Force 0.025 0.008 0.32 3 CON Gastroc Medial Head Force 0.028 0.007 0.25 4 CON Gastroc Medial Head Force 0.022 0.005 0.227 5 CON Gastroc Medial Head Force 0.047 0.01 0.212 7 CON HMB Gastroc Medial Head Force 0.039 0.01 0.256 8 CON HMB Gastroc Medial Head Force 0.047 0.011 0.234 9 CON HMB Gastroc Medial Head Force 0.057 0.012 0.210 10 CON HMB Gastroc Medial Head Force 0.05 0.013 0.26 11 EX Gastroc Medial Head Force 0.029 0.007 0.241 12 EX Gastroc Medial Head Force 0.041 0.008 0.195 13 EX Gastroc Medial Head Force 0.022 0.005 0.227 15 EX Gastroc Medial Head Force 0.02 0.008 0.4 16 EX HMB Gastroc Medial Head Force 0.035 0.007 0.2 17 EX HMB Gastroc Medial Head Force 0.049 0.01 0.204 18 EX HMB Gastroc Medial Head Force 0.026 0.005 0.192 19 EX HMB Gastroc Medial Head Force 0.027 0.006 0.222 20 EX HMB Gastroc Medial Head Force 0.044 0.009 0.204

165

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Gastroc Lateral Head Force 0.04 0.009 0.225 2 CON Gastroc Lateral Head Force 0.035 0.007 0.2 3 CON Gastroc Lateral Head Force 0.05 0.01 0.2 4 CON Gastroc Lateral Head Force 0.043 0.01 0.232 5 CON Gastroc Lateral Head Force 0.045 0.01 0.222 7 CON HMB Gastroc Lateral Head Force 0.04 0.008 0.2 8 CON HMB Gastroc Lateral Head Force 0.045 0.008 0.177 9 CON HMB Gastroc Lateral Head Force 0.051 0.011 0.215 10 CON HMB Gastroc Lateral Head Force 0.03 0.006 0.2 11 EX Gastroc Lateral Head Force 0.062 0.017 0.274 12 EX Gastroc Lateral Head Force 0.035 0.008 0.228 13 EX Gastroc Lateral Head Force 0.056 0.014 0.25 14 EX Gastroc Lateral Head Force 0.05 0.01 0.2 15 EX Gastroc Lateral Head Force 0.048 0.01 0.208 16 EX HMB Gastroc Lateral Head Force 0.046 0.011 0.239 17 EX HMB Gastroc Lateral Head Force 0.035 0.007 0.2 18 EX HMB Gastroc Lateral Head Force 0.038 0.01 0.263 19 EX HMB Gastroc Lateral Head Force 0.034 0.007 0.205 20 EX HMB Gastroc Lateral Head Force 0.033 0.007 0.212

166

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Gastroc Medial Head Non-Force 0.063 0.015 0.238 2 CON Gastroc Medial Head Non-Force 0.02 0.008 0.4 3 CON Gastroc Medial Head Non-Force 0.037 0.008 0.216 4 CON Gastroc Medial Head Non-Force 0.055 0.011 0.2 5 CON Gastroc Medial Head Non-Force 0.06 0.014 0.233 7 CON HMB Gastroc Medial Head Non-Force 0.05 0.011 0.22 8 CON HMB Gastroc Medial Head Non-Force 0.045 0.009 0.2 9 CON HMB Gastroc Medial Head Non-Force 0.037 0.009 0.243 10 CON HMB Gastroc Medial Head Non-Force 0.031 0.007 0.225 11 EX Gastroc Medial Head Non-Force 0.039 0.007 0.179 12 EX Gastroc Medial Head Non-Force 0.036 0.009 0.25 13 EX Gastroc Medial Head Non-Force 0.025 0.005 0.2 14 EX Gastroc Medial Head Non-Force 0.052 0.011 0.211 15 EX Gastroc Medial Head Non-Force 0.031 0.006 0.193 16 EX HMB Gastroc Medial Head Non-Force 0.057 0.013 0.228 17 EX HMB Gastroc Medial Head Non-Force 0.058 0.012 0.206 18 EX HMB Gastroc Medial Head Non-Force 0.05 0.01 0.2 19 EX HMB Gastroc Medial Head Non-Force 0.042 0.009 0.214 20 EX HMB Gastroc Medial Head Non-Force 0.052 0.013 0.25

167

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Gastroc Lateral Head Non-Force 0.049 0.013 0.265 2 CON Gastroc Lateral Head Non-Force 0.044 0.012 0.272 3 CON Gastroc Lateral Head Non-Force 0.045 0.012 0.266 4 CON Gastroc Lateral Head Non-Force 0.036 0.009 0.25 5 CON Gastroc Lateral Head Non-Force 0.032 0.008 0.25 7 CON HMB Gastroc Lateral Head Non-Force 0.033 0.008 0.242 8 CON HMB Gastroc Lateral Head Non-Force 0.059 0.014 0.237 9 CON HMB Gastroc Lateral Head Non-Force 0.035 0.01 0.285 10 CON HMB Gastroc Lateral Head Non-Force 0.044 0.01 0.227 11 EX Gastroc Lateral Head Non-Force 0.036 0.009 0.25 12 EX Gastroc Lateral Head Non-Force 0.039 0.01 0.256 13 EX Gastroc Lateral Head Non-Force 0.032 0.008 0.25 14 EX Gastroc Lateral Head Non-Force 0.031 0.008 0.258 15 EX Gastroc Lateral Head Non-Force 0.06 0.015 0.25 16 EX HMB Gastroc Lateral Head Non-Force 0.067 0.016 0.238 17 EX HMB Gastroc Lateral Head Non-Force 0.03 0.008 0.266 18 EX HMB Gastroc Lateral Head Non-Force 0.031 0.009 0.290 19 EX HMB Gastroc Lateral Head Non-Force 0.04 0.01 0.25 20 EX HMB Gastroc Lateral Head Non-Force 0.048 0.015 0.312

168

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Soleus Non-Force 0.028 0.007 0.25 2 CON Soleus Non-Force 0.019 0.005 0.263 3 CON Soleus Non-Force 0.021 0.006 0.285 4 CON Soleus Non-Force 0.038 0.008 0.210 5 CON Soleus Non-Force 0.025 0.006 0.24 7 CON HMB Soleus Non-Force 0.03 0.007 0.233 8 CON HMB Soleus Non-Force 0.037 0.007 0.189 9 CON HMB Soleus Non-Force 0.026 0.007 0.269 10 CON HMB Soleus Non-Force 0.03 0.007 0.233 11 EX Soleus Non-Force 0.028 0.007 0.25 12 EX Soleus Non-Force 0.044 0.011 0.25 13 EX Soleus Non-Force 0.041 0.008 0.195 14 EX Soleus Non-Force 0.024 0.006 0.25 15 EX Soleus Non-Force 0.024 0.008 0.333 16 EX HMB Soleus Non-Force 0.024 0.006 0.25 17 EX HMB Soleus Non-Force 0.034 0.008 0.235 18 EX HMB Soleus Non-Force 0.026 0.007 0.269 19 EX HMB Soleus Non-Force 0.018 0.007 0.388 20 EX HMB Soleus Non-Force 0.021 0.006 0.285

169

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Soleus Force 0.034 0.008 0.235 2 CON Soleus Force 0.038 0.009 0.236 3 CON Soleus Force 0.04 0.008 0.2 4 CON Soleus Force 0.032 0.008 0.25 5 CON Soleus Force 0.049 0.013 0.265 7 CON HMB Soleus Force 0.04 0.01 0.25 8 CON HMB Soleus Force 0.037 0.008 0.216 9 CON HMB Soleus Force 0.025 0.006 0.24 10 CON HMB Soleus Force 0.023 0.006 0.260 11 EX Soleus Force 0.032 0.007 0.218 12 EX Soleus Force 0.02 0.005 0.25 13 EX Soleus Force 0.035 0.009 0.257 14 EX Soleus Force 0.031 0.007 0.225 15 EX Soleus Force 0.039 0.01 0.256 16 EX HMB Soleus Force 0.032 0.008 0.25 17 EX HMB Soleus Force 0.03 0.007 0.233 18 EX HMB Soleus Force 0.024 0.005 0.208 19 EX HMB Soleus Force 0.03 0.008 0.266 20 EX HMB Soleus Force 0.033 0.01 0.303

170

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Plantaris Non-Force 0.035 0.01 0.285 2 CON Plantaris Non-Force 0.023 0.005 0.217 3 CON Plantaris Non-Force 0.036 0.009 0.25 4 CON Plantaris Non-Force 0.024 0.005 0.208 5 CON Plantaris Non-Force 0.037 0.009 0.243 7 CON HMB Plantaris Non-Force 0.025 0.006 0.24 8 CON HMB Plantaris Non-Force 0.041 0.009 0.219 9 CON HMB Plantaris Non-Force 0.019 0.005 0.263 10 CON HMB Plantaris Non-Force 0.043 0.012 0.279 11 EX Plantaris Non-Force 0.044 0.01 0.227 12 EX Plantaris Non-Force 0.042 0.006 0.142 13 EX Plantaris Non-Force 0.059 0.014 0.237 14 EX Plantaris Non-Force 0.029 0.006 0.206 15 EX Plantaris Non-Force 0.051 0.011 0.215 16 EX HMB Plantaris Non-Force 0.049 0.012 0.244 17 EX HMB Plantaris Non-Force 0.041 0.008 0.195 18 EX HMB Plantaris Non-Force 0.051 0.011 0.215 19 EX HMB Plantaris Non-Force 0.047 0.011 0.234 20 EX HMB Plantaris Non-Force 0.049 0.013 0.265

171

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Plantaris Force 0.065 0.013 0.2 2 CON Plantaris Force 0.082 0.019 0.231 3 CON Plantaris Force 0.075 0.019 0.253 4 CON Plantaris Force 0.055 0.014 0.254 5 CON Plantaris Force 0.031 0.008 0.258 7 CON HMB Plantaris Force 0.06 0.013 0.216 8 CON HMB Plantaris Force 0.047 0.01 0.212 9 CON HMB Plantaris Force 0.031 0.007 0.225 10 CON HMB Plantaris Force 0.023 0.006 0.260 11 EX Plantaris Force 0.043 0.009 0.209 12 EX Plantaris Force 0.046 0.009 0.195 13 EX Plantaris Force 0.048 0.009 0.187 14 EX Plantaris Force 0.023 0.004 0.173 15 EX Plantaris Force 0.05 0.012 0.24 16 EX HMB Plantaris Force 0.041 0.009 0.219 18 EX HMB Plantaris Force 0.043 0.01 0.232 19 EX HMB Plantaris Force 0.026 0.005 0.192 20 EX HMB Plantaris Force 0.03 0.006 0.2

172

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Quadriceps Muscle Group Non-Force 0.037 0.01 0.270 2 CON Quadriceps Muscle Group Non-Force 0.041 0.011 0.268 3 CON Quadriceps Muscle Group Non-Force 0.05 0.012 0.24 4 CON Quadriceps Muscle Group Non-Force 0.036 0.01 0.277 5 CON Quadriceps Muscle Group Non-Force 0.044 0.012 0.272 7 CON HMB Quadriceps Muscle Group Non-Force 0.06 0.017 0.283 8 CON HMB Quadriceps Muscle Group Non-Force 0.036 0.01 0.277 9 CON HMB Quadriceps Muscle Group Non-Force 0.055 0.014 0.254 10 CON HMB Quadriceps Muscle Group Non-Force 0.031 0.011 0.354 11 EX Quadriceps Muscle Group Non-Force 0.044 0.014 0.318 13 EX Quadriceps Muscle Group Non-Force 0.041 0.012 0.292 14 EX Quadriceps Muscle Group Non-Force 0.037 0.012 0.324 15 EX Quadriceps Muscle Group Non-Force 0.028 0.008 0.285 16 EX HMB Quadriceps Muscle Group Non-Force 0.052 0.013 0.25 17 EX HMB Quadriceps Muscle Group Non-Force 0.037 0.01 0.270 18 EX HMB Quadriceps Muscle Group Non-Force 0.039 0.01 0.256 19 EX HMB Quadriceps Muscle Group Non-Force 0.053 0.013 0.245 20 EX HMB Quadriceps Muscle Group Non-Force 0.032 0.008 0.25

173

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Quadriceps Muscle Group Force 0.063 0.013 0.206 2 CON Quadriceps Muscle Group Force 0.044 0.009 0.204 3 CON Quadriceps Muscle Group Force 0.066 0.014 0.212 4 CON Quadriceps Muscle Group Force 0.034 0.006 0.176 5 CON Quadriceps Muscle Group Force 0.046 0.011 0.239 7 CON HMB Quadriceps Muscle Group Force 0.038 0.009 0.236 8 CON HMB Quadriceps Muscle Group Force 0.059 0.013 0.220 9 CON HMB Quadriceps Muscle Group Force 0.029 0.007 0.241 10 CON HMB Quadriceps Muscle Group Force 0.039 0.01 0.256 11 EX Quadriceps Muscle Group Force 0.032 0.008 0.25 12 EX Quadriceps Muscle Group Force 0.033 0.009 0.272 13 EX Quadriceps Muscle Group Force 0.053 0.012 0.226 14 EX Quadriceps Muscle Group Force 0.033 0.007 0.212 15 EX Quadriceps Muscle Group Force 0.035 0.005 0.142 16 EX HMB Quadriceps Muscle Group Force 0.054 0.014 0.259 17 EX HMB Quadriceps Muscle Group Force 0.045 0.011 0.244 18 EX HMB Quadriceps Muscle Group Force 0.04 0.008 0.2 19 EX HMB Quadriceps Muscle Group Force 0.036 0.009 0.25 20 EX HMB Quadriceps Muscle Group Force 0.031 0.007 0.225

174

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Semitendinosus force 0.034 0.009 0.264 2 CON Semitendinosus Non-Force 0.04 0.011 0.275 3 CON Semitendinosus Non-Force 0.058 0.014 0.241 4 CON Semitendinosus Non-Force 0.041 0.011 0.268 5 CON Semitendinosus Non-Force 0.054 0.014 0.259 7 CON HMB Semitendinosus Non-Force 0.047 0.013 0.276 8 CON HMB Semitendinosus Non-Force 0.031 0.009 0.290 9 CON HMB Semitendinosus Non-Force 0.051 0.014 0.274 10 CON HMB Semitendinosus Non-Force 0.06 0.009 0.15 11 EX Semitendinosus Non-Force 0.068 0.017 0.25 12 EX Semitendinosus Non-Force 0.049 0.013 0.265 13 EX Semitendinosus Non-Force 0.066 0.017 0.257 14 EX Semitendinosus Non-Force 0.066 0.015 0.227 15 EX Semitendinosus Non-Force 0.05 0.012 0.24 16 EX HMB Semitendinosus Non-Force 0.028 0.006 0.214 17 EX HMB Semitendinosus Non-Force 0.043 0.01 0.232 18 EX HMB Semitendinosus Non-Force 0.051 0.014 0.274 19 EX HMB Semitendinosus Non-Force 0.053 0.013 0.245 20 EX HMB Semitendinosus Non-Force 0.029 0.009 0.310

175

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Semitendinosus force 0.034 0.009 0.264 2 CON Semitendinosus Force 0.046 0.011 0.239 3 CON Semitendinosus Force 0.1 0.026 0.26 4 CON Semitendinosus Force 0.033 0.008 0.242 7 CON HMB Semitendinosus Force 0.051 0.012 0.235 8 CON HMB Semitendinosus Force 0.053 0.016 0.301 9 CON HMB Semitendinosus Force 0.057 0.011 0.192 10 CON HMB Semitendinosus Force 0.048 0.013 0.270 11 EX Semitendinosus Force 0.022 0.003 0.136 12 EX Semitendinosus Force 0.08 0.02 0.25 13 EX Semitendinosus Force 0.024 0.005 0.208 14 EX Semitendinosus Force 0.045 0.008 0.177 15 EX Semitendinosus Force 0.023 0.003 0.130 16 EX HMB Semitendinosus Force 0.029 0.006 0.206 17 EX HMB Semitendinosus Force 0.072 0.016 0.222 18 EX HMB Semitendinosus Force 0.021 0.003 0.142 19 EX HMB Semitendinosus Force 0.022 0.001 0.045 20 EX HMB Semitendinosus Force 0.045 0.009 0.2

176

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Semimembranosus Non-Force 0.109 0.026 0.238 2 CON Semimembranosus Non-Force 0.107 0.025 0.233 3 CON Semimembranosus FORCE 0.045 0.01 0.222 3 CON Semimembranosus Non-Force 0.067 0.019 0.283 4 CON Semimembranosus Non-Force 0.076 0.017 0.223 5 CON Semimembranosus Non-Force 0.054 0.014 0.259 7 CON HMB Semimembranosus Non-Force 0.031 0.006 0.193 8 CON HMB Semimembranosus Non-Force 0.055 0.011 0.2 9 CON HMB Semimembranosus Non-Force 0.066 0.015 0.227 10 CON HMB Semimembranosus Non-Force 0.075 0.017 0.226 11 EX Semimembranosus Non-Force 0.077 0.017 0.220 12 EX Semimembranosus Non-Force 0.033 0.007 0.212 13 EX Semimembranosus Non-Force 0.018 0.005 0.277 14 EX Semimembranosus Non-Force 0.051 0.008 0.156 15 EX Semimembranosus Non-Force 0.084 0.019 0.226 16 EX HMB Semimembranosus Non-Force 0.035 0.009 0.257 17 EX HMB Semimembranosus Non-Force 0.058 0.011 0.189 18 EX HMB Semimembranosus Non-Force 0.026 0.006 0.230 19 EX HMB Semimembranosus Non-Force 0.035 0.009 0.257 20 EX HMB Semimembranosus Non-Force 0.051 0.012 0.235

177

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Semimembranosus Force 0.057 0.013 0.228 2 CON Semimembranosus Force 0.023 0.005 0.217 4 CON Semimembranosus Force 0.039 0.009 0.230 5 CON Semimembranosus Force 0.056 0.014 0.25 7 CON HMB Semimembranosus Force 0.057 0.013 0.228 8 CON HMB Semimembranosus Force 0.061 0.014 0.229 9 CON HMB Semimembranosus Force 0.046 0.012 0.260 10 CON HMB Semimembranosus Force 0.024 0.006 0.25 11 EX Semimembranosus Force 0.068 0.014 0.205 12 EX Semimembranosus Force 0.042 0.009 0.214 13 EX Semimembranosus Force 0.026 0.004 0.153 14 EX Semimembranosus Force 0.038 0.009 0.236 15 EX Semimembranosus Force 0.099 0.021 0.212 16 EX HMB Semimembranosus Force 0.04 0.009 0.225 17 EX HMB Semimembranosus Force 0.042 0.008 0.190 18 EX HMB Semimembranosus Force 0.046 0.01 0.217 19 EX HMB Semimembranosus Force 0.048 0.01 0.208 20 EX HMB Semimembranosus Force 0.022 0.004 0.181

178

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 2 CON Biceps Femoris Non-Force 0.051 0.011 0.215 3 CON Biceps Femoris Non-Force 0.052 0.015 0.288 4 CON Biceps Femoris Non-Force 0.032 0.008 0.25 5 CON Biceps Femoris Non-Force 0.087 0.024 0.275 7 CON HMB Biceps Femoris Non-Force 0.037 0.008 0.216 8 CON HMB Biceps Femoris Non-Force 0.07 0.017 0.242 9 CON HMB Biceps Femoris Non-Force 0.087 0.021 0.241 10 CON HMB Biceps Femoris Non-Force 0.07 0.017 0.242 11 EX Biceps Femoris Non-Force 0.05 0.012 0.24 12 EX Biceps Femoris Non-Force 0.073 0.017 0.232 13 EX Biceps Femoris Non-Force 0.04 0.008 0.2 14 EX Biceps Femoris Non-Force 0.055 0.014 0.254 15 EX Biceps Femoris Non-Force 0.059 0.014 0.237 16 EX HMB Biceps Femoris Non-Force 0.083 0.02 0.240 17 EX HMB Biceps Femoris Non-Force 0.046 0.011 0.239 18 EX HMB Biceps Femoris Non-Force 0.047 0.008 0.170 19 EX HMB Biceps Femoris Non-Force 0.032 0.006 0.187

179

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Biceps Femoris Force 0.081 0.018 0.222 3 CON Biceps Femoris Force 0.048 0.011 0.229 4 CON Biceps Femoris Force 0.144 0.033 0.229 5 CON Biceps Femoris Force 0.157 0.035 0.222 7 CON HMB Biceps Femoris Force 0.055 0.012 0.218 8 CON HMB Biceps Femoris Force 0.045 0.011 0.244 10 CON HMB Biceps Femoris Force 0.163 0.036 0.220 11 EX Biceps Femoris Force 0.2 0.044 0.22 12 EX Biceps Femoris Force 0.121 0.022 0.181 14 EX Biceps Femoris Force 0.174 0.04 0.229 15 EX Biceps Femoris Force 0.037 0.008 0.216 16 EX HMB Biceps Femoris Force 0.09 0.02 0.222 17 EX HMB Biceps Femoris Force 0.119 0.028 0.235 18 EX HMB Biceps Femoris Force 0.196 0.045 0.229 19 EX HMB Biceps Femoris Force 0.107 0.021 0.196 20 EX HMB Biceps Femoris Force 0.124 0.028 0.225

180

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Gluteus Medius Force 0.033 0.008 0.242 2 CON Gluteus Medius Force 0.061 0.014 0.229 3 CON Gluteus Medius Force 0.056 0.013 0.232 4 CON Gluteus Medius Force 0.054 0.015 0.277 5 CON Gluteus Medius Force 0.053 0.012 0.226 7 CON HMB Gluteus Medius Force 0.051 0.012 0.235 8 CON HMB Gluteus Medius Force 0.029 0.006 0.206 9 CON HMB Gluteus Medius Force 0.042 0.008 0.190 10 CON HMB Gluteus Medius Force 0.035 0.006 0.171 11 EX Gluteus Medius Force 0.04 0.008 0.2 12 EX Gluteus Medius Force 0.043 0.009 0.209 13 EX Gluteus Medius Force 0.046 0.009 0.195 14 EX Gluteus Medius Force 0.065 0.014 0.215 15 EX Gluteus Medius Force 0.037 0.008 0.216 16 EX HMB Gluteus Medius Force 0.03 0.007 0.233 17 EX HMB Gluteus Medius Force 0.048 0.009 0.187 18 EX HMB Gluteus Medius Force 0.052 0.012 0.230 19 EX HMB Gluteus Medius Force 0.032 0.007 0.218 20 EX HMB Gluteus Medius Force 0.056 0.012 0.214

181

Wet Dry Animal Force/Non- Weight Weight Number Treatment Muscle Force Leg (mg) (mg) Ratio 1 CON Gluteus Medius Non-Force 0.097 0.026 0.268 2 CON Gluteus Medius Non-Force 0.173 0.043 0.248 3 CON Gluteus Medius Non-Force 0.124 0.03 0.241 4 CON Gluteus Medius Non-Force 0.128 0.036 0.281 5 CON Gluteus Medius Non-Force 0.101 0.026 0.257 8 CON HMB Gluteus Medius Non-Force 0.06 0.014 0.233 9 CON HMB Gluteus Medius Non-Force 0.101 0.024 0.237 10 CON HMB Gluteus Medius Non-Force 0.092 0.022 0.239 11 EX Gluteus Medius Non-Force 0.119 0.025 0.210 12 EX Gluteus Medius Non-Force 0.09 0.022 0.244 13 EX Gluteus Medius Non-Force 0.113 0 14 EX Gluteus Medius Non-Force 0.116 0.027 0.232 15 EX Gluteus Medius Non-Force 0.165 0.066 0.4 16 EX HMB Gluteus Medius Non-Force 0.098 0.022 0.224 17 EX HMB Gluteus Medius Non-Force 0.131 0.03 0.229 18 EX HMB Gluteus Medius Non-Force 0.1 0.025 0.25 19 EX HMB Gluteus Medius Non-Force 0.094 0.022 0.234 20 EX HMB Gluteus Medius Non-Force 0.069 0.025 0.362

182

Wet Dry Animal Weight Weight Number Treatment Muscle (mg) (mg) Ratio 2 CON Wrist Flexors 0.076 0.019 0.25 3 CON Wrist Flexors 0.13 0.032 0.246 4 CON Wrist Flexors 0.123 0.031 0.252 7 CON HMB Wrist Flexors 0.109 0.025 0.229 8 CON HMB Wrist Flexors 0.091 0.021 0.230 9 CON HMB Wrist Flexors 0.081 0.02 0.246 10 CON HMB Wrist Flexors 0.085 0.022 0.258 11 EX Wrist Flexors 0.102 0.028 0.274 12 EX Wrist Flexors 0.147 0.035 0.238 13 EX Wrist Flexors 0.083 0.022 0.265 14 EX Wrist Flexors 0.112 0.021 0.187 15 EX Wrist Flexors 0.053 0.012 0.226 16 EX HMB Wrist Flexors 0.14 0.038 0.271 18 EX HMB Wrist Flexors 0.04 0.009 0.225 19 EX HMB Wrist Flexors 0.092 0.017 0.184 20 EX HMB Wrist Flexors 0.088 0.022 0.25

183 Gastrocnemius Medial Head Non-Force Measured Leg Total Total Total Phosphorylated Total Phosphorylated Total Total Total Protein Protein Total Protein Protein Total Protein Protein Protein Animal Relative to Relative to Activity of Relative to Relative to Activity of Relative to Relative to Relative to Number Treatment Standard Standard Protein Standard Standard Protein Standard Standard Standard pFoxO3A MAFbx / Akt pAKT pAkt/Akt FoxO3A pFoxO3A /FoxO3A atrogin-1 MuRF1 GAPDH 1 CON 1.8248704 2.2289596 1.2214345 3.2726941 1.7985329 0.5495573 1.7123635 0.8074157 0.8454534 2 CON 1.5276959 2.0464492 1.3395659 2.4006176 0.7413796 0.3088287 1.6678693 1.4094463 0.8355427 3 CON 1.1488028 1.5500193 1.3492475 1.4926506 1.2477247 0.8359121 0.9970321 7.3010353 0.9267083 4 CON 0.8710615 1.3161907 1.5110193 1.7044109 1.1255604 0.6603809 0.8185379 5.4967808 1.0063300 5 CON 1.0121769 1.9582717 1.9347128 1.2768691 0.7204579 0.5642379 0.9002867 2.8408404 0.9144641 7 CON HMB 0.6727299 0.9520856 1.4152567 1.2436266 0.8657374 0.6961393 0.4721230 3.6910602 0.7950499 8 CON HMB 1.3500119 1.4894227 1.1032664 0.8896483 1.0522181 1.1827349 0.9343578 1.2803052 0.8877384 9 CON HMB 0.9181415 1.5834282 1.7246014 0.8618605 1.3996644 1.6240034 0.8548631 1.1113511 0.7031860 10 CON HMB 5.0111737 1.3384268 0.2670885 1.3135242 1.5675901 1.1934230 1.2022517 0.9710018 1.0096888 11 EX 1.3904511 2.0318945 1.4613204 2.4159746 0.6502835 0.2691599 1.4106323 0.7149733 0.8475454 12 EX 1.4394712 1.2116557 0.8417367 2.1260568 1.1924477 0.5608729 1.5857653 1.0683226 1.0303791 13 EX 0.8524696 1.5237479 1.7874514 1.5009470 0.8661036 0.5770381 0.7174916 4.6243787 0.8032013 14 EX 1.3570297 1.9416745 1.4308269 2.3447849 1.8586636 0.7926798 1.7695974 4.3100919 1.0315444 15 EX 1.7485332 2.4261826 1.3875531 2.3169780 0.9436139 0.4072606 1.0616411 2.0628571 0.9940512 16 EX HMB 1.4527285 1.6315055 1.1230629 1.6954098 0.5893433 0.3476111 0.6443358 1.8974654 0.8495424 17 EX HMB 1.5160032 2.7849642 1.8370437 1.6894914 2.8178928 1.6678941 1.5383421 0.6791389 1.0278577 18 EX HMB 1.6974385 2.0340687 1.1983166 1.2878941 1.8582698 1.4428747 1.3941138 0.8804103 0.7270153 19 EX HMB 3.9169731 2.0217151 0.5161422 1.8515597 1.8221851 0.9841352 1.5036844 1.3650189 1.1714838 20 EX HMB 1.9192445 0.5939823 0.3094875 0.8049781 1.3722429 1.7046960 0.7123362 0.6087465 0.9106372 184 Quadriceps Muscle Group Non-Force Measured Leg Total Total Total Phosphorylated Total Phosphorylated Total Total Total Protein Protein Total Protein Protein Total Protein Protein Protein Animal Relative to Relative to Activity of Relative to Relative to Activity of Relative to Relative to Relative to Number Treatment Standard Standard Protein Standard Standard Protein Standard Standard Standard pFoxO3A/ MAFbx / Akt pAKT pAkt/Akt FoxO3A pFoxO3A FoxO3A atrogin-1 MuRF1 GAPDH 1 CON 4.7966043 2.4033736 0.5010573 5.1985998 2.3554899 0.4531008 1.7422675 0.6330965 0.9855891 2 CON 4.5816530 1.7494063 0.3818286 5.6037227 1.7452250 0.3114403 0.9631510 0.8656812 0.8544938 3 CON 2.0567392 4.3636942 2.1216565 1.6182297 1.5521535 0.9591676 1.1850141 0.8478735 1.0039607 4 CON 2.9483361 2.5452373 0.8632792 1.6152023 1.3405674 0.8299687 0.6210434 0.8632944 0.7304037 5 CON 1.7780235 1.7472874 0.9827133 5.0453022 1.0312972 0.2044074 1.0708292 1.3959668 0.8374694 7 CON HMB 1.1847566 1.0184438 0.8596228 1.6928517 0.4895540 0.2891889 0.4602222 0.7969367 0.7924990 8 CON HMB 2.7406833 1.5781920 0.5758389 2.8014599 1.2582717 0.4491486 1.2097893 0.8371565 0.8581087 9 CON HMB 1.1544124 0.9897438 0.8573572 1.7832862 1.2898099 0.7232770 0.7647279 0.7415266 0.9479196 10 CON HMB 2.2468833 1.9155012 0.8525148 2.3146041 1.4825424 0.6405166 4.5574745 3.7093087 1.0071554 11 EX 5.6698932 1.2511248 0.2206611 2.3379174 1.4234553 0.6088561 0.5604296 0.6823678 1.1206114 13 EX 4.1575360 1.6947949 0.4076441 3.0673125 1.5924891 0.5191806 0.8159539 0.4725397 0.9280826 14 EX 1.3401388 2.7300948 2.0371731 1.2598134 0.9685250 0.7687845 0.3877720 0.7548905 0.9138557 15 EX 3.5467826 2.9401858 0.8289727 2.1039346 1.2050997 0.5727838 1.2021407 1.3654220 1.2916593 16 EX HMB 1.4217537 1.5015977 1.0561588 3.6606296 0.7390776 0.2018990 0.6278644 0.9342336 0.9174599 17 EX HMB 1.6050777 1.4653397 0.9129401 2.3907251 0.7413865 0.3101095 0.5102157 0.7967531 0.7292454 18 EX HMB 1.6214205 1.5294227 0.9432610 1.8663939 1.1275246 0.6041193 0.8547982 0.9584491 0.6316652 19 EX HMB 2.1438703 1.3851882 0.6461157 3.9610902 1.4066841 0.3551255 0.7933772 0.9279928 1.1998964 20 EX HMB 1.4784513 0.8526594 0.5767247 1.0734717 0.7967396 0.7422083 2.5943045 2.0086054 0.7330545

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215 VITA

Geoffrey Josef Solares was born in California. After finishing high school he continued his studies at California State University Northridge. It was at this college that he received both a Bachelor of Science and Bachelor of Arts degree in kinesiology and psychology, respectively in 2009. Following his undergraduate graduation he began graduate school at California State University Northridge, and began working in the laboratory of Benjamin B. Yaspelkis, III. In 2011, he received his Masters of Science degree in kinesiology. In January 2012, he moved with his wife to Austin Texas and began his doctoral studies at The University of Texas at Austin in the laboratory of Dr. John Ivy. In 2017, following Dr. Ivy’s retirement, he transferred to the laboratory of Dr. Roger P. Farrar where he concluded his doctoral studies.

Email address: [email protected] This dissertation was typed by Geoffrey J. Solares

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