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2018 The Effect of Resistance Exercise and Protein Timing on Lipolysis and Fat Oxidation in Resistance-Trained Women Brittany Rose Allman

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COLLEGE OF HUMAN SCIENCES

THE EFFECT OF RESISTANCE EXERCISE AND PROTEIN TIMING ON

LIPOLYSIS AND FAT OXIDATION IN RESISTANCE-TRAINED WOMEN

BRITTANY ROSE ALLMAN

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

2018

Brittany Rose Allman defended this dissertation on April 4, 2018.

The members of her supervisory committee were:

Michael J. Ormsbee

Professor Directing Dissertation

Robert J. Contreras

University Representative

Jeong-Su Kim

Committee Member

Lynn B. Panton

Committee Member

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

ACKNOWLEDGEMENTS

I want to thank my Dad, Jerome for supporting me through this 11-year journey of education. I also want to thank my Mom, Sheila and my sisters, Abby, Melissa and Jamie for their support and love. Each of you are so different and contribute to my life in different ways. I cannot thank you enough. My best friend, Amber Kinsey has supported my academically and throughout my life journey for the past five years, and I am incredibly indebted to her. I want to thank my advisor, Dr. Michael Ormsbee for his patience and understanding through my learning process, and for his support on and off the court. Also, my dissertation committee, Dr. Jeong-Su Kim, Dr. Lynn Panton, and Dr. Robert Contreras for answering questions and always having an open door. Dr. Hickner provided the backbone for the technique that we used for this project and was always willing to lend a helping hand over the phone or in person. Maggie Morrissey was my right-hand girl during data collection and I am so thankful because she was able to tell me what I needed before I could even get the words out! Thank you for being my partner! A big shout-out to my research team in the Human Performance and Sports Nutrition Lab at FSU – without them, the moving parts of this project would have been a mess! Retrospectively, I would like to thank the Director and Co-Director of the Ronald E. McNair Post Baccalaureate Achievement Program, Dr. Calvin Masilela and Dr. Hillary Staples. Without the support of this program when I was an undergraduate at Indiana University of Pennsylvania, I would not have even thought I could go to graduate school. I would also like to thank all of my friends past and present. Without each of your unique qualities that you bring to my life, my life would be a little less fun. Thank you for everything! I also want to thank FrieslandCampina and Dymatize Nutrition for their financial support in this study.

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TABLE OF CONTENTS

List of Tables ...... vi List of Figures ...... vii List of Abbreviations ...... viii Operational Definitions ...... xii Abstract ...... xiv

CHAPTER ONE: INTRODUCTION ...... 1

1.1 Background ...... 1 1.2 Purpose ...... 4 1.3 Specific Aims and Research Hypothesis...... 4 1.4 Assumptions ...... 6 1.5 Delimitations ...... 6 1.6 Limitations ...... 6

CHAPTER TWO: REVIEW OF LITERATURE ...... 8

2.1 Adipose Tissue Physiology ...... 8 2.2 Muscle Physiology ...... 25 2.3 The Effects of Sex on Fat and Muscle Metabolism ...... 30 2.4 The Effects of Exercise on Fat and Muscle Metabolism ...... 46 2.5 Nutrient Timing in Conjunction with Exercise ...... 58 2.6 Summary ...... 75

CHAPTER THREE: METHODS ...... 77

3.1 Participants ...... 77 3.2 Research Design ...... 77 3.3 Procedures ...... 79 3.4 Statistical Analysis ...... 89

CHAPTER FOUR: RESULTS AND DISCUSSION ...... 90 4.1 Results ...... 90 4.2 Discussion ...... 99

APPENDICES ...... 111

A. Institutional Review Board Approval of Study Protocol ...... 111 B. Informed Consent ...... 115 C. Health and Medical History Questionnaire ...... 122 D. Food Log ...... 125 E. Physical Activity Record ...... 128 F. Visual Analogue Scale for Fatigue and Soreness ...... 133 G. Visual Analogue Scale for Hunger, Satiety and Fullness ...... 135

H. Pregnancy Consent Form ...... 137 I. Modified Resistance Training Rate of Perceived Exertion Scale ...... 138

References ...... 139

Biographical Sketch ...... 170

LIST OF TABLES

Table 1. Effects of pre-sleep consumption of a small meal or snack ...... 67

Table 2. Descriptive characteristics of participants measured at baseline (N=13) ...... 90

Table 3. Average dietary intake for 24 hours before each visit ...... 91

Table 4. VAS measurements of fatigue and soreness of the upper and lower body before PLA- PRO and PRO-PLA condition visits ...... 91

Table 5. Sleep quantity and quality measurements ...... 92

Table 6. Dietary intake during PLA-PRO and PRO-PLA conditions ...... 93

Table 7. Metabolism measurements at baseline and post resistance exercise ...... 95

Table 8. Plasma biomarkers at baseline, mid resistance training, and post resistance exercise….97

Table 9. Overnight interstitial glycerol concentrations in PLA-PRO and PRO-PLA conditions...97

Table 10. Measurements of resting energy expenditure after the pre-sleep pre-sleep supplement and the next morning compared to baseline in PLA-PRO and PRO-PLA conditions...... 98

Table 11. Plasma markers at baseline, immediately post pre-sleep supplement, and next morning in PLA-PRO and PRO-PLA conditions ...... 99

LIST OF FIGURES

Figure 1. De novo lipogenesis pathway ...... 11

Figure 2. Mechanisms of TAG storage in WAT...... 12

Figure 3. Steps preceding beta-oxidation in the muscle cell ...... 15

Figure 4. The steps of beta-oxidation ...... 16

Figure 5. The insulin tyrosine-kinase receptor cascade for translocation of glucose transporter 4 (GLUT-4) ...... 20

Figure 6. Mobilization of fatty acids due to the interaction between perilipin and HSL in an adipocyte ...... 23

Figure 7. Stimulation of mTOR by limited oxygen, limited energy, and amino acids ...... 28

Figure 8. Effects of myostatin on myofiber synthesis ...... 30

Figure 9. Steroidogenesis ...... 32

Figure 10. Timeline of study ...... 79

Figure 11. Timeline Aim 1 ...... 80

Figure 12. Timeline Aim 2 ...... 81

Figure 13. The effect of resistance exercise on interstitial glycerol concentration ...... 94

Figure 14. Individual interstitial glycerol concentration responses to resistance exercise...... 95

Figure 15. Energy expenditure, respiratory exchange ratio, and fat oxidation before and after resistance exercise ...... 96

Figure 16. SCAAT lipolytic during PLA-PRO and PRO-PLA conditions ...... 98

Figure 17. Plasma glucose, insulin, glycerol, and NEFA concentrations before, during and after resistance exercise during PLA-PRO and PRO-PLA conditions...... 102

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LIST OF ABBREVIATIONS

1RM, 1 repetition maximum; 3β-HSD, 3β-hydroxysteroid dehydrogenase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; α-AR, alpha adrenergic receptor; ACC, acetyl CoA carbozylate; ACL, adenosine triphosphate citrate lyase; AKT, protein kinase B; AMP, adeonosine monophosphate; ATGL, adipose triglyceride lipase; ATP, adenosine triphosphate; β-AR, beta adrenergic receptor; BMI, body mass index; BP, blood pressure; cAMP, cyclic adenosine monophosphate; CAT-1, carnitine acyltransferase-1; CAT-2, carnitine acyltransferase-2; CHO, carbohydrate; CHY, chylomicron;

CO 2, carbon dioxide; DAG, diacylglycerol; Deptor, DEP-domain-containing mTOR-interacting protein; DHEA, dehydroepiandrosterone; DXA, dual energy x-ray absorptiometry; E2, estrogen; Epi, epinephrine; ETC, electron transport chain; ER, estrogen receptor; Erα, estrogen receptor alpha; Erβ, estrogen receptor beta; ET, endurance training;

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FA, fatty acid;

FADH 2, flavin adenine dinucleotide; FAS, fatty acid synthase; FATP4, fatty acid transport protein 4; FFA, free fatty acid; FoxO, Forkhead box O; GβL, mammalian lethal with Sec13 protein 8; GAP, GTPase-activating protein; GDP, guanosine diphosphate; GH, growth hormone; GLUT4, glucose transporters; GLY, glycerol; GTP, guanosine triphosphate; HOMA-IR, the homeostatic model of insulin resistance; HR, heart rate;

HR max , maximum heart rate; HSL, hormone sensitive lipase; IDL, intermediate density lipoprotein; IRS-1, insulin receptor substrate 1; LBM, lean body mass; LDL, low density lipoprotein; LEU, leucine; LPL, lipoprotein lipase; MAG, monoacylglycerol; MAPK, mitogen-activated protein kinase; MGL, monoacylglycerol lipase; MPB, muscle protein breakdown; MPS, muscle protein synthesis; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of rapamycin complex 2;

NADH, nicotineamide-adenine dinucleotide; NADH+H, nicotineamide-adenine dinucleotide plus hydrogen ion; NE, norepinephrine; NEFA, non-esterified fatty acids;

O2, oxygen; OC, oral contraceptives; P4, progesterone; P450, cytochrome P450; PHD, pyruvate dehydrogenase; PI3K, phosphatidyl inositol kinase-3;

PIP 2, 4,5-bisphosphonate;

PIP 3, 3,4,5-bisphosphonate; PKA, protein kinase A; PLA, non-caloric placebo; PRAS40, proline-rich Akt substrate 40 kDa; PRO, protein; Raptor, regulatory-associated protein of mammalian target of rapamycin; REDD1, DNA damage response 1; REE, resting energy expenditure; RER, respiratory exchange ratio; Rheb, Ras homolog enriched in brain; RMR, resting metabolic rate; RQ, respiratory quotient; RT, resistance training; SCAAT, subcutaneous abdominal adipose tissue; SLC1A5, solute carrier family 1 member 5 neutral amino acid transporter; SLC3A2, solute carrier family 3 member 2; SLC7A5, antiport solute carrier family 7 member 5; T, testosterone; TAG, triacylglycerols; TBC1D4, TBC1 domain family 4;

TEE, thermic effect of exercise; TEF, thermic effect of food; TSC, tuberous sclerosis complex; TSC1, hamartin; TSC2, tuberin; VLDL, very low density lipoproteins;

VO 2max , maximal oxygen consumption; WAT, white adipose tissue;

OPERATIONAL DEFINITIONS

Anabolic: tissue building processes; opposite of catabolic (235)

Beta oxidation: energy production from a fatty acid; cyclic series of steps that cleaves off double carbon units from free fatty acids, which are used to form acetyl CoA to enter the Kreb’s cycle (235)

Body mass index (BMI): index of body fatness; equal to body weight in kilograms divided by height in meters squared (235)

Casein (micellar): labeled a slow digesting protein because it clots in the stomach which slows gastric emptying and subsequently prolongs postprandial plasma amino acid appearance (46)

Catabolic: tissue degradation processes; opposite of anabolic (235)

De novo lipogenesis: production of fat from non-fat sources including amino acids and glucose (111)

Esterification: process of triacylglycerol synthesis; attaching three fatty acids to a glycerol 3- phosphate backbone (235)

Ethanol: an alcohol; used to monitor local blood flow in adipose tissue as it is not readily metabolized. Blood flow is inversely related to the ratio of ethanol outflow to inflow (152)

Gluconeogenesis: production of glucose from non-carbohydrate sources; reverse of glycolysis (235)

Glycolysis: breakdown of a glucose molecule into two molecules of pyruvate (235)

Lipogenesis: formation of fatty acids from acetyl CoA; occurs in adipose or liver tissue (235)

Lipolysis: breakdown of triglycerides into glycerol and fatty acids (235)

Metabolism: sum and product of all anabolic and catabolic energy transformations in a living organism

Muscle protein balance: the product of muscle protein synthesis and muscle protein breakdown

Muscle protein synthesis: building of muscle tissue

Muscle protein breakdown: breakdown of muscle tissue

Non-esterified fatty acids (NEFA): free fatty acids (235)

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Palmitate: final product of fatty acid synthesis (lipogenesis); acts as precursor fatty acid to build longer molecules

Postprandial: time period following the ingestion of nutrients (123)

Resistance training: use of resistance during body movements to induce muscular contraction, which builds the strength, anaerobic endurance, and muscular hypertrophy

Resting metabolic rate (RMR): energy needed to sustain physiological functions at rest; accounts for 60-75% of total daily energy expenditure (235)

Thermic effect of food (TEF): postprandial increase in energy expenditure; accounts for 15-30% of total daily energy expenditure (235); highest after protein consumption and lowest after fat consumption (328)

Triacylglycerol (TAG): molecule composed of three non-esterified fatty acids a glycerol 3- phosphate molecule (235)

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ABSTRACT

The number of women participating in resistance training has increased from 14.4% in 1994 to 21.0% in 2010 (418). Resistance training is known to enhance body composition (20); however, the effects of an acute bout of resistance exercise (RE) on subcutaneous abdominal adipose tissue (SCAAT) glycerol release (lipolysis) and whole-body substrate utilization have only been documented in men (272). In fact, both RE and protein (alone and in combination) have been shown to improve lipolysis and fat oxidation leading to improved overall body composition (20). Mounting evidence suggests there are metabolic benefits of pre-sleep protein (PRO) consumption (65, 101, 190, 193, 274, 276). However, only one study directly assessed the effects of pre-sleep PRO consumption on SCAAT lipolysis and whole-body substrate utilization, and it was performed in overweight/obese men (190). Furthermore, few pre-sleep PRO studies to date have been directly compared to PRO consumed at other times of the day (17). Thus, it is difficult to interpret if the benefit is from pre-sleep feeding or simply increased daily PRO intake. PURPOSE: Therefore, the purpose of the current study was to assess the effects of RE (Aim 1) and pre-sleep versus daytime PRO consumption (Aim 2) on SCAAT lipolysis and whole-body substrate utilization in resistance-trained women. For Aim 1, a one-way ANOVA was used to analyze changes in interstitial GLY concentrations, metabolic rate, and plasma biomarkers around RE compared to baseline (BL) (BL, Mid-RE, post-RE). For Aim 2, a repeated measures ANOVA was used to compare the differences in interstitial GLY concentrations, metabolic rate, and plasma biomarkers between PRO-PLA and PLA-PRO conditions. If a significant finding was noted, a Tukey HSD post-hoc analysis was used to locate where the difference existed. Data were analyzed using SPSS (Version 25) with significance set at p<0.05, and are presented as mean ± standard error (SE). METHODS : Thirteen healthy, resistance-trained, eumenorrheic women (age, 22±3 years) volunteered to participate in the study. Participants reported to the laboratory on five occasions: pre-testing and familiarization of maximal testing (Visit 1), maximal testing (Visit 2), familiarization (Visit 3), and two experimental visits (Visits 4 and 5). For each of the experimental visits, participants came to the laboratory in a fasted state, and microdialysis probes were inserted into the SCAAT to measure lipolysis. Participants then performed a full-body RE session consisting of the following exercises in this order: squat, bench press, Romanian deadlift, bent-over row, shoulder press, reverse lunges. After RE on each of the experimental visit days, participants were randomized to consume either daytime PRO (30

xiv grams of casein protein) 30 minutes post-RE and pre-sleep non-caloric, sensory-matched placebo (PLA, 0 grams of casein protein) (PRO-PLA), or daytime PLA and pre-sleep PRO (PLA-PRO), switching the order of the supplements on the following visit. Participants slept in the laboratory for overnight assessment of lipolysis and dietary intake was controlled by providing participants with breakfast, lunch and dinner (Vale Food Inc., Tallahassee, FL) based on their calculated caloric needs. Resting energy expenditure (REE) and respiratory exchange ratio (RER) were measured at baseline, post-RE, post pre-sleep supplement and the next morning of PLA-PRO and PRO-PLA conditions. Fasted blood samples were collected from the antecubital vein on three occasions for Aim 1: 1) baseline, 2) mid-RE, and 3) post-RE. In addition, blood samples were collected on three occasions for Aim 2: 1) 30 minutes after the daytime supplement (fed); 2) 30 minutes after the pre-sleep supplement (fed), and; 3) the next morning (fasted). Non- esterified fatty acids (NEFA), glycerol, glucose, and insulin were measured for Aim 1 and 2, while catecholamines (CATs) and growth hormone (GH) were measured only for Aim 1. RESULTS : After RE, REE (baseline: 1554±193; post-RE 1772±257 kcal/d; p=0.001) and fat oxidation (FatOx) (baseline: 5.64±0.23; post-RE: 7.57±0.34 g/hr; p<0.001) significantly increased (Aim 1, n=13). Additionally, SCAAT interstitial glycerol concentration was significantly higher at mid-RE (1177.4±667.1 µM, p=0.049) and post-RE (1197.3±1063.4 µM, p=0.01), compared to baseline (596.7±452.3 µM) (n=13). There were no significant changes in plasma biomarkers NEFA, glycerol, glucose, insulin, CATs, or GH. There was a significant increase in REE in both groups compared to baseline but no difference between groups. There was a significant increase in FatOx in PLA-PRO only after consuming the nighttime supplement (baseline: 5.64±0.23 g/min; PLA-PRO: 6.59±0.32 g/min, p=0.02), but no differences between PLA-PRO and PRO-PLA conditions. There were no other differences in lipolysis, metabolic measures, or plasma biomarkers between PRO-PLA or PLA-PRO conditions. RE increased lipolysis and FatOx mid-RE and post-RE in resistance-trained women. There were no differences in fat metabolism throughout sleep between PLA-PRO and PRO-PLA. CONCLUSION : Our findings indicate that RE improves fat metabolism potentially mediated by increases in CATs and GH. Consuming daily PRO 30 min post-RE or 30-min pre-sleep has no additional influence on fat metabolism (does not blunt overnight lipolysis) in resistance-trained women. This study was supported by a research grant from Friesland Campina® and Dymatize® Nutrition.

CHAPTER ONE

INTRODUCTION

1.1 Background

Optimal dietary practices are becoming increasingly integrated with exercise because of the beneficial effects of both practices independently. Nutrition and exercise independently (21, 272) and combined (19) are known to affect fat metabolism and body weight. Specifically, our laboratory has found that resistance training (RT) alone increases lipolytic rate (rate of mobilization of triacylglycerols from fat cells) in resistance-trained men (277); however, the impact of RT on lipolytic rate in resistance-trained women has not been evaluated. Not only does RT alone affect fat metabolism, but protein (PRO) intake alone is known to exert positive effects on lipolytic rate of the subcutaneous abdominal adipose tissue (SCAAT) (1), contributing to improvements in body composition (177). Additionally, although the combination of RT and added PRO has shown promising results on body weight and body composition and metabolism (19), one interesting and new area of focus for research is how eating specifically before bed influences body composition and metabolism. However, no studies have examined the effects of pre-sleep feeding on these variables in resistance trained women. Even though pre-sleep eating has been a common practice in fit individuals for years, it has attracted significant attention in more recent years, particularly for the onslaught of media claiming the negative implications on body weight and overall health. The physiological basis to support the negative claims of pre-sleep eating is multi-fold. For instance, consumption of identical macronutrient composition meals in the evening hours compared to other times of the day elicits significantly lower measures of the thermic effect of food and satiety (64, 328), which ultimately leads to higher calorie intake throughout the day (64). Taken together, these data may suggest potential negative metabolic effects and subsequent body composition changes with pre- sleep eating. Further, adverse health consequences of consuming a large part of daily calories in the evening hours have been found in various populations (73, 415) and in epidemiological data (14, 28, 92). Specifically, individuals with disordered pre-sleep eating may be particularly affected. Neuroendocrine studies have found low levels of plasma melatonin and leptin, as well as high levels of cortisol in obese women with pre-sleep eating syndrome compared to healthy control

1 participants (363), indicating the inability to suppress pre-sleep appetite, and likely the consequential accumulation of fat (280). In addition, individuals with disordered pre-sleep eating display an elevated 24 hour respiratory quotient, indicating less reliance on fat oxidation and more on carbohydrate oxidation, compared to their healthy counterparts (119). Although these populations may be normal weight (41), in a review (415) it was concluded that obesity may be causally related to disordered pre-sleep eating, such that, upon onset, calorically dense food choices often lead to the development of obesity. Thus, it is plausible to assume that normal weight individuals partaking in the chronic practice of dysregulated pre-sleep eating will eventually gain weight. In addition to individuals with disordered pre-sleep eating, night shiftworkers with wake cycles during the evening hours demonstrate similar negative health effects (73, 232). Compared to day shiftworkers, night shiftworkers tend to have an increased risk of overweightness and abdominal obesity (222, 224), as well as impaired glucose tolerance (181, 223). Reductions in total daily energy expenditure and the thermic effect of dinner were found to be associated with weight gain and obesity when night shiftwork was simulated in a six-day experiment in normal weight women (239). The strong association between lack of sleep and increased body mass index (BMI) (103) combined with dysregulated sleep patterns of night shiftworkers and individuals with disordered pre-sleep eating, clearly subject these populations to negative metabolic and body composition ramifications (41, 270). However, the very high caloric intake and carbohydrate- and fat-rich (not PRO) macronutrient profiles of consumed meals are the main issues with rationalizing the negative outcomes experienced in these populations to the general population. Chronically, these patterns of dietary consumption predispose these populations to weight gain and abdominal obesity (14, 18, 118), and negative cardiometabolic outcomes such as elevated insulin concentration and markers of insulin resistance (73, 415). However, it is highly unlikely that the general population practices these same dietary habits. Even more, compared to consumption of large amounts of carbohydrate- and fat-rich foods, the practice of pre-sleep eating much less calorically-dense PRO snacks has been widely utilized in athletic populations such as bodybuilders. Often, these populations partake in heavy resistance training, and thus, added PRO is used to anecdotally maintain metabolism during times in which a catabolic state may prevail (sleep).

In terms of pre-sleep PRO consumption, importantly, metabolic rate (measured by next- morning resting metabolic rate) is not negatively affected (148, 193, 195). Rather studies have even shown elevations in these measures (228, 276), improved respiratory quotient (RQ) (or no change) to reflect greater reliance on fat (228, 276), as well as enhancements in muscle recovery through an increase in muscle protein synthesis and whole body protein turnover (316, 355), positive effects on markers of appetite (193, 195, 228, 276), and no effects on body weight (over 4 weeks) (276). However, even though studies of pre-sleep eating have shown promising metabolic and body composition outcomes, the limiting factor in each of the aforementioned studies is the lack of methodologically matching for PRO timing (190, 193, 228, 276, 316, 355), with the exception of one study (17). For instance, in a study from our laboratory, overweight and obese men consumed casein PRO or a non-nutritive placebo before bed (191). Not only did the PRO group consume more PRO, but there was no daytime PRO consumption trial to make effective comparisons. Likewise, in a study by Res et al., (316) after a RT protocol, participants consumed a bolus of either PRO or carbohydrate placebo before bed. Although both groups consumed a 24-hour standardized diet providing 1.2 g/kg of body weight in PRO, the PRO group consumed 40 g more PRO than the carbohydrate placebo group. In total, no studies have compared a pre-sleep PRO feeding trial to a daytime PRO feeding trial, and therefore, ultimately, it is unclear whether any reported benefits of the published pre-sleep feeding studies were a consequence of the addition of PRO calories (regardless of the time of day), or the consumption of PRO at night. Thus, to make effective comparisons and conclusions, it is imperative to study the metabolic effects of dietary PRO consumed at night versus during the day. Additionally, use of the microdialysis technique to measure localized fatty acid mobilization from the SCAAT depots and fat oxidation (FatOx) of those fatty acids after pre- sleep PRO consumption has only been utilized in one study from our laboratory (no exercise intervention) in overweight and obese men (191). In this study, although there were no significant changes in lipolysis or FatOx with pre-sleep PRO feeding compared to a non-nutritive placebo, we did not observe an attenuation in fat mobilization with this acute dietary practice (191). Therefore, research is warranted to provide further insight and clarity on the extent to which RT and PRO timing (daytime versus pre-sleep) affects SCAAT lipolysis and FatOx in resistance-trained women.

1.2 Purpose

To determine the effects of RE and PRO timing (Placebo [PLA]-PRO and PRO-PLA conditions) on SCAAT lipolysis and whole-body substrate utilization in resistance-trained women.

1.3 Specific Aims and Research Hypothesis

1.3.1 Specific Aim 1

Determine the extent to which a full-body, acute RE protocol affects SCAAT lipolysis and whole-body substrate utilization in resistance-trained women compared to baseline (independent of Aims 2 and 3). To address Aim 1, we assessed and compared SCAAT lipolysis using microdialysis at three time points: 1) baseline (one sample, collected 30 minutes before RE); 2) mid-RE (collected ~25 minutes into RE); and 3) post-RE (collected immediately upon finishing RE) over Visits 4 and 5 (average of two experimental visits, for a N of 13). In addition, we assessed and compared whole-body substrate utilization using open-circuit indirect calorimetry at baseline (30 min), and post-RE (30 min) on Visits 4 and 5. A one-way analysis of variance was used to compare the time points. If a significant finding was noted, a Tukey HSD post-hoc analysis was used to locate where the difference existed.

1.3.2 Hypothesis for Aim 1

There will be a significant increase in SCAAT lipolysis at mid-RE and post-RE compared to baseline, and there will be a significant increase in whole-body substrate utilization (to reflect greater reliance on fat as fuel) post-RE compared to baseline.

1.3.3 Specific Aim 2

Determine the extent to which pre-sleep versus daytime PRO consumption affects SCAAT lipolysis and whole-body substrate utilization in resistance-trained women compared to baseline, independent of RE (Aim 1). To address Aim 2, after RE, participants were randomized to consume either daytime PRO 30 minutes post-RE and pre-sleep PLA 30 minutes pre-sleep (PRO-PLA), or daytime PLA and pre-sleep PRO (PLA-PRO), switching the order of the supplements on the following visit. We assessed and compared SCAAT lipolysis using microdialysis during PRO-PLA and PLA-

PRO conditions at six time points: 1) baseline (one sample); 2) throughout sleep (four samples, collected every two hours); and 3) the next morning (one sample, collected before probe removal). In addition, we assessed and compared whole-body substrate utilization using open- circuit indirect calorimetry during PRO-PLA and PLA-PRO conditions at three time points: 1) baseline (30 min); 2) immediately following the pre-sleep supplement (30 min); and 3) the next morning (30 min). A repeated measures analysis of variance was used to compare the differences between the time points in PLA-PRO and PRO-PLA conditions. If a significant finding was noted, a Tukey HSD post-hoc analysis was used to locate where the difference existed.

1.3.4 Hypothesis for Aim 2

There will be no differences in SCAAT lipolysis or whole-body substrate utilization overnight or into the following morning between PRO-PLA and PLA-PRO conditions.

1.3.5 Specific Aim 3

Determine the extent to which a full-body acute RE protocol and pre-sleep versus daytime PRO consumption affect plasma biomarkers of fat metabolism compared to baseline measures. To address Aim 3, fasted blood samples were collected from the antecubital vein on three occasions for Aim 1: 1) baseline, 2) mid-RE, and 3) post-RE. In addition, blood samples were collected on three occasions for Aim 2: 1) 30 minutes after the daytime supplement (fed), 2) 30 minutes after the pre-sleep supplement (fed), and 3) the next morning (fasted). Growth hormone (GH) and catecholamines (CATs) were only measured around RE (baseline, mid-RE, post-RE), and glycerol, non-esterified fatty acids (NEFA), insulin, and glucose were measured at all time points (baseline, mid-RE, post-RE, after daytime supplement, after nighttime supplement, next morning). A one-way analysis of variance was used to analyze GH, CATs, glucose, insulin, NEFAs, and glycerol around RE. A repeated measures analysis of variance was used to compare the differences in GH, CATs, glucose, insulin, NEFAs, and glycerol between the time points in PRO-PLA and PLA-PRO conditions. If a significant finding was noted, a Tukey HSD post-hoc analysis was used to locate where the difference existed. 1.3.6 Hypothesis for Aim 3

Plasma GH, CATs, glycerol and NEFA will increase mid-RE and post-RE compared to baseline, while glucose and insulin will remain the same. Plasma biomarkers will not be different between PRO-PLA and PLA-PRO conditions.

1.4 Assumptions

1. Participants maintained the same diet and physical activity habits throughout their commitment to the study. 2. Participants consumed the provided meals at correct times before their visit to the laboratory. 3. Caloric intake on the microdialysis visits was standardized by providing each participant with meals from Vale Foods and supplements (Dymatize® Nutrition and FrieslandCampina®) which equated to their caloric needs based on their resting metabolism results at baseline and their activity levels. 4. Participants did not consume any other calories than those provided on visit days. 5. Participants provided their best effort on maximal testing. 6. A comfortable sleeping environment was provided for participants. 7. Research personnel accurately measured and recorded all variables. 8. Laboratory equipment provided accurate measurements over repeated visits to the laboratory.

1.5 Delimitations

1. Participants were healthy, resistance-trained (squat 100% of their body weight, bench 2 press 70% of their body), normal weight (BMI = 18.5-25.0 kg/m ; body fat <33.0%), normally menstruating (on or off oral contraceptives) females ages 18-35 years. Body composition was measured by Dual Energy X-ray Absorptiometry (DXA). 2. Between visits, participants maintained their habitual dietary intake and physical activity to minimize influence on the dependent variables. 3. Participants were eligible for participation in this study determined by telephone pre- screenings. 1.6 Limitations 1. Only normal weight and BMI, resistance-trained women were included in the present study. Thus, reported results may not be generalizable to other populations.

2. Although it is impossible to obtain direct measures of metabolism in human visceral fat, simultaneous measurement of SCAAT lipolysis (microdialysis) and whole-body metabolism (indirect calorimetry) provided valuable insight on the effects of pre-sleep PRO consumption on fat metabolism. 3. Although provided based upon caloric need and activity level, provided meals may not have given the same number of calories or macronutrient composition that participants were acclimated to. 4. Although participants were instructed to consume only provided meals and supplements on experimental visits, it is possible that compliance to these standards may not have been met.

CHAPTER TWO REVIEW OF LITERATURE

2.1 Adipose Tissue Physiology

Fat (adipose) tissue is a form of connective tissue found in and around organs of the body (247). It was traditionally thought to be a passive depot for energy storage; however, this notion is no longer valid (247). Adipose tissue is in fact an extremely active metabolic and endocrine organ with a highly complex structure, releasing various hormones and factors, including, but not limited to autocrine, growth, and inflammatory factors (6, 186).

2.1.1 Adipose Tissue

Adipose tissue consists of fat cells (adipocytes), the functional units responsible for energy storage, as well as immature adipocytes, connective tissue matrix, immune cells, nerve cells, stromovascular cells and blood vessels (107). An adipocyte is the storage depot for fats, including both triacylglycerols (TAG) and long-chain fatty acids (FA) (> 95%), some of which (TAG) are concentrated into lipid droplets (8, 247). TAG are insoluble in water, highly concentrated within the adipocyte with no negative effects on fluid movement, and extremely calorically dense (9.3 kcal per g), making them a very efficient form of energy storage (247).

Of interest to this review, fat storage depots include visceral compartments (in between organs of the abdomen), and subcutaneous compartments (underneath the skin). There are also three different types of adipose tissue: brown, white and beige (407). Brown adipose tissue is found in specific areas of the body (neck and interscapular regions in newborns and possibly perirenal in adults) (140) and is highly metabolic, producing heat. Beige adipose tissue (brown- in-white, or brite adipose tissue) develops and resembles white adipose with low expression of mitochondrial proteins, but shares some of the same activators and intercellular signaling pathways (cyclic adenosine monophosphate, cAMP) as brown adipose tissue (139). White adipose tissue (WAT) is a large storage depot for excess TAG in the diet, whereby large amounts are associated with obesity-related disorders (332). Therefore, this review will focus on WAT.

2.1.1.1 Structure and function of white adipose tissue. WAT adipocytes are very large, and typically range from 50-150 µm in diameter (247). WAT is the most common type of

8 adipose tissue, and adipocytes found in this tissue function as a long-term energy storage source (247). When WAT cells are found in an isolated state, they are spherical in shape, yet they become polyhedral (develop flat sides with edges) when they are packed in clusters within the connective tissue (247), such as with the onset of obesity (16). WAT cells contain one large droplet of concentrated TAG in the cytoplasm, thus they are termed unilocular (247). Importantly, immature WAT cells also contain immature, smaller lipid droplets in the cell cytoplasm, indicating the storage potential of adipocytes (247).

2.1.1.2 Blood supply and nervous innervation to white adipose tissue. WAT contains a series of nerves and blood vessels (247). Blood supply from the subcutaneous fat is emptied directly into systemic circulation (229). The production of the CATs norepinephrine (NE) and epinephrine (Epi) through the sympathetic nervous system is highly important in maintenance of homeostasis in WAT, while parasympathetic stimulation of WAT is minimal (354). Activation of the sympathetic nervous system leads to mobilization of energy stores from adipocytes (see sections 1.1.1.3 and 1.1.4.2).

2.1.1.3 Receptors on the adipocyte cell membrane. There are multiple receptors on the cell membrane of an adipocyte. Alpha-adrenergic receptors (α-AR) and beta-adrenergic receptors (β-AR) are the most important receptors involved in fat mobilization and storage. The stimulatory balance of these receptors determines the final overall metabolic outcome in adipose tissue. The α-AR and β-AR that affect WAT metabolism are G-protein coupled receptors that respond to the binding of NE and Epi (202). When this binding occurs, various conformational changes occur in the alpha (α), beta (β) and Gama (γ) subunits of the heterotrimeric G-protein complex, dependent upon the specific receptor involved. The β-AR isoforms (β 1-AR, β 2-AR, β 3-

AR) are found on the cell membrane of human adipocytes; however, β 3-AR are much less active in humans (more active in rodents) (24). All β-AR are coupled to the stimulatory (Gα s) subunit, which ultimately stimulates lipolysis, or the hydrolysis of stored TAG into free fatty acids (FFA) and glycerol (GLY). Binding and the consequent conformational change of the complex releases the Gα s subunit from the complex with the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). The Gα s subunit then binds and activates the adenylyl cyclase enzyme, which initiates the conversion of adenosine triphosphate (ATP) to the second messenger cAMP. Subsequently, the cAMP-dependent enzyme protein kinase A (PKA) is phosphorylated.

PKA can phosphorylate many other proteins, including hormone-sensitive lipase (HSL) which accelerates lipolysis (24) and provides the rate-limiting step in TAG breakdown (202), and adipose TAG lipase (ATGL), which catalyzes the first step in the hydrolysis of TAG into DAG and FFA (203). In contrast to the β-AR, the α-AR isoform involved in fat metabolism (α 2-AR) is coupled to the inhibitory alpha subunit (Gα i) of the G-protein complex, inhibiting the production of cAMP from ATP in the reaction previously discussed, ultimately attenuating downstream lipolysis (202). In WAT, α2-AR and β-AR perform antagonistic-mediated functions (202), with each receptor classified by differing affinity and sensitivity (see section 1.1.4.2).

2.1.2 Components of Fat Metabolism

The primary purpose of adipose tissue is to store TAG in adipocytes, and to release FFA and GLY into the circulation for energy production. The amount of stored TAG is a reflection of the relationship between chronic energy intake and energy expenditure, and thus reflects the balance between fat storage and fat mobilization (107). Three main processes metabolically regulate this balance: 1) lipogenesis, or the esterification of TAG (storage); 2) lipolysis, or the mobilization of adipocyte storage constituents, and; 3) beta-oxidation, or the use of FFA as energy through metabolic reactions.

2.1.2.1 Storage: lipogenesis. Lipogenesis occurs in both the liver and the adipose tissue, and includes the conversion of acetyl coenzyme A (acetyl-CoA) to FFA, as well as the esterification of TAG from FFA and a GLY molecule. Acetyl-CoA is a byproduct of beta- oxidation as well as glycolysis (breakdown of carbohydrate [CHO] into glucose molecules). Glucose from CHO breakdown initiates lipogenic responses by stimulating the pancreatic release of insulin (storage hormone), while inhibiting the release of glucagon (187) (see sections 1.1.4.1 and 1.5.1).

Exogenous sources (dietary intake) as well as endogenous sources (de novo lipogenesis) of TAG contribute to fat deposition. In humans, de novo lipogenesis, or the enzymatic pathway by which excess CHO is converted into fat in the liver occurs during periods of chronic overfeeding with a high-CHO diet (146) (Figure 1). This process begins with the production of pyruvate from glycolysis and the consequent entrance of pyruvate into the mitochondria (111). Pyruvate is converted into acetyl CoA by pyruvate dehydrogenase, and then acetyl CoA enters

10 the Kreb’s cycle (111). Acetyl CoA is then transported across the mitochondrial membrane as a part of citrate because the CoA portion cannot cross the inner mitochondrial membrane (111). Acetyl CoA can then be regenerated from citrate in the cytosol by ATP citrate lyase (111). Citrate then activates acetyl CoA carboxylate, the enzyme that catalyzes the conversion of acetyl CoA to malonyl CoA (rate-limiting step of FA synthesis) (111). Lastly, FA synthase catalyzes the elongation process, until a 16-carbon FA molecule (palmitate) is produced (111). Palmitate is a precursor molecule for creation of longer chained FA. These newly formed FA can then re esterify with glycerol-3-phosphate to form TAG, which are released from the liver as very-low- density lipoproteins (VLDL). VLDL transport the stored TAG to various tissues.

Figure 1. De novo lipogenesis pathway. De novo lipogenesis, or the enzymatic pathway by which excess CHO is converted into fat in the liver occurs during periods of chronic overfeeding with a high CHO diet. This process begins with the production of pyruvate from glycolysis and the consequent entrance of pyruvate into the mitochondria. Pyruvate is converted into acetyl CoA by pyruvate dehydrogenase (PDH), and then acetyl CoA enters the Kreb’s cycle. Acetyl CoA is then transported across the mitochondrial membrane as a part of citrate because the CoA portion cannot cross the inner mitochondrial membrane. Acetyl CoA can then be regenerated Figure 1 continued. from citrate in the cytosol by ATP citrate lyase (ACL). Citrate then activates

Figure 1 continued. acetyl CoA carboxylate (ACC), the enzyme that catalyzes the conversion of acetyl CoA to malonyl CoA (rate-limiting step of FA synthesis). Lastly, FA synthase (FAS) catalyzes the elongation process, until a 16-carbon FA molecule (palmitate) is produced. Palmitate is a precursor molecule for creation of longer chained FA (111).

Figure 2. Mechanisms of TAG storage in WAT. Postprandially, chylomicrons (CHY) form in the intestinal enterocytes from dietary removal of GLY, long-chain FFA, cholesterol, monoacylglycerols (MAG) and diacylglycerols (DAG). Likewise, short-chain FFA are directly absorbed from the small intestine and sent into the portal circulation to the liver, which synthesizes TAG from the short-chain FFA and glycerol, packaging them as VLDL to be sent back out into circulation, and potentially stored in WAT. Lastly, in the adipocyte, glucose is used to synthesize FFA and contribute to the FA pool through the creation of acetyl CoA. In the adipocyte, FA combine with GLY phosphate and re-esterify into TAG, contributing to the TAG pool in the lipid droplets (247).

There are three ways that TAG can be stored in WAT (Figure 2). First, postprandially, chylomicrons form in the intestinal enterocytes from dietary removal of GLY, long-chain FFA, cholesterol, monoacylglycerols and diacylglycerols. Chylomicrons are spherical lipoproteins that contain TAG, phospholipids, proteins, and cholesterol, and are responsible for the transport of lipids from the intestines into the lymphatic system and consequently the blood stream to be potentially stored in WAT or utilized in the muscle. Likewise, short-chain FFA are directly absorbed from the small intestine and sent into the portal circulation to the liver. TAG is synthesized in the liver from the short-chain FFA and GLY, and packaged as VLDL, which are sent into circulation to potentially be stored in WAT or utilized in the muscle. However, TAG from chylomicrons and VLDL cannot enter the capillary endothelium of tissues and thus must be hydrolyzed into NEFA and GLY by lipoprotein lipase (which is bound to the capillary endothelium of these tissues). NEFA can then enter tissues (i.e. adipocytes and muscle cells) or circulate in the blood, bound to the transporter protein, albumin (204). Once in the adipocyte, the FFA can combine with GLY phosphate to be re-esterified into a TAG, and then stored in the lipid droplet of the adipocyte (247). Lastly, in the adipocyte, glucose can be used to synthesize FFA and GLY, and consequently stored (247).

2.1.2.2 Mobilization: lipolysis. Lipolysis is the hydrolysis of TAG in an adipocyte to GLY and three FFA. Lipolysis occurs in a 1:3 ratio (GLY:FFA); however, the release of these constituents from the adipocyte does not always occur in this same ratio (24). The variability in lipolytic and release ratios of GLY and FFA occurs because some of the FFA stay in the adipocyte to be re-esterified into TAG (lipogenesis) (104). The adipocyte and circulating lipoproteins, such as CHY, release GLY and FFA. In each of these instances, the FFA are released by lipolysis and enter the blood bound to the protein carrier albumin (205) to either be available for cellular uptake and immediate energy production in the muscle tissue or for storage in the adipose tissue (123). The residual constituents of the original CHY are called CHY remnants, which travel to the liver to be converted into TAG and packaged into VLDL (123). VLDL will reenter circulation and will be broken down into low-density lipoproteins (LDL) and FFA by the enzyme lipoprotein lipase (123). These FFA are again taken up by the muscle for metabolism through FA transporters such as CD36 and family of FA transport protein 4 (FATP4) or they can be stored in the adipose tissue (123). GLY can neither enter nor be subsequently utilized by the adipocyte or muscle cell due to the lack thereof the enzyme GLY kinase, which

13 catalyzes the conversion of GLY to glycerol-3-phosphate (which can then be re-esterified). However, the liver does indeed possess GLY kinase, and thus, GLY can be taken up, and either broken down in glycolysis, or used for gluconeogenesis (to form new glucose) (123). In the adipocyte, in the presence of insulin, glucose entrance (see section 1.1.4.1) and consequent conversion to glucose-3-phosphate can initiate re-esterification processes without a GLY precursor. Specifically, in a reversible reaction requiring the enzyme glycerol-3-phosphate dehydrogenase, glucose is converted to glycerol-3-phosphate through the reduction of the glycolysis intermediate dihydroxylacetone phosphate.

2.1.2.3 Fatty Acid Beta-oxidation. Beta-oxidation occurs in the mitochondria, and is the catabolic process by which two carbon units from the carboxyl end of the FA (in the form of acetyl-CoA) are cleaved and ultimately enter into the Kreb’s cycle (123). In the Kreb’s cycle, nicotinamide adenine dinucleotide plus a hydrogen (NADH+H +) and flavin adenine dinucleotide + (FADH 2) are produced (123). NADH+H and FADH 2 are substrates oxidized in the electron transport chain (ETC) to produce the oxidized form of each (NADH and FAD, respectively). When these processes occur, ATP, which is a high-energy molecule is created and used to fuel cells (123).

Figure 3 depicts the steps preceding beta-oxidation in the muscle cell. First, in the muscle cell, each FFA must be activated before it enters the mitochondria. Activation of the FFA by coenzyme A requires the enzyme cytoplasmic acyl-CoA synthetase, and consumption of two high-energy phosphate bonds, yielding adenosine monophosphate (AMP) from ATP (123). Once in the cytoplasm, carnitine acyltransferase-1 transports the activated FFA (acyl-CoA) joined to carnitine across the outer membrane of the mitochondria, while carnitine acyltransferase-2 transports it across the inner membrane and releases the fatty acyl-CoA from the carnitine into the mitochondrial matrix (123).

Once the fatty acyl-CoA is inside the mitochondria, it can enter beta oxidation, where two carbon acetyl-CoA molecules are repeatedly cleaved from the carboxyl end of the FFA (123, 315) (Figure 4). The fatty acyl-CoA first undergoes oxidation, whereby the enzyme acyl-CoA dehydrogenase inserts a double bond, using an FAD molecule and consequently producing an

FADH 2 that will be sent to the electron transport chain (ETC) (123, 315) for ATP production. The molecule then undergoes a hydration phase (adding a water molecule) in which enoyl-CoA

14 hydratase converts the molecule to an alcohol (β-hydroxyacyl CoA) (123, 315). The alcohol then undergoes oxidation by NAD+ (producing an NADH molecule) using β-hydroxyacyl CoA dehydrogenase to make a carbonyl (β-ketoacyl CoA) (123, 315). Finally, acyltransferase inserts a CoA and cleaves two carbons from the β end, leaving a FA that is two carbons shorter and the two-carbon acetyl-CoA (123, 315). This sequence of events is repeated, as each FADH 2 molecule produces 2 ATP in the ETC, each NADH molecule produces 3 ATP in the ETC, and each acetyl-CoA is oxidized further in the Kreb’s cycle, producing 12 ATP (123, 315).

Figure 3. Steps preceding beta-oxidation in the muscle cell. When the GLY molecule separates from the three FFA, each FFA must be activated, requiring the enzyme cytoplasmic acyl-CoA synthetase, and consumption of two high-energy phosphate bonds, yielding AMP from ATP. Once the fatty acyl CoA is in the cytoplasm, carnitine acyltransferase-1 (CAT-1) transports the fatty acyl-CoA joined to carnitine across the outer membrane of the mitochondria, while carnitine acyltransferase-2 (CAT-2) transports it across the inner membrane and releases the fatty acyl CoA from the carnitine into the mitochondrial matrix to enter beta-oxidation (123).

Figure 4. The steps of beta-oxidation. The fatty acyl-CoA undergoes oxidation, whereby the enzyme acyl-CoA dehydrogenase inserts a double bond, using an FAD molecule and producing an FADH 2 (sent to the ETC for ATP production). The molecule then undergoes a hydration phase in which enoyl CoA hydratase converts the molecule to β-hydroxyacyl CoA. The alcohol then undergoes oxidation by NAD+ (producing an NADH molecule) using β-hydroxyacyl CoA dehydrogenase to make β-ketoacyl CoA. Finally, acyltransferase inserts a CoA and cleaves two carbons from the β end, leaving a FA that is two carbons shorter and the two-carbon acetyl CoA. This sequence of events is repeated, as each FADH 2 molecule produces 2 ATP in the ETC, each NADH molecule produces 3 ATP in the ETC, and each acetyl CoA is oxidized further in the Kreb’s cycle, producing 12 ATP (123).

2.1.3 Enzymatic Influence on Fat Metabolism

2.1.3.1 Hormone-sensitive lipase and adipose TAG lipase. The primary rate-limiting enzyme of lipolysis is HSL. This enzyme is regulated by hormones, hence its name (156). HSL activity, activated by the adenylyl cyclase-cAMP-protein kinase cascade, is enhanced by many factors including, but not limited to glucagon, CATs, and GH (49, 254) in order to mobilize fats

16 to be utilized for energy. Of importance, HSL is inhibited by insulin (49). Many of these hormone-metabolism interactions will be discussed in a later section (see section 1.1.4). In addition, adipose TAG lipase (ATGL) catalyzes the first step in the hydrolysis of TAG into DAG and FFA (203), providing the first step in lipolysis. Both HSL and ATGL are activated by PKA.

2.1.3.2 Lipoprotein lipase. One of the most influential hormones in lipolysis and lipogenesis, lipoprotein lipase (LPL), also catalyzes the mobilization of TAG but favors storage of TAG in adipocytes. LPL is responsible for partitioning the uptake of FFA from TAG between different tissues. LPL catalyzes the hydrolysis of TAG from circulating chylomicrons and VLDL in the blood, leaving a chylomicron remnant and FFA (123). LPL also acts on the TAG, and transports FFA into the adipocyte, contributing to the FA pool, whereas GLY cannot be utilized and is therefore shuttled back into circulation to be sent back to the liver (123). In the blood, LPL also catalyzes the conversion of VLDL to intermediate density lipoprotein (IDL), and IDL to LDL, with the removal of a TAG at each step (123). The cleaved TAG components, FFA, diacylglycerol (DAG), monoacylglycerol (MAG) can enter the muscle cell through FA transporters such as CD36 and family of FA transport protein 4 (FATP4). As discussed above, the FFA and GLY can then be reformed in the adipocyte and stored in the lipid droplet by the uptake of FFA and esterification to glycerol 3-phosphate (123).

2.1.4 Hormonal Interactions in Lipolysis, Lipogenesis and Beta Oxidation

Many hormones influence fat metabolism in an adipocyte, apparent by the strong diurnal changes in fat metabolism throughout the day (121).

2.1.4.1 Insulin . Insulin is arguably the most important factor in fat metabolism due to its influence on regulation of lipogenesis and lipolysis. Uptake of glucose into adipocytes, FA synthesis from the transported glucose into the adipocytes, and the re-esterification pathway require insulin (108, 247). Insulin synthesizes and stimulates LPL (247, 329), which, as discussed previously promotes storage of TAG in an adipocyte by removing TAG from CHY, VLDL and IDL (123). Likewise, insulin inhibits HSL, significantly reducing the release of FFA for energy production (247). Insulin performs this action through activation of cAMP-specific phosphodiesterase, which inhibits and reduces the amount and influence of cAMP (49, 198), the upstream second messenger responsible for activation of HSL. With covalent modification, such

17 as phosphorylation, insulin can then activate lipogenic and glycolytic enzymes such as pyruvate dehydrogenase, acetyl-CoA carboxylase, and GLY phosphate acyltransferase which are enzymes involved in glucose uptake and subsequent FA synthesis (49, 187). Pyruvate dehydrogenase transforms pyruvate into acetyl-CoA, which stimulates the lipogenesis pathway. Acetyl-CoA carboxylase catalyzes the irreversible conversion of acetyl-CoA to malonyl-CoA, a substrate for FA synthesis. GLY phosphate acyltransferase produces CoA by catalyzing an acyl group transfer from a fatty acyl-CoA to glycerol-3-phosphate.

Each of these actions is dependent on the presence of glucose and therefore reliant on the ability of insulin to facilitate the uptake of glucose by translocation of the glucose transporter 4 (GLUT-4) (174). This process begins with the insulin receptor, which is a tyrosine kinase receptor with two extracellular α subunits wherein insulin attaches, and two transmembrane β subunits containing tyrosine-binding sites (Figure 5). With the binding of insulin to the α subunits of the insulin receptor on the adipocyte surface, a conformational change occurs whereby the two tyrosine kinase monomers become a dimer (175). This process activates the tyrosine binding sites, which are attached to the β subunits of the receptor (175). Then, ATP is hydrolyzed into ADP and inorganic phosphate (175). The inorganic phosphate binds to all tyrosine binding sites, fully activating the tyrosine kinase dimer (175). Insulin receptor substrate 1 (IRS-1), found inside the adipocyte binds to a phosphorylated tyrosine binding site (175). IRS- 1 then serves as a binding and activation site for phosphoinositol-3-kinase (PI3K) (175). PI3K may also bind directly to a phosphorylated tyrosine-binding site (175). PI3K can then phosphorylate phosphatidylinositol 4,5-bisphosphonate (PIP 2), which is anchored to the membrane, to phosphatidylinositol 3,4,5-bisphosphonate (PIP 3) (175). PIP3 can then bind and activate protein kinase B (AKT) (175). AKT can then phosphorylate TBC1 domain family 4 (TBC1D4), which inhibits the GTPase-activating protein (GAP) (175). This interaction allows the Rab protein to change from its GDP- to GTP-bound state, causing the translocation of intercellular GLUT-4 vesicles to the cell membrane by phosphorylation, allowing glucose to enter the cell (175).

2.1.4.2 Catecholamines. Both Epi and NE are important modulators of adipocyte lipolysis (396). Mechanistically, NE and Epi are released from nerve endings and attach to either β1-, β 2-, and β 3-AR to promote lipolysis, or α 2-AR to inhibit lipolysis. Upon attachment to

18 the β 2-AR, as previously discussed, cAMP is stimulated (53, 199), leading to the phosphorylation of PKA, which subsequently phosphorylates perilipin and HSL (Figure 6). Perilipins are proteins that assimilate on the surface of lipid droplets, and act as a protective coating, preventing the mobilizing actions of lipase enzymes. Upon phosphorylation and thus activation, perilipins activate and translocate HSL, and also release the protective coating surrounding the lipid droplet, allowing HSL to hydrolyze TAG into FFA and DAG (30, 203, 247). PKA also activates adipose TAG lipase (ATGL), which catalyzes the first step in the hydrolysis of TAG into DAG and FFA (203). HSL can then hydrolyze DAG into FFA and MAG, and these molecules are then released into the cytoplasm of the adipocyte. Monoacylglycerol lipase (MGL) then hydrolyzes MAG into GLY and FFA. Compared to the attachment of catecholamines to the β 2-AR, upon attachment to the α 2-AR, inhibition of lipolysis occurs through inhibition of cAMP production, and thus attenuated downstream phosphorylation of the previously mentioned proteins responsible for lipolysis.

Important feedback mechanisms are in place to control cAMP production. A negative feedback system provides β 1- and β 2-AR desensitization in response to elevations in cAMP and HSL activation (143). Human in vitro studies have shown that β-AR desensitization occurs due to down-regulation of receptors (58). However, desensitization in response to NE (the initial catalyst in this cascade) does not occur after a physiological and sustained sympathetic nervous system activation, such as with aerobic exercise training, allowing the effects to be sustained (75). Compared to β-AR responsiveness, the α-AR is quite refractory. Neither short term nor chronic exposure to catecholamines or α 2-AR agonists changes the α-AR number on the adipocyte (58, 283, 390).

The affinity of these receptors to the CATs is important to consider, as well. CATs have a lower affinity for β 3-AR, compared to β 1- and β 2-AR (202). Also, CATs have a higher affinity for the antilipolytic α 2-AR compared to the lipolytic β 1-AR, 2-AR, and 3-AR (202). Interestingly, skeletal muscle lipolysis is more responsive to Epi compared to NE (306). Therefore, it is critical to maintain optimal concentrations of these receptors on the cell surface, as the affinity of the CATs to the AR is disproportional, favoring antilipolytic actions.

2.1.4.3 Growth hormone. GH exerts both acute (256) and chronic (24) effects on fat metabolism. GH attenuates lipogenesis and promotes lipolysis, resulting in fat loss (99, 247). It

19 has been shown that there is a significant interaction between GH and circulating FFA (p<0.01), as circulating FFA increases dramatically after a GH spike (0 min: 0.64 ± 0.05 mmol/L; 330 min: 1.21 ± 0.12 mmol/L, p<0.01), indicating the large influence of GH on lipolysis (256). These effects, as well as increased fat oxidation rates are experienced with pulsatile and continuous GH administration in humans (255, 257). The mechanism of GH-triggered lipolysis is unclear, but may be due to its effects on activation of adenylyl cyclase and consequent activation of HSL, activation of β-AR, enhancement of the effects of the CATs, or inhibition of phosphodiesterase (84, 279). Additionally, GH causes a decrease in insulin sensitivity and consequently down- regulates the enzyme FA synthase, which attenuates both glucose uptake and FA synthesis (414).

Figure 5. The insulin tyrosine-kinase receptor cascade for translocation of glucose transporter 4 (GLUT-4). The binding of insulin to the α subunits of the insulin receptor on the adipocyte surface causes a conformational change whereby the two tyrosine kinase monomers become a dimer. This process activates the tyrosine binding sites, which are attached to the β subunits of the receptor and ATP is hydrolyzed into ADP and inorganic phosphate (P). The P binds to all tyrosine-binding sites, fully activating the tyrosine kinase dimer. Insulin receptor substrate 1 (IRS-1), found inside the adipocyte binds to a phosphorylated tyrosine binding site. IRS-1 then serves as a binding and activation site for phosphoinositol-3-kinase (PI-3-K). PI-3-K can then

Figure 5 continued. phosphorylate phosphatidylinositol 4,5-bisphosphonate (PIP 2), which is anchored to the membrane, to phosphatidylinositol 3,4,5-bisphosphonate (PIP 3). PIP3 can then bind and activate protein kinase B (AKT). AKT can then phosphorylate TBC1 domain family 4 (TBC1D4), which inhibits the GTPase-activating protein (GAP). This allows Rab protein to change from its GDP- to GTP-bound state, causing the translocation of intercellular GLUT-4 vesicles to the cell membrane by phosphorylation and consequent entrance of GLU into the cell (174).

2.1.4.4 Autocrine factors: prostaglandins, adenosine, and cytokines. Prostaglandins and adenosine are inhibitors (23, 319), while cytokines are initiators of lipolysis (136).

Prostaglandins and adenosine elicit inhibition of lipolysis through the Gα i protein pathway

(similar to the α 2-AR) (24). Cytokines, particularly tumor necrosis factor alpha produced in large amounts by adipocytes and cells in the adipose tissue (24) have a very powerful effect on stimulation of basal lipolysis (98). Cytokines activate mitogen-activated protein kinase (MAPK), leading to a decrease in production of and phosphorylation of perilipin. Perilipin is a coating protein that, as previously discussed, prevents hydrolysis of the TAG in the adipocyte by HSL, thus, phosphorylation and therefore activation of this protein exposes the adipocyte to the lipolytic actions of HSL.

2.1.4.5 Glucagon. Glucagon is a stimulator of lipolysis, displayed by the suppression of FFA and GLY appearance with induced hypoglucaconemia (by somatostatin treatment as a perturbation used to inhibit endogenous glucagon release), and conversely, the large increase in FFA and GLY appearance with induced hyperglucagonemia (by infusing 1.3 ng of glucagon per kg/min to induce physiological hyperglucagonemia) in healthy male humans (60). In humans, glucagon exerts its effects on lipolysis through the same mechanism as the CATs, using its own specific glucagon receptor, and ultimately stimulating cAMP production and consequent phosphorylation of HSL via PKA (245).

2.1.4.6 Ghrelin. Ghrelin is orexigenic (appetite stimulator), and thus has a strong impact on appetite and energy balance, due in part to ghrelin’s effects on GH release. Infusion of ghrelin strongly stimulates GH release from the anterior pituitary in a dose-dependent manner in healthy male adults (366). In these healthy adult males, infusion of graded ghrelin in 0.2, 1.0, and 5.0 mcg/kg doses elicited a significant GH response (43.3 ± 6.0, 81.5 ± 12.7, and 107.0 ± 10.7 ng/mL, respectively) (367). Likewise, food intake is stimulated with attachment of ghrelin 21 to the GH secretagogue receptor in the anterior pituitary on the neuropeptide Y/agouti-related peptide-producing neurons of the arcuate nucleus (128). In rats, fat oxidation is decreased with four days of two-200 mg subcutaneous injections of ghrelin (26). Researchers found that ghrelin increased body weight in these rats, which was not mediated by elevated insulin or calorie intake (26). Further, with ghrelin injections, TAG content increased, while AMP-activated protein kinase (stimulator of fat oxidation) decreased in the liver (26). Interestingly, TAG concentration in the muscle was reduced (26), and thus, TAG deposition seems to be tissue-specific with ghrelin infusion in rodents. In humans, the effects of ghrelin on lipolytic rate and fat oxidation are conflicting and depot-specific (323, 389). With ghrelin infusion, lipolytic rate has been shown to transiently increase in subcutaneous (abdominal and femoral) depots (388), and decrease in visceral depots (324). However, in the former study (388), participants were hypopituitary, which may have affected the outcome. Compared to the previously mentioned rodent model, fat oxidation with ghrelin infusion remains unchanged or decreases depending upon the fat depot and the population being assessed. Overall, the effects of ghrelin on lipolysis and beta-oxidation in humans are inconsistent.

Two of the preproghrelin gene-derived peptides include acyl ghrelin and des-acyl ghrelin (79). Des-acyl ghrelin was once considered a degradation product of acyl-ghrelin with no biological activity, due to the absence of effects in small doses on the receptor (200). However, these thoughts are no longer considered valid. Des-acyl ghrelin may be implicated in obesity, as low des-acyl ghrelin levels have been reported with no differences in acyl ghrelin in obese compared to lean individuals (281). Even more, an elevated acyl ghrelin/des-acyl ghrelin ratio is found in insulin-resistant compared to insulin-sensitive individuals (357). Des-acyl ghrelin can either support or antagonize ghrelin’s activities (80), or act completely independent, indicating that it is important to discern the different types of ghrelin when interpreting findings.

2.1.4.7 Leptin. Leptin is the antagonist of ghrelin. Leptin is released from WAT, and is known as the “satiety hormone”, which functions as a regulator of appetite by communicating with the hypothalamus under normal conditions (247). In rodent models, leptin also inhibits lipogenesis, stimulates the release of GLY from the adipocytes, and increases enzymatic markers of FFA oxidation (393). For example, in normal lean Zucker diabetic fatty rats (with normal functioning leptin receptors), leptin infusion increased GLY release with no effect on FFA

22 release (393). Conversely, in obese Zucker diabetic fatty rats with dysfunctional leptin receptors, leptin infusion had no effect on GLY or FFA release (393). These effects are purportedly due to the attenuation of leptin receptors and expression of FA synthase mRNA with leptin infusion (393). The attenuation in FA synthase mRNA would attenuate both glucose uptake and FA synthesis (414).

Figure 6. Mobilization of fatty acids due to the interaction between perilipin and HSL in an adipocyte. Upon attachment to the β-AR, cAMP is stimulated leading to the phosphorylation of PKA, subsequently phosphorylating perilipin and HSL. Upon phosphorylation and thus activation, perilipins activate and translocate HSL, and release their protective coating that surrounds the lipid droplet, allowing HSL to hydrolyze TAG into FFA and DAG. PKA also activates adipose TAG lipase (ATGL), which catalyzes the first step in the hydrolysis of TAG into DAG and FFA. HSL can then hydrolyze DAG into FFA and MAG, which are released into the cytoplasm of the adipocyte. Monoacylglycerol lipase (MGL) hydrolyzes MAG into GLY and FFA (116).

In humans, the relationship between leptin and fat metabolism is similar, yet dependent upon body composition. In non-diabetic obese men, after 16 weeks of dietary intervention and 8 weeks of weight maintenance (tested when participants lost an average of 19.2 kg), researchers found that leptin concentrations were inversely related to the amount of weight lost at the 8-, 16- and 24-week marks (385). Likewise, leptin levels were inversely correlated with resting- and 24- hour respiratory quotient (ratio of carbon dioxide [CO 2] being produced to oxygen [O2] being consumed at the cellular level), indicating that higher levels of leptin were related to a greater reliance on fat oxidation (386). Therefore, higher levels of weight loss contribute to lower levels of leptin, and thus a higher respiratory quotient, indicating less fat oxidation (and more CHO oxidation). In lean participants, the relationship between leptin levels and respiratory quotient was not present; however, there was an inverse relationship between leptin and 24-hour energy expenditure when adjusted for fat mass, but not lean mass (386). These findings indicate that, in obese humans, weight loss-stimulated attenuation of leptin compromises fat oxidation; however, these findings are not true in their lean counterparts.

Insulin also stimulates leptin release. However, the sensitivity of the leptin response to insulin is depot specific, and has been found to be correlated to the lipolytic state of the SCAAT (measured by dialysate glycerol concentrations), but not femoral depots (102).

2.1.4.8 Adiponectin. Adipose tissue is the only organ by which adiponectin is secreted. Circulating levels of adiponectin are inversely related to level of adiposity, as plasma concentrations of adiponectin are found to be significantly lower in obese adipose tissue compared to nonobese adipose tissue (22). In nondiabetic humans, plasma adiponectin levels are strongly associated with insulin sensitivity index, but not insulin secretion (185). The insulin- sensitizing effects of adiponectin likely contribute to the increased FA oxidation rates in skeletal muscle (410). Mechanistically, adiponectin increases FA oxidation through activation of 5’ adenosine monophosphate-activated protein kinase (410). In addition to its effects on fat oxidation, in a mouse model with elevated circulating adiponectin, VLDL-TAG catabolism is increased through an increase in LPL activity, and thus fasting plasma TAG levels are decreased (305).

22.1.4.9 Glucocorticoids: cortisol. Cortisol is the most common glucocorticoid, which binds to the glucocorticoid receptor. Cortisol is responsible for both lipogenic and lipolytic

24 actions in visceral and subcutaneous depots, respectfully. Lipogenic actions of cortisol in visceral depots are elevated due to the high concentrations of glucocorticoid receptors in the visceral fat, and less in subcutaneous fat (312). For example, in instances of high and/or prolonged cortisol concentrations such as times of chronic high stress and Cushing’s Syndrome, fat deposition in abdominal visceral fat is favored (219) through the cortisol-mediated redirection of TAG from subcutaneous adipose depots to visceral adipose depots. Impairments in cortisol levels are also found in obese, insulin resistant individuals due to an inability to convert cortisone to cortisol, and thus compensatory amplified reactivation of cortisol and storage of TAG in subcutaneous WAT (310).

2.2 Muscle Physiology

Muscle tissue is an extremely active metabolic organ, and proteins within muscle tissue are constantly renewed and replaced. The process of rebuilding or renewing protein is termed protein synthesis, and the process of degrading proteins is termed protein breakdown. Specific to skeletal muscle tissue, the terms muscle protein synthesis (MPS) and muscle protein breakdown (MPB) are used. The overall balance of protein synthesis and breakdown contributes to net protein balance, or protein turnover (87). During energy balance, such as when rest and nutrition are adequate to supplement exercise training volume and intensity, protein synthesis and breakdown are in balance. When protein breakdown exceeds protein synthesis, a catabolic state ensues, whereby tissues are broken down. When protein synthesis exceeds protein breakdown, an anabolic state follows, whereby growth of tissue occurs. The ability of an organism to rebuild and achieve an anabolic state is quintessential for viability, and thus, dysfunction of protein turnover can have serious negative implications.

2.2.1 Protein Synthesis

The mammalian target of rapamycin (mTOR) signaling pathway regulates protein synthesis (Figure 7). This pathway culminates various intracellular and extracellular signals in order to regulate cell growth, metabolism, survival, and proliferation (207). The mTOR protein is an intracellular protein, belonging to the PI3K family (207). Two multi-protein complexes are present within this system: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (207). However, mTORC1 is active during protein synthesis and cellular growth and metabolism; thus, this system will primarily be discussed. mTORC1 has five complex subunits 25

(Figure 7), including: 1) mTOR, the catalytic subunit; 2) regulatory-associated protein of mTOR (Raptor); 3) mammalian lethal with Sec13 protein 8 (GβL); 4) proline-rich Akt substrate 40 kDa (PRAS40); and 5) DEP-domain-containing mTOR-interacting protein (Deptor) (207, 289) (Figure 7). Conversely, mTORC2 is required for protein kinase B/Forkhead box O (Akt-FoxO) signaling (system controlling protein breakdown), and will therefore be discussed in the protein breakdown section.

2.2.1.1 Stimulation of mTORC1 . mTORC1 is stimulated by three main signals: energy status, oxygen availability, and amino acids (207). Stimulation of the mTORC1 complex occurs through inhibition of the tuberous sclerosis complex (TSC) (except amino acids, which occurs independently of TSC) (207) (Figure 7). TSC is a GTPase activating protein and a protein dimer composed of hamartin (TSC1) and tuberin (TSC2). Upon inhibition of TSC (i.e. attachment of growth factors such as insulin), TSC is unable to interact with the active form (bound to GTP) of the protein Ras homolog enriched in brain (Rheb) (and consequently unable to perform its GTPase actions) (339), subsequently keeping the Rheb protein active, stimulating mTORC1. Conversely, activation of TSC (i.e. limited oxygen availability and energy) makes the Rheb protein inactive and unable to stimulate mTORC1. Thus interaction with TSC negatively regulates the mTORC1 signaling pathway (164).

Energy status affects stimulation of mTORC1. Energy status as a means of measuring intracellular downstream effects is quantified using the ATP:ADP ratio, with a low ratio indicating energy depletion. Energy availability is regulated through the master sensor 5’ AMP- activated protein kinase (AMPK) (137). AMPK phosphorylation due to energy depletion attenuates mTORC1 activation by: 1) phosphorylating TSC2 and inactivating Rheb (164), as well as 2) directly phosphorylating and inactivating the Raptor subunit of the mTORC1 (125). Each of these mechanisms occurs to attenuate growth and proliferation of cells in times of low nutrient and energy availability.

Oxygen levels also affect stimulation of mTORC1. A reduction in oxygen availability causes a reduction in ATP, and thus, as discussed earlier, a decrease in the ATP:ADP ratio. A decrease in this ratio stimulates the phosphorylation and activation of AMPK, and consequent phosphorylation and inhibition of the TSC complex, decreasing mTORC1 signaling (218). Hypoxia also regulates DNA damage response 1 (54), which releases the 14-3-3 proteins from

TSC2, blocking mTORC1 signaling (82). Likewise, hypoxia directly inhibits mTOR signaling through other mechanisms beyond the scope of this review (35, 215).

Lastly amino acids, in particular leucine (LEU), are potent stimulators of the mTORC1 pathway (124). Entry of LEU into the cell requires another amino acid, glutamine which enters the cell through the solute carrier family 1 member 5 neutral amino acid transporter (262). Upon entry into the cell, glutamine is transported back out of the cell in exchange for a LEU through the heterodimeric transporters antiport solute carrier family 7 (cationic amino acid transporter, y+ system, member 5) and solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 SLC3A2 (262). Albeit inconclusive, the mechanism of intracellular activation of mTORC1 by LEU is independent of the TSC complex (267), unlike most of the other upstream regulators of mTORC1. Stimulation of mTORC1 in the presence of amino acids occurs through binding of the Raptor subunit of the mTORC1, consequently relocating the mTORC1 to a region that contains the activated Rheb (338). As discussed previously, activated Rheb activates the mTORC1 pathway. Importantly, without amino acids, other stimulators of mTOR (i.e. growth factors) are ineffective (207).

2.2.1.2 Downstream effects of mTORC1 . Particular to muscle anabolism and catabolism, mTORC1 promotes growth and proliferation of muscle cells through increasing anabolic processes (i.e. MPS), as well as attenuating catabolic processes (i.e. MPB) (207). Regulation of MPS occurs via phosphorylation of various downstream proteins, ultimately stimulating the components of translation regulation of protein synthesis: cap-dependent translocation, translation elongation, mRNA biogenesis, and ribosome biogenesis. The most effective way to affect translation of new proteins is to assume control over the initiation of translation, as it is the rate-limiting step of protein synthesis. When activated, mTORC1 can phosphorylate many proteins specific to muscle anabolism and catabolism.

mTOR is not only important in promoting anabolic pathways, but also inhibits catabolic pathways, as phosphorylation of two components (i.e. serine/threonine-protein kinase ULK1 and autophagy-related protein 13) stimulates autophagy. Autophagy, the catabolic process by which autophagosomes sequester and degrade intracellular components, is accelerated during times of low nutrient availability to provide energy to sustain anabolism. Direct inhibition of mTORC1 increases autophagy, while direct stimulation attenuates the process (69).

2.2.2 Protein Breakdown

2.2.2.1 Stimulators of muscle protein breakdown . Most of the stimulators of MPB function via dephosphorylation of AKT. This process consequently inhibits TSC, and ultimately accelerates Rheb phosphorylation, keeping it in an active state (preventing mTORC1 activation).

Figure 7. Stimulation of mTOR by limited oxygen, limited energy, and amino acids. Stimulation of the mTORC1 complex occurs through inhibition of the tuberous sclerosis complex (TSC), composed of hamartin (TSC1) and tuberin (TSC2). Low energy, and thus low ATP:ADP ratio phosphorylates 5’ AMP-activated protein kinase (AMPK) and consequently attenuates mTORC1 activation by phosphorylating TSC2 and inactivating Rheb, as well as directly phosphorylating and inactivating the Raptor subunit of the mTORC1. The reduction in oxygen causes a reduction in the ATP:ADP ratio, stimulating the phosphorylation and activation of AMPK, which phosphorylates and inhibits the TSC complex, decreasing mTOR signaling. Low oxygen also regulates DNA damage response 1 (REDD1), which releases the 14-3-3 proteins from TSC2, blocking mTORC1 signaling. Likewise, hypoxia can directly disrupt mTOR signaling. Lastly amino acids (LEU) affect mTOR activation. LEU enters the cell (requires glutamine) through the solute carrier family 1 member 5 neutral amino acid transporter (SLC1A5). Upon entry into the cell, glutamine is transported back out of the cell in exchange for LEU through the heterodimeric transporters antiport solute carrier family 7 (cationic amino acid transporter, y+ 28

Figure 7 continued. system, member 5) (SLC7A5) and solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 (SLC3A2). Binding of the raptor subunit of the mTOR complex causes relocation of the protein complex to a region of the cell that contains activated Rheb (207).

FoxO transcription factors are imperative in the process of MPB. External stimuli such as growth factors and insulin stimulate the FoxO transcription factors through the insulin/PI3K/Akt signaling pathway, consequently changing subcellular localization of the FoxO protein complex to initiate effects. The insulin pathway produces phosphoinositide-dependent kinase 1, which phosphorylates and activates AKT and serum- and glucocorticoid-induced protein kinase 1 (207). Serum- and glucocorticoid-induced protein kinase 1 sequesters FoxO factors into the cytoplasm by phosphorylating three conserved residues, preventing FoxO- dependent transcription (38, 55, 207). With mTORC2 inhibition, serum- and glucocorticoid- induced protein kinase 1 is completely abolished; thus, the FoxO system is highly activated (113). Lastly, the myostatin protein, which is predominantly expressed and secreted by skeletal muscle, negatively regulates muscle growth and hypertrophy (Figure 8). Myostatin inhibits many factors involved in the activation and differentiation of satellite cells (162, 237, 238). Satellite cells enable the growth and regeneration of muscle cells. Myostatin is known to inhibit AKT phosphorylation in mice (238) and elderly men with sarcopenia (208). Thus, through inhibition of AKT phosphorylation, myostatin allows TSC2 to keep Rheb in the inactive state, thereby decreasing mTORC1 signaling, while also stimulating the FoxO system.

2.2.2.2 Downstream effects of muscle protein breakdown . FoxO transcription factors, specifically FoxO1 and FoxO3 control MPB. These FoxO proteins regulate energy homeostasis of the muscle through regulation of glycolytic and lipolytic changes, as well as metabolism in the mitochondria (340). FoxO proteins also regulate MPB through changes in the ubiquitin- proteasome and autophagy-lysosomal pathways (340). Mechanistically, FoxO proteins form a forkhead motif that binds to DNA, ultimately inhibiting MPS and increasing MPB. FoxO proteins may be inhibited and consequently translocated out of cell nuclei through phosphorylation of other factors, ultimately negating the effects listed above. When they are translocated to the nucleus, FoxO proteins transcribe and up-regulate the atrophy genes atrogin-1

29 and muscle ring finger-1 (341), which utilize ubiquitination, or the covalent attachment of a chain of ubiquitin regulatory proteins to catalyze proteolysis (degradation of proteins to small peptides) (197)2.3 The Effects of Sex on Fat and Muscle Anabolism and Catabolism

Figure 8. Effects of myostatin on myofiber synthesis. Myostatin inhibits many factors and steps, and subsequently inhibits the activation and differentiation of satellite cells. Myostatin inhibits Akt phosphorylation, allowing TSC2 to keep Rheb in the inactive state, thereby decreasing mTOR signaling, while also stimulating the FoxO system (334).

2.3 The Effects of Sex on Fat and Muscle Metabolism 2.3.1 Physiology of Sex Hormones

Figure 9 depicts the cascade of the conversion of cholesterol to the sex hormones. The precursor for sex hormone synthesis is cholesterol. The cytochrome P450scc enzyme converts cholesterol into pregnenolone. Pregnenolone can then either enter a cascade of reactions to synthesize testosterone (T) and then estrogen (E2), or it be converted to progesterone (P4).

The primary male sex hormones are androgens (specifically T), secreted primarily by the testes. Males also produce E2 and P4, albeit to a lesser extent. Normal reproductive function requires efficient communication between the hypothalamus, the pituitary gland, and the testes, which together comprise the hypothalamic-pituitary-gonadal axis. The hypothalamus releases gonadotropin releasing hormone in a pulsatile fashion, which signals the release of lutenizing hormone and follicle stimulating hormone from the gonadotrophs of the anterior pituitary gland. Lutenizing hormone and follicle stimulating hormone catalyze the production of sex hormones, in addition to many other functions. The primary female sex hormones are E2 and P4 (secreted by the ovaries). Females also produce T in very small amounts. Importantly, T in females exerts less impactful physiological effects compared to males; thus, E2 and P4 will be the focus of the discussion on sex hormones in females.

The blood transports each of the sex hormones differently, in a bound or unbound state. E2 and P4 are transported in the blood bound to protein carriers including sex hormone-binding globulin, which binds E2 to a lesser affinity compared to androgens, and corticosteroid-binding globulin (also known as transcortin), which binds P4 (>90%) (406). Likewise, about 44% of circulating T is bound to SHBG, about 50% is bound to albumin, and about 2-3% is freely circulating (free T) (86). Binding of hormones to SHBG makes them biologically unavailable, whereas freely circulating hormones and hormones bound to albumin are biologically available (387).

2.3.1.1 Testosterone production. In males, the anterior pituitary gland as part of the hypothalamic-pituitary-gonadal axis synthesizes lutenizing hormone, which stimulates the production of T from the interstitial Leydig cells surrounding the seminiferous tubules of the testes. Production of T produces negative feedback to both the pituitary gland and the hypothalamus. The enzyme 5α-reductase in the testes and adrenal glands converts T into dihydrotestosterone. In females, to a lesser degree, theca cells of the ovary secrete T into the blood stream. T can be converted to dihydrotestosterone in the skin, and E2 in the adipose tissue (130). Additionally, the ovarian hilar interstitial cells secrete androgens, and thus display similar characteristics of the Leydig cells from the male testes (130).

Figure 9. Steroidogenesis. Early steroidogenesis in the adrenal cortex, Leydig cells of the testes, and theca cells of the ovaries (medium gray highlight). Cholesterol is converted to pregnenolone by cytochrome P450 (P450). Pregnenolone is either converted to progesterone by 3β- hydroxysteroid dehydrogenase (3β-HSD), or to 17OH pregnenolone by 17, 20 lyase. 17OH pregnenolone is converted to dehydroepiandrosterone (DHEA) by 17, 20 lyase. The enzyme 17, 20 lyase also converts progesterone to 17OH P4, and then 17OH progesterone to androstenedione. DHEA can also be converted to androstenedione through 3β-HSD. Androstenedione is converted to testosterone by 17β-hydroxysteroid dehydrogenase (17β-HSD). The two-cell hypothesis, or conversion of androgens from the theca cells to estrogens in the granulosa cells occurs in the ovaries by the enzyme aromatase (black highlight). Androstenedione can be converted into estrone, and testosterone can be converted to estradiol, with both actions mediated by aromatase. T is converted into dihydrotestosterone in peripheral tissues such as the prostate, skin, and epididymis of the testes by the enzyme 5 α-reductase (light gray highlight). P4 is ultimately converted to the mineralocorticoid, aldosterone through a series of reactions catalyzed by 21 hydroxylase, 11β-hydroxylase, and aldosterone synthase. 17OH progesterone is converted to 11 deoxycortisol by 21 hydroxlase, and then to cortisol by 11β- hydroxylase. Cortisol and cortisone are involved in a reverse reaction whereby cortisol is converted to cortisone by aldosterone synthase, and cortisone is converted to cortisol by 11β- hydroxylase (244).

2.3.1.2 Estrogen production. In males, the aromatase enzyme in sizable quantities in the Leydig cells of the testes convert T to E2 (61). Albeit unable to independently stimulate T production in the Leydig cells, attenuation of E2 in males has been show to inhibit in vitro production of T (7), owing to the critical role of E2 in production of T. In females, lutenizing hormone and follicle stimulating hormone released from the anterior pituitary gland aids in synthesis of E2 from cholesterol in the ovary by way of the two-cell model of steroidogenesis (130). Lutenizing hormone binds with lutenizing hormone receptors on the theca cells of the ovary, ultimately converting cholesterol into androstenedione and T through a series of reactions (130). Some androstenedione may be converted to T by the enzyme 17β-hydroxysteroid- dehydrogenase. Both androstenedione and T are transferred to the granulosa cells of the ovary, which are rich in aromatase (130). Then with the attachment of follicle stimulating hormone to the follicle stimulating hormone receptor on the granulosa cells, androstenedione and T are converted to E2 through the aromatase enzyme (130). 17β-estradiol is the most prevalent and potent E2 in humans, and the metabolites of this form of E2 (estrone and estriol) are much weaker agonists (145). In terms of feedback systems, E2 provides both negative and positive feedback to the pituitary gland (130). Negative feedback of E2 occurs when E2 levels are low during the follicular phase, while positive feedback occurs when E2 concentrations are high near the end of the follicular phase to trigger elevated production of follicle stimulating hormone and lutenizing hormone from the anterior pituitary and consequently more E2 production.

2.3.2 Physiological Changes throughout the Menstrual Cycle

The menstrual cycle is the repetitive process of ovarian follicle maturation, ovulation, and development of and possibly the sloughing-off of the endometrial lining of the uterus (pending fertilization or lack thereof, respectively). The normal menstrual cycle in humans (28 days) is characterized by two phases (56). The first phase, the follicular phase (days 1-13) is separated into two phases: the early follicular phase (days 1-4) when menstruation occurs, and the late follicular phase (days 5-13) (56). The follicular phase is characterized by significantly low levels of lutenizing hormone, as well as E2 and P4 (56). However, the secretion of follicle stimulating hormone from the anterior pituitary gland rises and peaks during this phase, which stimulates the growth of the ovarian follicle (aggregation of cells encapsulating an ovum, or egg), as well as the release of E2 from the dominant developing follicle (56). The secretion of

E2 from the dominant follicle inhibits growth of other follicles, as well as follicle stimulating hormone production, while concomitantly stimulating the development of the endometrium (mucus and blood lining in the uterus) and secretion of lutenizing hormone (56). This surge in lutenizing hormone triggers ovulation around day 14, marking the beginning of the second phase of the menstrual cycle: the luteal phase (days 15-28, separated into early-, mid-, and late-luteal phases), which is characterized by significantly elevated E2 and P4, and decreased follicle stimulating hormone and lutenizing hormone (56). During this phase, rupture of the matured follicle creates a corpus luteum (glandular endocrine encapsulating structure), which secretes E2 and P4 (56). E2 and P4 production by the corpus luteum in the luteal phase inhibits lutenizing hormone and follicle stimulating hormone release, while P4 thickens the lining of the endometrium (56). The corpus luteum degrades over time, and subsequently, P4 levels slowly fall (56). The endometrium cannot be maintained without the presence of P4, and the lining is therefore sloughed off (menstruation) (56). Because follicle stimulating hormone is no longer inhibited by P4, the menstrual cycle begins again (56). If fertilization occurs, the cells surrounding the developing embryo begin to produce human chorionic gonadotropin, which maintains the corpus luteum so that P4 production continues until the placenta (connects developing fetus to the uterine wall, allowing for nutrient uptake without mixing of blood) can produce its own P4.

2.3.3 Effect of Oral Contraceptives on Synthesis of Endogenous Hormones

Oral contraceptives (OC) are the most widely used form of contraceptives (28% or 10.6 million women) (176), and are composed of synthetic E2 and P4 in differing doses. Generally, the type of synthetic E2 remains the same throughout different forms of OC, whereas type and dose of P4 changes. When ingested chronically, OC activate a negative feedback system signaling the brain to suppress production of endogenous E2 and P4 (129). Suppression of these endogenous hormones decreases the occurrence of ovulation and thickens the cervical mucus and endometrium, making transport of sperm and implantation less likely (129). OC are ingested daily for three weeks, followed by a week of a PLA pill wherein menstruation generally occurs (129). However, it is important to note that the various forms, formulations, and generations of OC may influence measures of physiological outcomes differently. Likewise, it is important to note that there are monophasic (active tablets containing the same dose of hormones) and

34 multiphasic formulations (developed to lower the dose of P4 throughout the active pills). Therefore, the exogenous E2 and P4 content of each formulation varies greatly, and thus, direct associations with fat and muscle metabolism are difficult to make (91).

2.3.4 Physiological Interactions and Responses of Sex Hormones

E2, P4, and T receptors are ligand-inducible transcription factors and nuclear receptors that are activated with binding (127). Upon binding, the receptors dissociate from the heat shock proteins and dimerize, enter the nucleus, and become transcription factors (127). The dimerized receptors bind to their respective response elements located at the promoter target genes on the DNA molecules (127). From that point, binding stimulates the transcription of genes, known as the genomic effects, taking place within ≥ 2 hours (127). In the unbound state, the E2, P4, and T nuclear receptors block transcription of proteins (127).

The sex hormones may also have nongenomic effects whereby they influence second messenger signaling cascades (127). These effects typically occur within seconds to minutes (127). The following sections will discuss the effects of each of these hormones on fat and muscle metabolism; however, supraphysiological doses of exogenous hormones are used in many of the following studies. Additionally, ovariectomized or castrated rodent models and/or in vitro human adipocyte or muscle cell cultures are often cited, making direct comparisons to humans difficult.

2.3.4.1 Estrogen: binding and physiological effects. There are two isoforms of estrogen (E2) receptors (ER), known as Eralpha (Erα) and Erbeta (Erβ) which respond to 17-β estradiol (266). It appears that both ER isoforms are influential in the anti-lipogenic activity of E2; however, precise effects and mechanisms are unknown (105, 144). There are clear differences in the expression of both isoforms between genders, as well as effects on fat metabolism (see section 1.3.7.2) (83). E2 is transported to adipocytes in both paracrine and endocrine fashions, and the adipocyte itself can produce E2 from an androgen precursor (365). Importantly, there is a high interspecies variability with fat metabolism actions of ER, and therefore, definitive conclusions are difficult to make.

The binding of E2 to the ER elicits multiple responses in the fat tissue, and is dependent upon attachment to the Erα or Erβ isoforms. Erα mediates the positive metabolic effects of E2,

35 including enhanced lipolysis, anti-lipogenesis, enhanced insulin sensitivity and glucose tolerance, and attenuation of body weight and adipose tissue mass accrual (105). These effects are clearly depot-specific (114), as a decrease in E2 with menopause is somewhat associated with an increase in visceral adipose tissue and a decrease in WAT (379), while E2 supplementation in male-to-female transsexuals (90) and postmenopausal women (217) is known to inhibit basal lipolysis and Epi-stimulated lipolysis in the subcutaneous adipose tissue, respectfully. However, one study has shown no effects on visceral adipocytes with ER supplementation in human women, but did indeed show an upregulation of antilipolytic α 2-AR and an attenuation of Epi-induced lipolysis in subcutaneous abdominal adipose tissue (SCAAT) through Erα (285). Thus, the effects of E2 are likely depot-specific and perhaps, the specific effects of E2 depend on the balance of the activated ER. Conversely, although Erβ is also known to decrease lipogenesis, its effects on lipolysis are unknown (105).

In a recent review by Foryst-Ludwig and Kintscher (105), researchers eloquently summarized the metabolic effects of each of these receptors in adipose tissue. Researchers found that Erα and Erβ have antagonistic effects on insulin sensitivity and glucose tolerance in WAT and skeletal muscle through GLUT-4 expression, as well as respiratory quotient (the ratio of carbon dioxide produced to oxygen used at the cellular level), whereby these factors are increased with Erα activation and decreased with Erβ activation (105). In addition, Erα activation increases food intake, while Erβ has no effect (105). Lastly, activation of Erα increases insulin secretion and pancreatic β-cell function, and hepatic insulin sensitivity, while it decreases gluconeogenesis primarily in the SCAAT (105). The effects of activation of Erβ on these factors is unknown (105).

E2 reduces adiposity through the utilization of lipids as fuel. With binding, fat oxidation in the muscle is elevated due to alterations in fuel allocation and enhancement of oxidative capacity, potentially mediated by up-regulation of muscle LPL (78). E2 enhances the pathways of fat oxidation in the muscle through upregulation of peroxisome proliferator activated receptor δ, and activation of AMPK, which regulates fat oxidation, FFA synthesis, and glucose uptake (78). AMPK down-regulates the expression of the lipogenic sterol regulatory element-binding protein 1 (78). Additionally, through inactivation of acetyl-CoA carboxylase, AMPK prevents the synthesis of a necessary metabolite in TAG synthesis (malonyl-CoA), consequently

36 enhancing carnitine palityltransferase-1 activity (78). This increase in activity promotes oxidation by transporting long chain FA into the mitochondria (78).

Although production of E2 in males is lower compared to females (males: 15-60 pg/mL; females: 30-370 pg/mL) (168), the relationship between E2 and fat metabolism is similar. Males, just like females, express the ER in adipose tissue (284). In a study using Erα knockout male mice, WAT weight in many depots surrounding organs increased by more than 100% due to decreased energy expenditure, but not necessarily an increase in food consumption (71, 144). Likewise, these male mice experienced adipocyte hyperplasia and hypertrophy, insulin resistance, and glucose intolerance similar to female Erα knockout mice (71). Interestingly, in male-to-female transsexual humans, one year of E2 treatment combined with anti-androgen treatment resulted in increases in adipocyte size and lower basal lipolytic rate (90). However, researchers noted that it was unclear whether these effects were mediated by the changing E2 or T milieu with treatment. E2 supplementation has also been shown to alter fuel utilization both at rest and during exercise (cycling for 90 min at 65% maximal oxygen uptake [VO 2max]), as fat oxidation is significantly enhanced while CHO oxidation is significantly decreased in recreationally active men supplemented with E2 (22 mg 17β-estradiol/d) for 8 days (132). However, neither fat mobilization (measured by GLY rate of appearance and disappearance) nor respiratory exchange ratio (RER) were shown to be affected with 8 days of ~3 mg/d of E2 supplementation in healthy, active men during exercise (90 min of cycling at 60% peak oxygen uptake [VO 2peak]) (62). In the latter study, it is important to note that fat mobilization and RER measures were not decreased with E2 supplementation, and thus, in men, although not all studies show an increase in fat metabolism, E2 will not decrease these measures with exercise.

As with the effects on fat metabolism, the most basic way to study the effects of E2 on muscle anabolism and catabolism is through either ovariectomization of rodents, or hormone replacement studies in post-menopausal females. In ovariectomized rats, supplemental E2 was able to increase skeletal muscle growth, likely due to its effect on skeletal MPS (378). Likewise, in post-menopausal females, hormone replacement therapy has been shown to preserve or add muscle mass by inhibiting menopausal-related MPB (291, 302). However, many studies refute this finding, as ovariectomized female rats (low E2 levels) have significantly higher fat free mass (compared to sham groups and ovariectomized groups supplemented with both E2 and P4),

37 mediated by increases in MPS, whereas supplementation with E2 and P4 attenuated these increases in muscle mass accrual and MPS (317, 378). E2 also attenuates MPB, as measured by reductions in 3-methylhistidine excretion in growing ovariectomized female rats (342); however, this measurement may not be the most accurate measurement of MPB (251).

Overall, receptor-dependent, E2 appears to have a direct anti-lipogenic and lipolytic impact on adipocytes, acting to increase lipolysis and fat oxidation, but can also act centrally to affect behavioral energy intake and expenditure. In muscle, supplemental E2 acts to enhance protein synthesis and attenuate MPB, as shown in ovariectomized rodent and post-menopausal females on hormonal replacement therapy models.

2.3.4.2 Testosterone: binding and physiological effects. T and its metabolite 5α- dihydrotestosterone activate the androgen receptor. T concentrations are greater in men (300- 1000 ng/dL) compared to women (6-86 ng/dL) (168), and there are higher densities of androgen receptors found in visceral adipose tissue compared to subcutaneous adipose tissue (42). Exposure of adipocytes (specifically visceral adipocytes) to T up-regulates the number of androgen receptors on the cell surface (286). Therefore, in human males there is a strong inverse relationship between T level and visceral body fat (382); however, no other correlations have been found when considering other fat depots in a 7.5 year follow-up study (382). Even in young human males (aged 20-29 years), visceral body fat is inversely correlated with bioavailable T levels (265).

T is highly lipolytic, as shown by changes in adiposity, lipolysis and fat oxidation rates with T suppression and supplementation. With T suppression (using a gonadotropin releasing hormone analog) in healthy, lean men, fat oxidation was significantly suppressed, (-31%; p=0.05) and adiposity was significantly elevated (pre: 19.2 ± 2.5% fat mass; post: 22.2 ± 2.5% fat mass, p=0.001) (233). Conversely, T treatment consistent shows the opposite findings. Fat mass was significantly decreased in hypogonadal males treated with 180 days of T gel at low (50 mg/day: -0.90 ± 0.32 kg) and high levels (100 mg/day: -1.05 ± 0.22 kg) (392). Further, substrate oxidation shifts with T treatment as one study has shown that after 6 months of transdermal T treatment in males with low to moderate T levels, basal fat oxidation is increased (5.65 mg/min/m 2, p=0.045) and basal CHO oxidation is decreased (-9.71 mg/min/m 2, p=0.046) (109). Lastly, while both abdominal and gluteal depot subcutaneous fat and adipocyte size were

38 significantly decreased in female-to-male transsexuals with T treatment, basal lipolytic rate was only significantly increased in abdominal, but not gluteal depots (90). These findings indicate that T is certainly lipolytic, and that there are likely regional metabolic variations.

From a mechanistic standpoint, T upregulates adrenoceptors, and thus, enhances lipolysis (50). In isolated rat adipocytes, T has been shown to stimulate the NE-stimulated lipolysis (134), and increase the number of lipolytic β-AR on the adipocyte (286). Specifically, this relationship occurs in a dose-dependent fashion through β-AR activation and adenylate cyclase formation (408). In order to clarify the lipolytic effects of the sex hormones, even when an aromatase inhibitor was administered, T-induced lipolysis was still significantly elevated, clearly indicating that E was not responsible for these lipolytic actions (408). Circulating androgens also tend to reduce LPL activity (230), and inhibit cortisol’s LPL-activating effects (43). These effects are accentuated in the presence of GH, as the combination of T and GH significantly increases fat metabolism to a greater extent than each of them, individually (314).

In terms of muscle anabolism and catabolism, T has robust effects. Importantly, in skeletal muscle tissue, the activity of 5α-reductase is minimal, thus the metabolite dihydrotestosterone is not nearly as effective, making T the main regulator of growth (400). The effects on muscle anabolism and catabolism by T are apparent, given the overuse and abuse of anabolic steroids containing T or derivatives of T (303). It is known that even with acute administration of injectable T or oral oxandrolone (synthetic T analogue), MPS is increased in normal, eugonadal males (348). However, acutely, MPB remains unchanged (348). Additionally, in hypogonadal males (37, 51, 184), as well as hypogonadal elderly males (371), T replacement therapy significantly increases muscle mass, indicating a strong, positive effect of T on MPS.

Overall, T increases lipolysis and attenuates accumulation of TAG in adipocytes. Low levels of T increase adipose tissue mass (specifically in the subcutaneous depots), whereas increased levels of T reduce total body adipose tissue. T also acutely increases MPS, but only increases muscle mass with chronic utilization.

2.3.5 Differences in Fat Metabolism between Sexes

2.3.5.1 Amount and distribution of adipose tissue. Even after correcting for BMI, adipose stores are greater in females compared to males, holding true across all races and cultures (304). Interestingly, the average body fat percentage (29 ± 2% body fat) for normal BMI females (18-25 kg/m 2) is very similar to the body fat percentage (32 ± 1% body fat) of obese males (BMI >30 kg/m 2) (264). In terms of depot storage, females tend to store more fat in the gluteal-femoral regions, whereas males tend to store more fat in visceral abdominal regions (210). In the abdominal region (in general), young men have significantly higher abdominal visceral adipose tissue stores (men: 122.93 ± 48.96 cm 2 vs. women: 103.71 ± 53.77 cm 2, p<0.05) and total body visceral adipose tissue stores (men: 5.23 ± 2.39 L vs. women: 3.61 ± 1.91 L, p<0.01), while young women store more subcutaneous adipose tissue (women: 427.90 ± 200.52 cm 2 vs. men: 253.96 ± 100.76 cm 2, p<0.05) (210). However, males tend to have a higher turnover of visceral fat compared to females, as rates of both lipolysis and lipogenesis in the visceral fat depots are greater in males compared to females (398). This difference is purportedly due to the absence of CAT-induced stimulation of splanchnic FA release in females compared to males (171).

2.3.5.2 Receptor expression. In terms of receptor concentrations, androgen and Erα/Erβ densities have been found to be more dependent on adipose tissue depot than sex, with higher mRNA concentrations of these receptors in visceral depots compared to subcutaneous depots (322). Likewise, lipolytic Erα expression is greater than antilipolytic Erβ regardless of depot or sex (322). Erα expression is not different between males and females; however, Erβ expression is greater in females compared to males in adipose tissue (83). In vitro , E2 is shown to upregulate both Erα and Erβ, whereas in males, only Erα is up-regulated (83). Therefore, in females, the Erα:Erβ ratio remains constant, while in males, this ratio can increase (83), clearly owing to the potential differences in resting fat metabolism in males compared to females.

Although E2 up-regulates α 2-AR in subcutaneous adipose tissue (resulting in attenuation of lipolysis) in both males and females, E2 does not affect α 2-AR levels in the visceral adipose tissue (285). Further, in subcutaneous adipose tissue, females have higher α 2-AR levels and consequently lower subcutaneous lipolytic rates compared to males (318). However, specific to the subcutaneous gluteal regions, β-AR activity is about the same between males and females

(318); therefore, it may be plausible to assume that much of the discrepancies in fat metabolism are due to location of α 2-AR.

In addition to the AR and ER, adipose tissue levels of androgen receptors also vary between the sexes. For both males and females, subcutaneous fat contains a lower amount of androgen receptors compared to visceral fat (322). Further, in females, E2 down-regulates androgen receptor expression (362). This point is further emphasized by the fact that females with polycystic ovarian syndrome (hyperandrogenic and produce pronounced endogenous concentrations of T) tend to accrue visceral fat to a comparable rate and amount compared to their healthy, male counterparts (313).

2.3.5.3 Hormonal differences affecting fat metabolism. As noted, normal ranges of T (men: 300-1000 ng/dL; women: 6-86 ng/dL) and E2 (women: 30-370 pg/mL, depending upon the phase of the menstrual cycle; men: 15-60 pg/mL) vary between the sexes (168). In vitro , E2 increases proliferation and differentiation of preadipocytes in both males and females, with effects greater in females compared to males (15). In females, as previously mentioned, E2 down-regulates the androgen receptor (362). Although E2 favors subcutaneous fat deposition in females, a dearth of E2 leads to visceral fat weight gain (304), shown by the fact that postmenopausal females have greater amounts of visceral fat compared to premenopausal females (370).

Due to the differences in E2 between males and females, females are more apt to use fat as fuel during sustained bouts of energy expenditure such as aerobic activities, whereas males are more likely to utilize amino acids and glucose (206). Interestingly, when given E2, the ability for males to metabolize amino acids and glucose during sustained exercise significantly decreases (132). Likewise, albeit a miniscule increase (10-20%), when postmenopausal females were supplemented with E2, FA rate of appearance was elevated (172) indicating enhanced utilization of fat. However, this small increase in FA rate of appearance in postmenopausal females is likely not transferable to a premenopausal population, as premenopausal women have normal levels of E2.

The metabolic actions of T in adipose tissue vary greatly between visceral and subcutaneous depots, as well as acute and chronic exposures. In young men, levels of visceral

41 and subcutaneous adipose tissue are inversely correlated with level of T, with the relationship between subcutaneous adipose tissue and T level accounted for by differences in sex-hormone binding globulin (265). Acutely, in cultured in vitro adipose precursor cells, administration of T increases the basal rate of lipolysis through elevations in β-AR number and activity of adenylate cyclase (408, 409). Chronically, as previously mentioned, in the visceral adipose tissue of women, long-term administration of T has a lipid-accumulating effect (89). Conversely in subcutaneous regions, T, when provided over two months, has been shown to increase the turnover of fat in obese males (230). In female rats, after an ovariectomy, T replacement was shown to increase the number of β-AR and cAMP-mediated stimulation of lipolysis, but not lipolysis stimulated by catecholamines; thus, T treatment after an ovariectomy is ineffective in restoring the total intercellular machinery needed for lipolysis (287). The lipolytic responses to Epi seem to be similar between males and females in vivo (171, 250). However, it seems that lower-body adipose tissue lipolytic rate is increased in males but not females, whereas upper- body adipose lipolytic rate is similar between the sexes (171, 250).

Cortisol production is also affected with differences in the sex hormone milieu, as females have an increased production of cortisol via the enzyme 11β-hydroxysteroid dehydrogenase type 1 (310). This effect is likely due to increased fat mass in females compared to males, as a similar effect is seen between obese males and females (310). Additionally, in vitro , E2 and glucocorticoids prompt leptin secretion from female adipocytes; however, male adipocytes do not respond (63).

Leptin and insulin, as previously discussed, regulate central and peripheral fat metabolism. Subcutaneous fat is more closely associated with leptin concentrations, and visceral fat is more closely associated with insulin concentrations (304). Peripherally, due to the higher levels of subcutaneous fat in females, leptin is generally better correlated with fat mass, while insulin is better correlated with fat mass in males (405). This effect can be seen as administration of intranasal insulin enhances fat weight loss and decreases feelings of hunger in males, while insulin administration enhances water weight gain and feelings of hunger in females (131). Responsiveness to these hormones seems to be mediated by the sex hormones, as E2 administration increases the sensitivity to central leptin and decreases the sensitivity to central insulin in male rats (67). Although any type of increase in fatness is associated with a decrease

42 in insulin sensitivity, insulin sensitivity in females is less affected with fluctuations in fat mass compared to males (353). This is likely due, again to the differences in the distribution of visceral and subcutaneous fat stores as these fat depots respond to insulin differently (88). Interestingly, regardless of amount of fat mass, females have higher levels of circulating leptin (278).

2.3.5.4 Fat metabolism . Males and females also differ in rate of fat mobilization, utilization of fat for metabolism, and the metabolic consequences of fuel restriction and excess (304). The differences in fat metabolism relative to sex are depot-specific. Rates of mobilization of FA are higher in the abdominal adipose tissue in females compared to males; however, in females, rates of FA mobilization are much lower from the femoral or gluteal adipose tissue (398).

With feeding, metabolic kinetics are also different between the sexes. After a glucose- rich meal, it is imperative to decrease endogenous production of VLDL-TAG to maintain lipid homeostasis, as postprandial CHY begin to release FA into circulation immediately after a meal. This effect is mediated by the glucose-induced rise in secretion of insulin, which suppresses VLDL-TAG production (214). With glucose infusion and subsequent hyperglycemia- hyperinsulinemia, the production of VLDL-TAG is decreased in lean and obese males and lean females, but not obese females, despite similar plasma FA responses in all groups (252). Therefore, in obese females, FA availability in the plasma may not be the key regulator of production of VLDL-TAG. This finding of impaired fat metabolism, albeit through different mechanisms, is consistent with other findings in obese versus lean females (213) and a combination of females and males (40), purportedly mediated by obesity-induced insulin resistance. Not surprisingly, when groups were split between insulin-sensitive and insulin- resistance subpopulations, it was found that large VLDL-TAG concentrations were attenuated after hyperinsulinemia in insulin-sensitive subjects, and remained the same in the insulin- resistance subjects (40) indicating dysregulation of blood lipid responses to a glucose load with obesity. Additionally, after feeding, in both males and females, rate of FA uptake is higher in the abdominal adipose tissue compared to the femoral or gluteal tissue; however, in the abdominal regions, FA uptake in males primarily moves to the visceral fat, while FA uptake in females primarily moves to the subcutaneous fat (398).

At rest, measurements of GLY rate of appearance (index of whole body lipolytic rate) was shown to be higher in females compared to males when matched for percentage of body fat (252). This effect was proposed to be due to higher concentrations of circulating insulin in males, and thus increased suppression of lipolysis (252). However, regional differences in sensitivity to insulin is different between the sexes, as females have a greater suppression of FA release during an oral glucose tolerance test (240), likely due to less insulin resistance of the upper body (visceral and abdominal) subcutaneous adipose tissue in females (prompting storage in these regions) (169). Interestingly, the females in these studies had more body fat than the males, and although higher amounts of body fat are associated with a decreased responsiveness of the adipose tissue to insulin (100), these females displayed higher suppression of FA release. Similarly, although GLY rate of appearance is greater in females compared to males in basal conditions, with acute fasting (22 hours) GLY rate of appearance is greater in males, likely due to amplified Epi levels with fasting (253). This relationship, again, may be a protective mechanism by which females have a blunted lipolytic response in order to prevent potentially harmful increases in circulating FA concentrations (356).

Likewise, although resting energy expenditure (REE) is tightly correlated with rate of appearance of FA, females tend to have higher rates of FA mobilization in relation with their energy requirements compared to males (263). As previously mentioned, this increase in FA mobilization may be of benefit to females in times of increased fuel requirements such as prolonged exercise; however, these high concentrations may compromise the health of the liver (236). Thus, to maintain some sort of FA homeostasis, it is reasonable to assume that increased sensitivity to the antilipolytic effects of insulin in females is a compensatory mechanism for the increased rate of basal flux of FA.

Also at rest, more circulating NEFA are shunted into re-esterification pathways in females compared to males (263). This would theoretically increase the rate at which females re- esterify FA. Also, although circulating levels of VLDL-TAG are similar between sexes, lean females have greater production rates of VLDL-TAG compared to lean males (252). Further, VLDL-TAG secretion rate in both males and females is shown to increase with increasing body fat (252). Even more, plasma levels of VLDL-TAG are inversely related to VLDL-TAG clearance in females, but positively associated with VLDL-TAG production in males (252).

Thus, it appears females hold on to fat substrates longer than males, maybe predisposing them to higher storage rates, as VLDL-TAG delivers TAG to adipocytes for storage.

2.3.6 Differences in Muscle Anabolism and Catabolism between Sexes

Males and females have different amounts and distributions of skeletal muscle mass, as well as different substrate metabolism by skeletal muscle.

2.3.6.1 Amount and distribution of muscle tissue. Males are known to have more total (men: 33.0 ± 5.3 kg, females: 21.0 ± 3.8 kg) and relative (men: 38.4 ± 5.1 kg, women: 30.6 ± 5.5 kg) skeletal muscle mass compared to females, with differences in the upper body compartments (men: 14.1 ± 2.6 kg, women: 8.4 ± 1.8 kg) (167). Although distribution of muscle fiber types is similar between the sexes, males have a significantly greater cross sectional area of muscle compared to females, as displayed by the disparities in size of the biceps brachii (men: 6632 mc 2, women: 3963 mc 2) and vastus lateralis (men: 7070 mc 2, women: 4040 mc 2) muscles (248). Likewise, the predominant muscle fiber type and the pattern of the area of occupation of each of the fibers may vary between sexes; however, this evidence is inconclusive (248, 337, 359).

2.3.6.2 Hormonal differences affecting muscle anabolism and catabolism. Hormones play a role in the differences in muscle protein metabolism, displayed by the disparities in the size of muscle between the sexes. In males, T levels are about 10-15-fold higher than concentrations in females, and because T is a strong anabolic hormone, males would theoretically have significantly higher levels of protein synthesis. However, it is seemingly difficult to examine the comparison of the effects of T between males and females, as there could be unwarranted side effects associated with exogenous administration of T in females. The potent differences in the effects of T can be observed during prepubertal times. Before puberty, boys and girls are similar in size and have the same amount of muscle mass (179, 307) and T concentrations (307). After puberty, T concentrations in boys drastically increase (307) with an associated increase in overall size and muscle mass (179); however, similar T and muscular changes are not observed in girls (307). Thus, the variations in hormones clearly contribute to the differences in lean body mass between the sexes.

2.3.6.3 Glucose metabolism in muscle between the sexes. It appears that during normoglycemic-hyperinsulinemic times at rest, whole body insulin sensitivity is 41% greater in

45 females (52 ± 6 μmol/kg muscle −1/min −1) compared to males (37 ± 3 μmol/kg muscle −1/min −1, p<0.05). Women also display a 47% higher rate of glucose uptake by femoral muscles (113 ± 10 μmol/kg muscle −1/min −1) compared to men (77 ± 7 μmol/kg muscle −1/min −1, p<0.01) (269). Therefore, women may theoretically have a metabolic advantage in energy flux into the muscle, as displayed by elevated glucose entry during rest.

2.3.6.4 Muscle protein metabolism . Although it may be assumed that larger muscle masses in males would be associated with higher basal MPS, research does not support this notion. For example, although males were found to have elevated absolute MPS, MPB, and net muscle protein balance in the post-absorptive state in the forearm compartment, once lean mass was corrected for, these changes were not apparent (166). Other studies have also confirmed that there are no differences between the sexes in MPS (295, 412), MPB (295), or net muscle protein balance (295). Likewise, males may catabolize more protein by about 30% (as measured by urea nitrogen excretion) compared to females with endurance training (ET) (65% VO 2max for 15.5 km) (369). LEU flux and oxidation are also greater in males compared to females during submaximal ET, as measured by a primed continuous infusion of L-[1-13C]LEU (292). Measurement of labeled radioisotopes and nitrogen balance are becoming more commonplace. However, like whole body protein metabolism, the dearth of research regarding the differences between males and females in basal muscle protein metabolism makes definitive conclusions difficult to make.

2.4 The Effects of Exercise on Fat and Muscle Metabolism

Exercise has a significant impact on fat and muscle metabolism. Specifically, this section will discuss the detrimental effects of inactivity, as well as the beneficial effects of ET and RT on fat and muscle anabolism and catabolism. Further, this section will also discuss the differences in fat and muscle anabolism and catabolism between varying intensities of ET and RT exercise.

2.4.1 Effects of Inactivity

The effects of inactivity on metabolism are so potent that they can be reported as soon as activity ceases. For example, when exercise volume is significantly decreased for as little as two weeks, individuals experience decreases in glucose tolerance, postprandial lipid metabolism, sensitivity of muscle LPL (reduces uptake of plasma TAG into muscle and consequently

46 utilization) (36), Epi-stimulated lipolysis (81), whole body lipolysis and skeletal muscle HSL activity (9), and lipolytic rate (155). Additionally, inactivity results in increases in insulin resistance (31, 32, 44), glucose uptake into the subcutaneous femoral adipose tissue (155), and visceral fat mass (271), and altered fat trafficking between adipose and muscle tissue (32, 33). Thus, the mobilization and consequent utilization of FA as an energy source is clearly affected. Interestingly, with inactivity, although there is an increase in sensitivity to the β-AR (27), plasma CATs are attenuated, which decreases consequent lipolysis (45). Physical inactivity also attenuates fat oxidation and there is a shift toward CHO oxidation. For example, in healthy, lean males, lipid oxidation is decreased by 37% and CHO oxidation is increased by 21% with three months of bed rest in the postprandial and post-absorptive phases (32). Clearly a decrease in lipid oxidation in combination with hyperinsulinemia will lead to an increase in liver de novo lipogenesis, an increase in VLDL production, and an increase in fat storage in ectopic locations (31), as dietary fat is shunted from being oxidized for energy to being stored as intramuscular fats (33, 76). This ectopic fat storage will contribute to metabolic inflexibility, or the inability to adapt fuel oxidation to fuel availability.

From a muscle anabolism and catabolism standpoint, it is well known that inactivity results in muscle loss. Using a 28 day bed rest model, it was found that healthy, male volunteers lost a significant amount of muscle mass, due to a negative net protein balance which was the result of an attenuation in MPS (282). In addition, it is known that housebound (inactive), elderly individuals display a negative nitrogen balance (-95 mmol/d) compared to healthy, active, elderly individuals who are in equilibrium (0 mmol/d) (57).

2.4.2 Effects of Endurance Exercise

2.4.2.1 Effects of acute endurance exercise on fat metabolism. Fat is a more ideal source of energy for ET (intensity-dependent, for example at <60% VO 2max , see section 1.2.4.3) because of its high energy density, and thus sustainability, compared to CHO. However, it is important to note that fats are not stored homogenously. Slow oxidative fibers contain significantly higher levels of stored TAG (7.1 ± 1.7 mmol/kg wet weight), compared to fast twitch fibers (4.2 ± 1.2 mmol/kg wet weight); whereas the reverse relationship is true for stored glycogen (slow oxidative fibers: 77.8 ± 18.0 mmol/kg wet weight; fast twitch fibers: 84.7 ± 19.0 mmol/kg wet weight) (336). These relationships are highly influential when considering

47 substrate utilization in ET versus RT. Importantly, during ET the muscle only takes up 2-4% of circulating FFA, with only a small fraction of that percent being oxidized for energy.

Not only does the rate of appearance of FA increase from rest to moderate intensity ET, but the amount of re-esterification dramatically decreases (70% at rest to about 25% with 30 minutes of low intensity [40% VO 2max ] ET) (404). The combination of these two changes produces a 6-fold increase in FA availability for oxidation (404), as FA get shunted from the adipose tissue to working muscles during ET (326). Further, both cross-sectional (383) and longitudinal (293) studies have shown that submaximal ET increases fat oxidation and decreases CHO oxidation. It also seems as though increased fat oxidation with submaximal ET is ubiquitous across populations as it increases in lean (3) and obese individuals (72), females, different racial populations (72), aged populations (352), and both trained and untrained individuals (110). Importantly, though, fat oxidation during ET is lower in untrained compared to trained individuals (110), African Americans compared to Caucasians populations (72), obese compared to lean individuals (72), and females compared to males (304).

Early studies have shown that after an ET session, muscle TAG concentrations decrease (120, 161), indicating that muscle TAG starts high and that the net breakdown of TAG in muscle after ET is quite large (161). Importantly, in terms of changes in TAG stores, modality of exercise needs to be considered, as no changes in TAG stores were experienced when utilizing a one-leg knee extensor protocol (188). This observation is speculated to be due to a blunted sympathetic response to this modality of exercise and thus, blunted metabolic action with smaller muscle groups compared to larger or multiple muscle group exercises. The lipolytic action in muscular depots, just as in other depots, is due to β-AR activation, as it has been shown that a blockade of these receptors attenuates GLY release (183), and β-AR agonist infusion enhances GLY release in contracting muscles (325).

However, when considering the interaction between CAT-stimulated antilipolytic α 2-AR and lipolytic β-AR, adipose TAG reserves are also affected. It has been shown that low intensity

ET-induced release of CATs stimulates the antilipolytic α 2-AR of subcutaneous adipose tissue, resulting in attenuated lipolytic responses in both healthy, untrained nonobese (117, 361, 364) and obese individuals (361). Thus, energy is being derived from other stores of TAG. However, because only a modest amount of energy is derived from stored TAG inside the muscle fibers, it

48 has been suggested that much of the energy is coming from adipocyte lipolysis between muscle fibers, indicated by increases in GLY concentrations of the muscle with 8 weeks of ET training (188). This concept certainly indicates that adipocytes between muscle fibers contribute to energy production during exercise.

Another factor that may affect fat oxidation with ET is the transport of FA across mitochondrial membranes. However, there are differences in movement between FA chain lengths, as long chain FA require the CAT-1 and CAT-2 (discussed previously), and medium chain FA use a less regulated complex or can freely diffuse into the cell (333). High intensity exercise and the associated decrease in pH attenuates fat oxidation, either independently by reducing the activity of CAT-1 (360), or by amplifying the sensitivity of CAT-1 to malonyl-CoA (FA synthesis mediator that inhibits basal CAT-1 activity) (4). Conversely, with lower intensity exercise, phosphorylation of the enzymes responsible for production of malonyl-CoA by AMPK significantly reduces the concentration of malonyl-CoA, and consequently allows the uptake and oxidation of FA in the mitochondria (241).

Contrary to theoretical logic concerning the differences in fat oxidation with ET versus RT, there seem to be minimal disparities. A recent review by Melanson et al., (241) concluded that, although there are multiple studies indicating the positive effects of ET on metabolism, ET does not significantly increase 24 hour fat oxidation rates. Indeed, 24-hour fat oxidation rates are similar to that of a RT session with similar energy expenditures (380). Overall, this lack of change in 24-hour fat oxidation rate may simply be due to the anti-lipolytic and β–oxidation effects of the insulinemic response to CHO consumption prior to exercise (241). Although preexercise feeding is ethically sound from a safety standpoint when conducting research, allowing participants to consume as little as 60 g of CHO (240 kcal) an hour before the exercise protocol significantly attenuates the lipolytic and fat oxidation response during the exercise (74), as well as up to 6 hours post consumption (258).

2.4.2.2 Effects of acute endurance exercise on muscle anabolism and catabolism. ET increases MPS acutely (~50-60%) (135); however, this increase in MPS is not associated with any significant changes in muscle mass. Likewise, net amino efflux has been found to be high after cycling (226), and muscle proteolysis is also found to be high after 45 minutes of treadmill walking at 40% VO 2max (349), indicating some effect of ET on MPB. However, these effects are

49 found to be transient in young individuals (only lasting about 10 minutes after exercise), and longer-lasting in older men (350). As previously mentioned, men catabolize about 30% more protein compared to women during ET (369).

2.4.2.3 Varying intensities of endurance exercise. As the intensity of exercise varies, so does lipolysis, beta-oxidation, MPS and MPB.

Varying intensities of endurance exercise on fat metabolism. Fat metabolism and substrate utilization change greatly with varying intensities of ET (326). Generally speaking, at lower intensities, fat oxidation surpasses CHO oxidation, and the opposite is true with high intensity ET (326). Typically, intensity will fall within one of these ranges: 1) low intensity:

<60% VO 2max ; 2) moderate intensity: 60-80% VO 2max ; and, 3) high intensity: >80% VO 2max .

During ET, the lipid sources available to working skeletal muscle are derived from: 1) VLDL-TAG; 2) intramuscular TAG, and; 3) long-chain FA bound to albumin from subcutaneous and visceral depot lipolytic reactions (241). Long chain FA are the predominant source of FFA energy with very low intensity ET (25% VO 2max ) (326) and low intensity ET (241). Specifically, plasma FFA utilization was found to be the highest with very low intensity ET (25% VO 2max :

25.8 ± 2.6 µmol/kg/min) compared to moderate (65% VO 2max : 22.8 ± 2.7 µmol/kg/min) and high intensity (85% VO 2max : 17.0 ± 3.4 µmol/kg/min) ET. With low intensity ET, there was also small contributions (~5%) from VLDL-TAG (241) and plasma- and intramuscular TAG (326). In addition to the uptake of plasma FA, the hydrolysis of intramuscular TAG is also a factor in FA oxidation. Muscle TAG lipolysis is minimally, if at all stimulated with ET at low intensities

(25% VO 2max : glycerol rate of appearance: 1.1 ± 0.7 µmol/kg/min). At moderate intensities

(65% VO 2max : 6.7 ± 1.2 µmol/kg/min) muscle TAG lipolysis is significantly higher compared to both low (25% VO 2max : glycerol rate of appearance: 1.1 ± 0.7 µmol/kg/min) and high intensities

(85% VO 2max : 4.9 ± 1.9 µmol/kg/min) (326). There is also other evidence to support these findings (161). With an increase in ET intensity, fats are oxidized less, while CHO oxidation increases. Romijn et al. (326) found that with moderate intensity ET (65% VO 2max ), fat oxidation contributes half of the total energy, and CHO oxidation contributes the other half.

Similarly, in a review by Saltin and Astrand., (335) it has been quantified that at 60-70% VO 2max

(moderate intensity ET), when the RER (proportion of volume of CO 2 generated to O 2 consumption using expelled air in the calculation) is below 0.9, lipids account for about 60% of

50 expended energy (335). With intense ET (72-85% VO 2max ), LPL is able to hydrolyze VLDL- TAG to a major extent (326).

Interestingly, Romijn et al. (326) reported that lipolytic rate was the same when cycling at

65% and 85% VO 2max , potentially the result of FA trapping in the adipose tissue due to attenuated adipose tissue blood flow with increased intensity of ET (154). These changes in FA source utilization are evidenced by drastically attenuated lipolytic rate and significantly elevated plasma FA release during the post-ET recovery period (326). Unchanged (220) or decreased (326) FFA concentrations with increased exercise intensity from moderate to high is due to attenuated FFA availability, as this value is a factor of muscle blood flow and plasma FFA concentration. This effect was found by examining an increase in fat oxidation with infusion of intralipid and heparin (designed to raise and maintain plasma FFA concentrations) during an ET session at 85% VO 2max (327). Therefore, the decrease in fat oxidation with increasing exercise intensity is due in part to the decrease in FFA availability and/or the inability of FFA to increase above resting levels, which is partly due to the decrease in intracellular pH, carnitine availability, and/or FA transporters (220, 327). Likewise, oxygen availability and utilization of energy systems are also influential determinants of fat oxidation.

Using methodology that allowed for analysis of more intensities of exercise with smaller increments, it was found that fat oxidation peaks at 62.5 ± 10.4% of VO 2max in moderately endurance trained individuals (66.9 ± 1.8 ml/kg/min) (2, 5), with a strong positive association between VO 2max and maximal fat oxidation rates (216). Further, with classification of training status into moderately- and highly-trained endurance athletes, the measures of peak fat oxidation are significantly different (3). The authors mentioned that, even with separation of training status, there are still large variations in maximal fat oxidation rates (3); thus, there are likely other contributing factors to the reported discrepancies. In addition to these findings, peak fat oxidation rates (women: 8.3 ± 0.2 mg/kg/FFM, men: 7.4 ± 0.2 mg/kg/FFM) occur at a higher intensity in females compared to males (women: 63.6 ± 0.9% HR max , men: 59.5 ± 0.8% HR max ) (384). Thus, peak fat oxidation occurs at different exercise intensities depending upon other factors such as training status (VO 2max ) and sex.

Further evidence for the utilization of TAG during ET is the attenuation in fasting TAG concentrations and elevation of LPL levels in the day following ET (180). Specifically,

51 clearance of intravenous fat is increased by 76% the morning after a marathon race (331). Similarly, when tested in females, uptake of TAG after ET was increased, regardless of training status (290).

Overall, ET induces the plasma clearance of TAG. Likewise, with low intensity ET, tissue uptake of plasma glucose as well as muscle glycogen oxidation decreases, while peripheral lipolysis and subsequent release of FFA into the plasma increases (326). Importantly, these effects are seen in trained participants. Overall, as the intensity of ET increases, the utilization of lipids as a substrate decreases; however, it seems that length of the ET session (as in long- distance running) and intensity of long-duration sessions significantly affects FFA availability.

Varying intensities of endurance exercise on muscle anabolism and catabolism. Just as with fat metabolism, intensity of ET affects muscle anabolism and catabolism. In healthy males, MPS is elevated 24-28 hours after high- (30 min at 60% maximum wattage), but not low- (60 min at 30% maximum wattage) intensity cycle ergometer ET in the fasted state (85). However, there are no studies that directly examined the effects of varying intensities of ET on MPB.

2.4.2.4 Effects of chronic endurance exercise training. Training status and amount of ET certainly affect both fat and muscle anabolism and catabolism.

Effects of chronic endurance exercise training on fat metabolism . Training status has a profound effect on one’s ability to metabolize TAG. Trained individuals display elevated fat oxidation rates compared to untrained individuals, even at the same absolute and relative ET intensities (110). This disparity is partially explained by the ability of trained individuals to sustain work at higher absolute work rates. Interestingly, there are even differences between various levels of trained individuals (3). Although the relative contribution of fat oxidation to the total energy expenditure is the same between moderately- and highly-trained individuals, oxidation rates are much higher in trained individuals, eluding to the differences in metabolic efficiency between training statues (3).

There are several physiological changes mediated by ET that allow for enhanced metabolic efficiency. Endurance exercise training increases vascularization to muscle tissue, which enhances LPL delivery and activity in the muscle (163). In men that ET only one leg per day for eight weeks at 65% VO 2max (163), muscle LPL activity was found to be significantly

52 higher in the ET leg compared to the non-exercise leg, which authors associated with improvements in capillary density (163). Therefore, trained muscle can harness higher amounts of FA from TAG-rich lipoproteins for energy with greater vascularization. Amount of Type I muscle fibers is also positively associated with LPL activity (165), and because endurance athletes have elevated amounts of these fibers (399), metabolic capacity of TAG handling is increased. Ultimately, there is direct evidence of enhanced TAG clearance rates in trained compared to untrained males and females (95, 297, 330). In addition, ET males have enhanced TAG removal rates after an oral fat challenge, as the area under the curve (plasma TAG concentration versus time) after an ET bout is lower in ET males compared to untrained males (70, 246). Additionally, trained individuals experience greater Epi-stimulated blood flow in subcutaneous tissue compared to sedentary individuals; however, trained individuals have the same lipolytic sensitivity to Epi compared to sedentary individuals (358). Thus, higher levels of blood flow and fat oxidation rates indicate a greater metabolic efficiency of the sympathetic response (specific to subcutaneous adipose tissue) to exercise in trained individuals.

Many of these training enhancements may be because of training status on improvement of TAG uptake to produce energy in the muscle. Trained individuals have the enhanced ability to oxidize intramuscular TAG for energy utilization (394). Henriksson et al., (147) reported that when participants trained on a one-legged bicycle for eight weeks (60 revolutions/min at 150- 225 Watts), fat uptake into skeletal muscle as well as fat oxidation were enhanced in the trained leg but not the untrained leg (147). Researchers associated these changes with increased mitochondrial capacity to oxidize TAG (147).

ET also has a profound impact on fat metabolism in deconditioned, sedentary and chronic disease populations. Postprandial TAG uptake can improve with ET in sedentary individuals, as coronary artery disease patients exhibited a significantly attenuated lipemic response with ≥3 months of ET (as a part of a cardiac rehabilitation program), compared to sedentary patients (411). Likewise, with weight loss, healthy, young males experienced a 49% improvement in the ability to clear TAG as well as an increase in LPL activity after an oral fat challenge (395). However, with twelve weeks of training in hyperlipidemic males, and 12 weeks of ET with walking in healthy, middle-aged females (138), there were no significant improvements in fat tolerance with an oral fat challenge. It is important to note that testing occurred 48-72 hours

53 after the last training session, indicating that timing of testing in these studies may not have been ideal. In addition, twelve weeks of ET in obese females did not promote beneficial alterations in lipolytic pathway gene expression, or improve the basal lipolytic response; however, it did reduce the actions of the CATs on the antilipolytic α-AR during the ET (320). These findings indicate that ET likely promotes significant alterations in fat metabolism in most populations that are predisposed to poor regulation of fat metabolism; however, all the mechanisms required to enhance metabolism may not be improved.

Effects of chronic endurance exercise training on protein turnover. With 4 weeks of ET

(30-45 minutes per day, 3-5 days per week, ≥65% HR max ) in previously unfit, healthy men and women, skeletal muscle protein turnover is significantly increased without changes in body mass or body composition (296). However, although there was a significant increase in MPS in the study, net protein synthesis decreased due to an increase in MPB (296). In contrast, another study found that a higher ET intensity (up to 45 minutes at 80% peak heart rate, 3-4 days per week) significantly elevated MPS with no effect on whole body protein turnover in healthy, previously untrained men and women (351). Thus, there seems to be inconclusive evidence on the effects of ET on muscle protein anabolism and catabolism.

2.4.3 Effects of Resistance Exercise

Compared to ET, the effect of RT on fat metabolism is not extensively studied. However, there are a multitude of studies examining the effects of RT on muscle anabolism and catabolism. Likewise, the rate of fat metabolism is commonly explored during the exercise perturbation with ET, while it is examined after the exercise perturbation with RT. Additionally, in general whereas fats are the main substrate utilized in low to moderate intensity ET and RT, CHO are the main substrate utilized in high-intensity ET and RT. This effect is shown as FFA levels remain elevated after a low-intensity RT bout (60-70% 1 repetition maximum [1RM], 12- 15 repetitions), indicating the preferential use of CHO over fats (301). Similar effects are shown with high-intensity RT (intermittent 30-s exhaustive exercise bouts targeting the quadriceps femoris muscle comprising 6–12 repetitions, interspersed with 60-s rest periods for 30 min) in bodybuilding young men (97), as plasma glycerol and FFA are increased. However, these authors concluded that the elevation in FFA likely indicated that lipolysis contributed to substrate utilization during high-intensity RT, whereas the aforementioned study concluded that

54 higher FFA levels indicated preferential use of CHO. Overall the differential effects of RT intensity on fat metabolism requires further elucidation.

2.4.3.1 Effects of acute resistance exercise on fat metabolism . Acute RT initiates various beneficial fat metabolism responses. Metabolic rate has been found to be elevated up to two hours post-RT (243), as well as into the morning after the evening training session (243). Likewise, after RT, RER, measured using indirect calorimetry (oxygen and carbon dioxide production) has been shown to decrease, indicating greater fat oxidation in RT-trained males (243, 277) and females (39), both immediately post- (345) and up to 43 hours post-RT (345). In one study, RER was 0.89 and 0.79 at pre- and post-RE, respectively (345). At 43 hours post-RT, there was still a significant difference in RER compared to pre-RE values (pre resistance exercise: 0.89 vs. 43 h post-resistance exercise: 0.84) (345). This decrement in RER is indicative of slightly elevated reliance on lipids as an energy substrate as well as slightly enhanced fat oxidation, rather than CHO oxidation. A note of caution is to interpret conservatively the impact of this relatively small attenuation in RER on whole body changes in fat mass.

However, results are slightly different when measuring fat oxidation using direct calorimetry in a room calorimeter for 24 total hours after a full-body 60-minute RT circuit (three sets of 10 repetitions and one set to failure at 70% exercise-specific 1RM) in non-obese (body fat: 19.4 ± 4.6%), moderately active (3-5 hours of exercise/wk), healthy adult males (242). In this study, it was reported that 24-hour CHO oxidation was higher in the RT (349.4 ± 22.8 g, p<0.05) and ET (370.2 ± 17.9 g, p<0.05) (60 minutes at 70% VO 2max ) groups compared to the no exercise group (249.3 ± 29.5 g) (242). However, fat and PRO oxidation were not different between RT, ET, and no exercise control groups (fat: RT–91.0 ± 10.4; ET–87.0 ± 12.6; no exercise–86.5 ± 16.7; PRO: RT–86.6 ± 8.85; ET–88.9 ± 7.5; no exercise–85.1 ± 7.2, p>0.05) (242). Even so, RER was higher during the RT session compared to the ET and no exercise sessions, indicating that there is a greater reliance on CHO as a fuel source with RT (242). Importantly, because the duration of the training trials was the same (60 minutes), differences in metabolism are likely due to differences in intensity.

The effect of chronic RT (16 weeks, 3 times/wk) in older (67 ± 1 yrs) females on 24-hour fat oxidation rates has also been studied. It is important to note that in this study, basal fat oxidation rate was much lower compared to healthy, young males and females (380). Significant

55 decreases in nonprotein RQ using indirect room calorimetry (metabolic exchange of gas ratio of

CO 2 being produced to O 2 being consumed) and CHO oxidation revealed an increase in fat oxidation (380). However, it is important to note the activity levels of the study participants in this study (sedentary) are far different from the active individuals previously discussed. Likewise, the chronic nature of this training study may have also affected outcome measures, as with training there is a shift toward greater fat oxidation, as previously discussed.

At the cellular level, acute RT has been shown to enhance lipolysis in both the SCAAT (272, 277), as well as gluteal (66) depots. Ormsbee et al. (272) found that SCAAT lipolysis and oxidation are enhanced in both lean (BMI: 20.9 ± 0.6 kg/m 2) and obese (BMI: 36.2 ± 2.7 kg/m 2 men, although lean men display significantly higher concentrations of GLY and fat oxidation. Interestingly, researchers concluded that the attenuation in SCAAT lipolytic response in obese males was not the result of β-AR activation by RT-induced CAT release; rather, changes were likely the result of suppressed GH (lean: 3.3 ± 0.7 ng/ml, obese: 1.0 ± 0.9 ng/ml, p=0.037) (272). Interestingly, in a different study, obese males were able to obtain similar lipolytic responses post-RT, only with a slightly delayed onset (66). However, the lean males in this study had elevated fat oxidation levels (66). It is important to note that both the lean and obese groups were sedentary, and thus, although both groups had similar RT-induced lipolytic responses, it seems that either: 1) the presence of obesity has implications on the utilization of fat for energy (as displayed by attenuated fat oxidation rates in obese vs lean somatotypes) and/or 2) lean individuals are more metabolically efficient in their ability to adapt substrate utilization with exercise. Additionally, these studies utilized men (both fit and overweight/obese) therefore, studies examining the same variables are warranted in women.

2.4.3.2 Effects of acute resistance exercise on muscle anabolism and catabolism . It is known that RT elicits an increase in MPS rate without an increase in whole body PRO breakdown in both young (24 yrs) and elderly (63-66 yrs) males and females (413). The time course for the positive metabolic effects of RT on MPS is such that it is elevated by 50% at four hours post-heavy RT (12 sets of 6- to 12-RM elbow flexion exercises), it is more than doubled (109%) 24 hours post-RT, and it is back to baseline around 36 hours post-RT (225). Changes in MPS after RT are at a significantly higher magnitude compared to MPB (294). However, MPB will exceed the anabolic nature of MPS if left in the fasted state (294, 403). In a study in

56 untrained young men, RT designed to increase lean body mass (12 wks, 5d/wk) reduced both fasted (167 ± 18 vs. 152 ± 17 μmol/ [kg LBM/h]) and fed state (197 ± 23 vs. 178 ± 21 μmol/ [kg LBM/h]) LEU turnover (measured by a primed constant infusion of [1-13 C] LEU) (p<0.01), while it increased dietary nitrogen retention measured by analysis of nitrogen balance (13.7 ± 8.1 vs. 33.4 ± 12.5 g/(kg LBM/d), p<0.01) (259). Therefore, one bout of RT significantly stimulated MPS, and, to a lesser extent, MPB, and these changes were exerted for up to 36 hours.

2.4.3.3 Effects of chronic resistance exercise. It is clear that chronic RT induces positive changes in body composition by promoting decreases in fat mass and increases in lean mass (29, 59, 66, 381). In terms of cellular metabolism, there are changes, as well.

Effects of chronic resistance exercise training on fat metabolism. Polak et al. (301) found that in obese (BMI: 32.7 ± 0.9 kg/m 2), insulin-resistant males, 12 weeks of dynamic RT has been shown to improve SCAAT β-AR sensitivity and insulin sensitivity. More importantly, though, was the restoration of the balance of activation of the lipolytic β-AR and the antilipolytic α-AR with chronic RT (301). Even further, in another study, researchers concluded that all lipid mobilizing pathways in SCAAT displayed improved sensitivity after RT (3 months, 3x/wk full- body dynamic RT), thereby improving the state of metabolic inflexibility (inability to switch from fat to CHO oxidation, particularly affected by insulin sensitivity) (112) reported in obese, insulin-resistant males (301). The metabolic inflexibility was improved through increased whole-body and adipose tissue insulin sensitivity, and consequently the enhanced utilization of CHO as an energy substrate, as well as increased adipose tissue β-AR lipolytic pathway and an improved functional balance of the β-AR and α-AR (301).

However, the effects of chronic RT on fat oxidation are less consistent. Neither 6 months of RT in young, lean, sedentary females (298), nor 3 months of RT in young, health males (52) produced improvements in fat oxidation, measured using a ventilated hood. However, one study found that 16 weeks of RT in healthy, older females could increase fat oxidation significantly (RER: 0.90 ± 0.01 to 0.82 ± 0.01, p<0.001) (380). Likewise, 6 months of minimal RT (only 11 minutes per session) in previously sedentary, overweight (BMI: 27.7 ± 0.5 kg/m 2) young adults significantly reduced the RQ during rest and sleep, indicating increased fat oxidation, but did not alter 24-hour fat oxidation rates (196). Thus, the chronic effects of RT on fat oxidation may be dependent upon training status and BMI.

Effects of chronic resistance exercise training on muscle anabolism and catabolism. With 8 weeks of unilateral RT (3 sessions/wk, 80% of 1RM on knee extension and leg press, progression from 2 to 4 sets), the acute responsiveness of MPS to RT was enhanced through attenuation of the increase in protein synthesis (not in the muscle) and the maintenance of MPS in sedentary, healthy young individuals in the fasted state (189). Thus, chronic RT clearly elicits improvements in net muscle protein balance.

2.5 Nutrient Timing in Conjunction with Exercise

Optimal nutrition will enhance the effects of an exercise regimen, apparent by changes in body composition with improvements in dietary habits. Unfortunately, in the literature, when examining the effects of eating before, during, and after exercise, the majority of studies examine factors such as recovery, muscle glycogen replenishment, and measures of soreness. The effects of nutrient timing on muscle anabolism and catabolism may be quite extensive; however, the effects on fat metabolism are minimal in the literature. Additionally, there are very few studies directly comparing the differences in metabolism with feeding before and after exercise.

Specific to this review, the addition of PRO enhances the metabolic effects of an exercise regimen. In overweight and obese adults (BMI: 28.6 ± 5.4 kg/m 2), evenly spaced whey PRO (20 g, 3x/d, for 16 wks) in combination with RT significantly decreased percent fat mass and increased percent lean mass (19). Interestingly, the decrease in percent fat mass with the combination of PRO and RT was not significantly greater than solely the addition of dietary PRO, indicating that PRO consumption by itself may produce comparable body composition results (19). However, only adding PRO to the diet does not allow for significant decreases in visceral adipose tissue, whereas the addition of RT does (19). These effects on body composition are even more amplified when adding the additional whey PRO to a multimode exercise program consisting of RT, interval training, stretching, yoga and Pilates (19).

Likewise, when combining 12 weeks of increased PRO intake as a part of a balanced diet (40% kcal from PRO) with high intensity ET and RT in people aged 26-60 years old, body weight, body fat percentage, and abdominal fat percentage are significantly decreased (20). Importantly, the improvements with these combined perturbations are greater compared to consumption of traditional PRO intakes (15-20% kcal from PRO) combined with moderate ET

58 training (20) indicating that the combination of increased PRO and RT can certainly amplify body composition outcomes. Therefore, added PRO is beneficial to metabolism.

2.5.1 Eating Before Exercise

Consumption of energy before exercise exerts powerful effects on fat and muscle metabolism. Foremost, compared to eating nothing, eating a large (910 kcal) mixed meal prior to low intensity (5 min of cycle ergometer exercise at 300 kpm/min) and high intensity exercise (cycling just below the anaerobic threshold) increases metabolic rate significantly higher in lean women (11%) compared to obese women (4%) (p<0.005) (346). Additionally, although the TEF was similar for 4 hours after the meal at rest between lean and obese females, during exercise, the thermic effect of food (TEF) was 2.54 times higher compared to rest in lean women, and only 1.01 times greater in obese women (p<0.005) (346). These same effects were found when comparing the effects of a 750-calorie mixed meal (14% PRO, 31.5% fat, and 54.5% CHO) consumed before exercise in lean and obese males (347). Thus, it seems as though obese males and females have impaired metabolic responsiveness to the combination of a meal and exercise, potentially due to impairments in food- and exercise-induced sensitivity to hormonal and neural stimulation (346).

The effect of a before exercise meal on fat metabolism was previously discussed in the fat metabolism section. Ingesting a fatty meal (oral fat challenge) before exercise enhances TAG removal rates such that the working muscles may more readily utilize them for energy; however, the effectiveness of TAG removal rates is dependent upon training status, with trained individuals being more efficient in this process (70, 246, 395). Likewise, it is known that a CHO-rich meal prior to exercise causes the release of insulin, which is a potent antilipolytic and lipogenic agent, blocking lipolysis, inhibiting glucagon release (187) and HSL activity (49), synthesizing and stimulating the release of LPL (247, 329), activating enzymes involved in glucose uptake and subsequent FA synthesis (49), and inhibiting cortisol (49). On the other hand, when compared to CHO ingestion, PRO ingestion exerts the opposite effects on fat metabolism. When obese adults ingested a PRO-rich snack (160 total kcals, 33.3 g of PRO, 3.0 g of fat) 30 minutes prior to cycle ergometer ET (below the anaerobic threshold), plasma GLY levels were significantly elevated compared to ingestion of a CHO-rich source (290 kcal, 47 g CHO, 9 g fat, 4.6 g PRO) prior to the ET bout (94). In this same study, when CHO were

59 provided further out from ET (120 minutes), compared to PRO ingestion 30 minutes prior to ET, lipolytic rates were comparable (94). Different insulinemic responses as well as preferential use of available fuel source to PRO, fat, and CHO macronutrient-dense snacks are likely the catalysts for changes in fat metabolism responses.

When examining MPS, an essential amino acid and CHO supplement (6 g of essential amino acid, 35 g of sucralose) provided before RT in healthy human participants increased MPS significantly greater than consumption immediately after exercise (377), as well as 1 and 3 hours post exercise (311). This amplified effect is likely due to the enhanced delivery of amino acids to target tissues prior to exercise (377). As previously mentioned, RT designed to increase lean body mass reduces both fasted and fed state LEU turnover, while it increases dietary nitrogen retention (259). Likewise, RT also decreases (p<0.01) MPB in both the fasted (165 ± 18 vs. 144 ± 17) and fed (111 ± 23 vs. 93 ± 20) states (259). Thus, regardless of feeding status, RT alters protein kinetics as well as muscle protein balance.

2.5.2 Eating After Exercise

Research regarding the effects of post-exercise energy consumption on fat metabolism is not well known (141, 157, 260). Newsom et al., (260) examined the effects of consumption of differing levels of energy intake after ET (~45 min of treadmill exercise followed by ~45 minutes of cycle ergometer exercise at 60-65% VO 2peak) on lipid metabolism in healthy, young (29 ± 1 yrs), nonobese (BMI ≤ 25 kg/m 2), sedentary (<2 hr activity/wk for ≥6 months) men. Participants were provided either a nutrient-balanced meal matching calories burned during exercise (52 kcal/kg fat free mass [FFM], 7.7 g CHO/kg FFM), a low CHO meal (52 kcal/kg FFM, 3.5 g CHO/kg FFM), or a low energy meal after exercise (38 kcal/kg FFM, 7.7 g CHO/kg FFM) (260). PRO intake was consistent among all groups (1.15 g/kg FFM) (260). Researchers found that the group that maintained an energy deficit after exercise had the highest rates of lipid mobilization, albeit non-significant (plasma FA: nutrient balanced, 0.31 ± 0.05 mmol/l; low CHO, 0.35 ± 0.9 mmol/l; low energy, 0.45 ± 0.07 mmol/l; p=ns) and FA mobilization and oxidation in muscle biopsy samples (260).

Harrison et al., (141) examined the effects of post-ET energy replacement on lipid metabolism in young (27 ± 4 yrs), recreationally active (VO 2peak: 46.8 ± 4.9 ml/kg/min) men.

ET consisted of cycling for 90 minutes at 70% VO 2peak, followed by ten 1-minute sprints separated by 1 minute of resting recovery (141). There were 3 conditions: 1) rest with no energy intake (control); 2) ET with no CHO replacement (exercise + energy deficit); 3) ET and consume 105% of the CHO oxidized during exercise (4.4 ± 0.2 g/kg body mass) at 0, 2 and 4 hours post exercise during the (exercise + balanced energy) (141). The day after each of these trials, participants returned for an oral fat tolerance test (per 2 m 2 body surface area: 97 g fat, 124g CHO, 1450 kcal) (141). Researchers found that postprandial serum TAG was significantly attenuated in the energy deficit group compared to control and energy balance trials at each time point after the oral fat tolerance test (p<0.05) (141). Likewise, serum FFA were significantly higher in the post exercise energy deficit group compared to the control group at 0, 2, 4 and 6 hours post oral fat tolerance test (p<0.05), with no effects in the energy balance group (141). Therefore, TAG uptake and metabolism is elevated in the hours after an ET session with postexercise low energy intake.

Similar findings were reported in another study. Holtz et al., (157) analyzed the effects of CHO replacement on fat metabolism in young (21 ± 2 yrs), overweight (body fat: 37 ± 3%) men and women, with 3 conditions: 1) no exercise (control); 2) exercise and energy balance with CHO restriction (17.8% ± 0.1% CHO); and 3) exercise and energy balance with no CHO restriction (76.6% ± 2.5% CHO). The exercise consisted of cycling at 70% VO 2peak to achieve 30% of total daily energy expenditure (157). Researchers found that fasting NEFA were significantly higher following the low CHO condition compared with the control group (p=0.046), but not when comparing the no CHO restriction and control conditions (157). Fat oxidation rates were also significantly higher in the CHO restriction (109.8 ± 10.5 mg/min) compared to the control condition (80.7 ± 9.6 mg/min) (p=0.04) (157). Thus, with CHO replacement post ET, lipid mobilization is affected for an extended period, whereas restricting CHO enhances lipid mobilization and utilization.

In regards to muscle protein balance, consumption of dietary PRO after both ET (160, 211) and RT (96, 376) stimulates MPS, inhibits MPB, and thus elevates accretion of muscle protein. Enhancements of the skeletal muscle adaptive response occur with acute- (160, 211, 376) as well as chronic- (96) ingestion of dietary PRO post-exercise. However, when comparing pre-exercise and post-exercise consumption, one study has found that MPS was unable to be

61 enhanced to the same extent when consuming 6g essential amino acids and 35g sucralose after exercise compared to before exercise. Even though this is the case, it is important to note that in that study, post exercise ingestion of nutrients did, indeed increase MPS, however, not to the same degree as preexercise ingestion of nutrients. In a review by Gibala (115), it was concluded that net protein balance during ET and recovery is improved when individuals consume PRO and CHO, compared to CHO alone.

The effect of timing of post exercise consumption of amino acids and PRO on MPS seems to be dose-dependent (48), and also amplified by the addition of a CHO (249). Specifically, Børsheim et al. (48) reported that consumption of 6g of essential amino acids provided at both 1 and 2 hours post lower body RT in healthy, young men and women elicited comparable MPS responses. Another study examined the effects of consumption of a supplement (6 g essential amino acids, 35 g sucralose) provided 1-hour after a bout of RT, and then consumption of a placebo drink 3 hours after a bout of RT, compared to the opposite (placebo and then supplement at 1 and 3 hours after, respectively) (311). MPS was reported to be the same whether the essential amino acid supplement was consumed at 1 hour or 3 hours post RT (311).

Esmarck et al., (96) investigated the effects of 12 weeks of RT (3x/wk) in combination with chronic consumption of a PRO supplement (10 g PRO, 7 g CHO, 3 g fat) ingested immediately post- compared to 2 hours post exercise on body composition in elderly (74 ± 1 yrs) males. Interestingly, in elderly males, the chronic timing of PRO consumption post exercise seems to matter, as quadriceps femoris cross sectional area and average fiber area were significantly increased when PRO was consumed immediately after exercise compared to 2 hours after exercise (96). This effect seems to hold true in young individuals as well. Cribb and Hayes (77) examined the effects of consumption of a PRO, creatine, and glucose drink (1 g/kg of body weight, 40 g whey isolate PRO, 43 g glucose, 7 g creatine monohydrate) before and after RT (4x/wk for 10 wks) compared to in the morning and evening time (at least 5 hours outside of the workout) in recreational male bodybuilders. Researchers reported an increase in lean mass through an increase in the cross-sectional area of type II fibers and contractile proteins in the group that consumed the supplement immediately before and after RT (77). Therefore, the window for optimal muscle protein balance is controversial; however, it appears timing of PRO

62 around RT may enhance the adaptations to RT in both young and elderly individuals, with the most optimal effects occurring when consuming the supplement immediately before and after exercise.

Type of PRO or amino acid profile does not seem to have a drastic effect on muscle protein balance. Tipton et al. (376) examined whether consumption of a full amino acid profile (mixed amino acid drink, 40 g) versus only essential amino acids (essential amino acid drink, 40 g) was imperative to optimize MPS in young, healthy males and females. Net muscle protein balance was not different between the groups, indicating that ingestion of only essential amino acids after exercise sufficiently induces MPS (376). Likewise, consuming 20 g of both whey and casein PRO has been found to produce similar elevations in muscle protein net balance, and consequently MPS after a bout of lower body RT in young, healthy individuals (375). Importantly, these similarities occurred despite significant differences in blood amino acid responses (375). However, one study found no changes in net muscle protein balance with ingestion of 20 g of whey PRO after lower body RT in healthy, non-RT males and females (374). Importantly, they also found no differences between ingestion of whey PRO before or after the bout of exercise (374). Thus, the authors concluded that the timing of consumption of intact PRO will not affect MPS to the extent of free amino acids combined with CHO, as in previous studies (249, 311).

2.5.3 Pre-sleep Eating

Pre-sleep eating is a relatively new concept that has been misinterpreted in the media through the mishandling of existing data that report negative effects of pre-sleep eating in individuals with pre-sleep eating disorders and night-shift workers. The physiological basis to support the negative claims of pre-sleep eating is multi-fold. For instance, consumption of identical macronutrient composition meals in the evening hours compared to other times of day elicits significantly lower measures of the TEF and satiety (64, 328), which ultimately leads to higher calorie intake throughout the day (64). Taken together, these data may suggest that there may be negative metabolic effects and subsequent body composition implications of eating in the evening hours.

Further, adverse health consequences of consuming a large part of daily calories in the evening hours have been found in various populations (73, 415) and in epidemiological data (14, 28, 92). Specifically, individuals with night eating syndrome and nocturnal eating/drinking syndrome may be particularly affected. Individuals with night eating syndrome are classified as consuming ≥50% of their daily calories after dinner, in addition to sleeplessness and morning anorexia with negligible caloric intake (415). Some studies have shown high daily caloric intake in this population (41), while others have not (11). However, this population consumes much more CHO and fat compared to their healthy counterparts (12). In addition to night eating syndrome, nocturnal eating/drinking syndrome is classified as repeated awakenings and the incapacity to return to sleep without eating or drinking (415). Neuroendocrine studies have found low levels of plasma melatonin and leptin, as well as high cortisol levels in obese women with pre-sleep eating syndrome compared to healthy control participants (363). These findings indicate the inability to suppress pre-sleep appetite. Not only would these hormones affect sleep and consequently energy intake, but they likely directly affect visceral fat accumulation, as chronically elevated cortisol levels are associated with negative implications on fat metabolism (previously discussed, see section 1.1.4.11) (280). In addition, individuals with night eating syndrome display an elevated 24-hour RQ, indicating less reliance on fat oxidation and more on CHO oxidation compared to healthy counterparts (119). Although individuals with night eating syndrome may be normal weight (41), in a review (415) it was concluded that obesity may be causally related to disordered pre-sleep eating, such that, upon onset, calorically dense food choices likely lead to the onset of obesity. Thus, it may be plausible to assume that normal weight individuals partaking in dysregulated pre-sleep eating will eventually gain weight.

In addition to individuals with disordered pre-sleep eating, night shiftworkers with irregular work hours demonstrate similar negative health effects (73, 232). Compared to day workers, night workers tend to have an increased risk of overweightness and abdominal obesity (223, 224), as well as impaired glucose tolerance, high TAG levels, low levels of HDL (the good cholesterol), and dyslipidemia (181, 223). Reductions in total daily energy expenditure and the thermic effect of dinner were found to be associated with weight gain and obesity when night shiftwork was simulated in a six-day study in normal weight women (239).

The strong association between lack of sleep and increased BMI (103) combined with dysregulated sleep patterns of night shiftworkers and individuals with night eating syndrome clearly subject these populations to negative body composition ramifications (41, 270). Taken together, these data indicate the potential for negative metabolic and body composition outcomes with dysregulated eating in the evening hours.

However, the very high caloric intake and CHO- and fat-rich (not PRO) macronutrient profiles of consumed meals is the main issue with rationalizing the negative outcomes experienced in these populations to the general population. Chronically, these patterns of dietary consumption predispose these populations to weight gain and abdominal obesity (14, 18, 118), and negative cardiometabolic outcomes such as elevated insulin concentrations and markers of insulin resistance (73, 415). However, it is highly unlikely that the general population practices these same dietary habits. Even more, compared to consumption of large amounts of CHO- and fat-rich foods, the practice of pre-sleep eating of much less calorically dense PRO snacks high in casein (i.e. cottage cheese and PRO powders) has been widely utilized in athletic populations such as bodybuilders. Often, these populations partake in heavy RT, and thus, added PRO is used to anecdotally maintain metabolism during times in which a catabolic state may prevail (sleep). Interestingly, although PRO is known to be lipolytic (94), antilipogenic (10), anabolic in muscle tissue (368), and effectively digested and absorbed at night (316), there are very few investigative studies assessing the effects of consumption of PRO at night (73, 118, 190, 193, 194, 228, 276, 316, 343, 355, 415) (Table 1). Importantly, though, recently there has been surmounting evidence of the absence of negative metabolic outcomes with pre-sleep consumption of small, low energy, nutrient-dense foods or single macronutrients such as a PRO or CHO (122, 193, 228, 276, 391) (Table 1). However, of these studies, only one study has controlled for the timing of PRO consumption by comparing the effects of pre-sleep to daytime PRO consumption (17).

Resting metabolism in the morning following a pre-sleep feeding is of concern with pre-sleep eating. Resting metabolism is the greatest contributor to daily energy expenditure (60-75%), and thus, any modifications of this measure, potentially during sleeping hours, will significantly affect total daily energy expenditure. Even so, it is known that pre-sleep metabolic rate is attenuated during sleep (182). However, a high PRO diet (36% of total daily energy

65 intake, compared to 15% of total daily energy intake) has been found to attenuate the suppression of pre-sleep metabolic rate during hypocaloric states in overweight and obese (BMI: 27.8-34.1 kg/m 2), middle-aged (31-57 yrs) men and women (397). Further, timing of PRO ingestion immediately before going to sleep has been shown to positively benefit next-morning REE, contrary to popular thinking. Specifically, in a crossover study, Madzima et al., (228) examined the effects of a single dose of 30 g of whey PRO (150 kcal; 30 g PRO, 3 g CHO, 2 g fat), 30 g of casein PRO (140 kcal; 30 g PRO, 3 g CHO, 1g fat), 33 g of CHO (150 kcal; 0 g PRO, 33 g CHO, 2 g fat), or non-nutritive PLA (0 kcal) consumed before bed in active (≥ 4d/wk and 50 min/d of self-reported moderate-to-vigorous physical activity for >12 months), normal weight (BMI: 25.8 ± 0.8 kg/m 2), college-aged (23.6 ± 1.0 yrs) males. Researchers found that next-morning REE was significantly higher in each of the PRO and CHO groups, but not the non-nutritive PLA group. This finding indicates that regardless of macronutrient type (excluding fats) a small pre- sleep snack may elicit favorable metabolic effects the morning after pre-sleep eating. Of particular interest in this study, the whey and CHO, but not casein elicited lower rates of estimated fat oxidation as measured with RQ compared to the non-nutritive PLA (228). This indicates that casein (like PLA) may be an ideal option to consume prior to pre-sleep sleep for preserving fat oxidation.

In a similar study in sedentary (< 2 times/wk in the last 6 months), overweight and obese (25.7-54.6 kg/m 2), young to middle-aged (18-45 yrs) females, researchers examined the impact of acute pre-sleep feeding of whey (150 kcal, 30 g whey PRO [50% blend of whey PRO isolate and concentrate], 4 g CHO, 1.5 g fat), casein (140 kcal, 30 g micellar casein PRO, 3 g CHO, 0.5 g fat), or CHO placebo (150 kcal, 0 g PRO, 34 g maltodextrin, 2 g fat) snacks provided 30 minutes before going to sleep (193). Researchers found that next-morning RMR and RQ tended to be elevated with both whey and casein PRO consumption compared to the CHO PLA, and whey PRO tended to elicit higher RMR compared to casein PRO, albeit all findings were insignificant (193). Surprisingly, the change in RQ and substrate utilization with PRO intake (whey: baseline–0.82 ± 0.01, post–0.85 ± 0.01; casein: baseline–0.84 ± 0.01, post–0.86 ± 0.01) indicated that CHO utilization was favored, and not fat, although these were very modest changes (193). However, it is important to mention that this study did not include a true non- nutritive PLA.

Table 1. Effects of pre-sleep consumption of a small meal or snack

Study Demographics Intervention(s) Effects Length Res et al. M 20g CAS + 60g CHO Acute With PRO: ↑ circulating AA 2012 22.7±0.8 years post RE, 40g CAS levels throughout night, ↑ 22.6±0.7 kg/m 2 30min before sleep, PL whole body PRO synthesis Recreationally consumed PL before rates, ↑ net PRO balance, ↑ active bed, bilateral lower mixed muscle PRO synthesis body RT rates Groen et M 40g labeled CAS or PL Acute With PRO: ↑ phenylalanine al. 74±1 during sleep via appearance rates, ↑ PRO 2012 25.6±0.8 nasogastric tube synthesis, ↑ overnight whole- body PRO balance, ↑ overnight muscle PRO fractional synthesis rates Hibi et al. F ~200kcal 13d ↔ sleeping metabolic rate, ↑ 2013 23±1 years (PRO:fat:CHO night, hunger and prospective to eat 20.6±2.6 kg/m 2 5:50:45) 13d and ↓ fullness at lunch with day pre-sleep snack, no other changes, ↔ insulin, adiponectin or leptin, ↔ RQ, CHO oxidation, or PRO oxidation b/t groups, ↓ fat oxidation in pre-sleep group Kinsey et F 30g whey, CAS, or Acute ↔ RMR/RQ, ↔ hunger, al. 18-45 years CHO satiety, desire to eat, ↑ 2014 25.7±54.6 kg/m 2 insulin and HOMA-IR in all Sedentary groups, ↔ leptin, adiponectin or cortisol Madzima M 38g whey, CAS, CHO, Acute ↑ REE after whey, CAS and et al. 2014 23.6±1.0 years or non-nutritive CHO, not PL, ↔ b/t groups, 25.8±0.8 kg/m 2 placebo ↓ RQ with PL compared to Active whey and CHO but not CAS, ↔ hunger, satiety or desire to eat, greater satiety with whey/CAS compared to CHO (ns) Ormsbee et F 30g whey, CAS, or 4 ↔ RMR/RQ, RMR tended al. 2015 18-45 years CHO, 2d RT, 1d HIIT weeks to ↑ with PRO/↓ with CHO 25.7±54.6 kg/m 2 (ns), ↔ in body comp or wt Sedentary b/t groups, ↑ morning satiety with CAS vs whey and PL, ↔ hunger or desire to eat, ↔ insulin, HOMA-IR, leptin, adiponectin, cortisol,

Table 1 continued Study Demographics Intervention(s) Effects Length Kinsey et M 30g CAS or noncaloric Acute ↔ RMR or RER b/t groups, al. 2016 27±2.2 years PL ↔ satiety or desire to eat 36.1±1.9 kg/m 2 between groups, ↔ insulin, Sedentary glucose, HOMA-IR, GH b/t groups, ↔ overnight SCAAT interstitial glycerol concentrations b/t groups, ↔ fat oxidation b/t groups Antonio et M/F 54g CAS (morning or 8 ↔ body composition or fat al. 2017 29.5±8.9 years evening) weeks mass <25.0 kg/m 2 Resistance trained

M, males; F, females; PRO, protein; PL, placebo; CHO, carbohydrate; CAS, casein; g, grams; c, cups; d, days; wks, weeks; RT, resistance training; HIIT, high-intensity interval training; ↔, no change; ↑, increase; ↓, decrease; RMR, resting metabolic rate; RQ, respiratory quotient; ns, not significant; REE, resting energy expenditure; b/t, between; RER, respiratory exchange ratio; HOMA-IR, homeostatic model assessment of insulin resistance; GH, growth hormone; SCAAT, subcutaneous abdominal adipose tissue; AA, amino acid

The beneficial effects of pre-sleep eating on metabolism seem to be similar when RT is also added. In one study, researchers combined the same dietary intervention (casein vs. whey vs. CHO) with RT (2x/wk) and high intensity interval training (1x/wk) in sedentary (< 2x/wk in the last 6 months), overweight and obese (25.7–47.5 kg/m 2), young to middle-aged (18-45 yrs) females (276). Researchers measured RMR and RQ via indirect calorimetry, and found no differences between supplement groups (276). However, again, there was no additional non- nutritive PLA group; thus, it appears the metabolic effects of PRO and CHO may be similar when added to a RT program. Thus, despite consuming an additional 150 calories (as either PRO or CHO), no negative resting metabolic impacts occur and in some instances, positive outcomes prevailed.

In addition to resting metabolism, muscle protein metabolism and specifically postexercise overnight muscular recovery may also be affected with the combination of pre-sleep eating and RT. Res et al., (316) studied healthy, young (PRO: 22.9 ± 0.7 yrs; PLA: 22.4 ± 0.7 yrs), recreationally active (PRO: 6.3 ± 1.1 hours/wk; PLA: 5.2 ± 1.0 hours/wk) normal-weight 68

(PRO: 22.7 ± 0.6 kg/m 2; PLA: 22.5 ± 0.8 kg/m 2) males that performed bilateral lower body RT (8 sets of 8 repetitions of the horizontal leg press and leg extensions, with 1 set at 55% 1RM, 1 set at 65% 1RM, and 6 sets at 75% 1RM, and a 2-minute rest between sets and ~5-minute rest between exercises) in the evening (testing beginning at 1830 hrs). Participants consumed 20g of casein PRO combined with 60g of CHO immediately after exercise, and then consumed 40g casein PRO 30 minutes before going to sleep (160 total kcals) (316). Researchers found significantly elevated levels of circulating amino acids throughout the night as well as into the next morning, elevated whole-body PRO synthesis rates by about 22% (PRO: 0.059 ± 0.005%/h, PLA: 0.048 ± 0.004%/h, p=0.05), and elevated net protein balance with PRO consumption when compared to a non-nutritive PLA (316). Thus, the addition of a pre-sleep PRO snack may significantly enhance overnight muscle recovery after RT.

Something to consider when analyzing metabolic measurements is the effect of sleep quality and quantity on insulinemic response, and consequently RMR. It has been shown that just five nights of sleep restriction of about four hours per night lowers RER (increases the reliance on fat oxidation), and concomitantly increases whole body and peripheral (not hepatic) insulin resistance and CAT and cortisol release (308). Researchers concluded that elevated cortisol and Epi levels likely contributed to insulin resistance by increasing lipolytic rate and circulating NEFA concentrations (308). The increase in insulin resistance with elevated FA levels is likely due to an increase in subcutaneous adipose tissue lipolysis, an attenuation of lipolysis from visceral adipose tissue, a shift toward hepatic and CHY release of TAG, and a shift of the metabolic set point toward favoring visceral adipose tissue storage. Thus, sleep quality and quantity may significantly impact metabolic activity; however, this study showed that there was no effect on RMR (308). Conversely, in an unpublished study by Kinsey et al., (190) participants did not meet the recommended sleep duration per night (7-8 hours) for the entirety of the study, which may have produced an additive negative effect on metabolic and hormonal responsiveness, independent of supplement ingestion. Even so, because there were no differences in sleep quality or quantity between pre-sleep trials of casein and PLA ingestion in the study, the consumption of a pre-sleep snack was not a confounding factor in sleep quality and quantity (190). However, except for this unpublished data, there are no published studies regarding pre-sleep feeding that noted sleep quality. Therefore, future research should include this measure.

Importantly, digestive kinetics are not affected with pre-sleep eating of PRO. Groen et al., (122) examined healthy, normoglycemic, elderly (74 ± 1 yrs) normal-weight (PRO: 26.0 ± 0.7 kg/m 2; placebo: 25.2 ± 0.8 kg/m 2) males, and provided a single intragastric bolus of 40 g (160 total kcals) of casein PRO to participants during sleep between 0200 and 0205 hours (122). Researchers found that MPS (PRO: 0.045 ± 0.002 %/hr; placebo: 0.029 ± 0.002 %/hr; p<0.05) and whole-body PRO balance (PRO: 11.8 ± 1.0 μmol phenylalanine/kg/hr; PLA: 0.3 ± 0.1 μmol phenylalanine/kg/hr; p<0.05) were greater with PRO ingestion compared to the non-nutritive PLA due to the increased amino acid availability, and plasma concentration and incorporation of phenylalanine, LEU and essential amino acids (indicated by amino acid tracer incorporation) (122). Therefore, PRO consumed at night not only enhances MPS, but is also effectively digested and utilized in the muscle.

Body composition may also be significantly altered with pre-sleep ingestion of a small snack. Ormsbee et al. (276) reported trends toward decreases in total fat mass and increases in lean mass in sedentary (< 2 times/wk in the last 6 months), but otherwise healthy, obese women (BMI: 25.7–47.5 kg/m 2) with 4 weeks of pre-sleep whey (30 g, 50% blend whey PRO isolate and concentrate, 4 g CHO, 1.5 g fat) and casein (30 g, micellar casein PRO, 3 g CHO, 0.5 g fat) PRO consumption (140-150 kcals/serving), when combined with ET (3x/wk), albeit not statistically significant. However, because body composition in all groups improved, these effects were likely the result of training. Nevertheless, this study also provides evidence that eating at night is not harmful or deleterious to improvements in body composition. Further, regarding changes in body fat percentage, the greatest reported response was in the whey PRO group (whey: Δ 2.4% body fat; casein: Δ 0.4%; CHO PLA: Δ 0.5%) (276). Even when PRO timing is controlled for, there seem to be no effects on body composition. Antonio et al (17) found that 4 weeks of casein consumption consumed in the morning (before 12:00 pm) or in the evening (~90 minutes or less before sleep) in resistance-trained men and women did not affect body composition or fat mass, measured using a BodPod®. Importantly, the Antonio study utilized a much higher dose of PRO (54 g CAS) in the evening compared to the Ormsbee study (30 g CAS), and there was still no effect. Therefore, over the long-term, PRO can be consumed in larger boluses with no negative effect on body composition. Other studies have also reported changes in body composition with pre-sleep eating, specifically changes in lean mass (355). In the longest pre-sleep feeding study to-date, Snijders et al. (355) had healthy, recreationally active (performed sports on a

70 noncompetitive basis for 2-5 hrs/wk), young (22 ± 1 yrs) males with no RT history (over the past 2 yrs) participate in a progressive, 12-week RT program (3 times/wk). Participants were randomly assigned to consume a PRO supplement (27.5 g PRO [13.75 g casein hydrosylate, 13.75 g casein], 15.0 g CHO, 0.1 g fat) or a noncaloric PLA immediately before sleep for 12 weeks. Quadriceps muscle cross sectional area (p<0.05) was significantly increased in the group that consumed the PRO supplement before sleep (355). Specifically, the percentage of type II muscle fibers was significantly higher with pre-sleep PRO ingestion (pre: 54% ± 6.3%, post: 65% ± 6 3%, p<0.05) (355). Therefore, the chronic practice of pre-sleep PRO ingestion may lead to enhancements in body composition through changes in body fat and in MPS.

Behavioral feeding and markers of satiety may also be affected with pre-sleep snack consumption. In a study by Waller et al., (391) healthy adults who engaged in pre-sleep eating behaviors were randomized into a cereal group (100-135 kcal, 2.0-6.0 g PRO, 23.0-32.0 g CHO, <0.5 g fat), or a non-cereal group (no change in diet) for 4 weeks. Over the 4 weeks, there was a significant correlation between compliant consumers of the post-dinner cereal and weight loss in these participants, likely due to the significant attenuation of total daily caloric intake with pre- sleep cereal consumption (391). In addition to the effects of pre-sleep eating on behavioral compliance and consequently weight loss, levels of satiety are also affected. The satiating effect of PRO has been observed in various studies (1, 122, 193, 276). Four weeks of pre-sleep ingestion of casein PRO in combination with RT training has been shown to significantly increase next-morning satiety and decrease desire to eat measures compared to whey PRO and CHO consumption in sedentary, obese females (193, 276). Additionally, pre-sleep intragastric administration of casein PRO compared to a PLA during sleep in elderly males produced a feeling of less hunger the next morning (122). Conversely, there were no significant differences in hunger, satiety, or desire to eat with acute ingestion of whey, casein or CHO in active college- aged males (228), or with casein ingestion in overweight and obese males (190). In fact, in obese males, the desire to eat was greater with casein consumption compared to baseline values (not compared to PLA consumption) (190). However, it is feasible to conclude that participants may have misconstrued “desire to eat” with “desire to chew”, as the 24-hour dietary standardization prior to testing consisted of solely liquid beverage ingestion (190). Thus, pre- sleep eating seems to have potential beneficial effects on levels of satiety. However, if changes

71 in behavioral feeding and markers of satiety result in long-term weight or body composition changes with pre-sleep PRO (not cereal) consumption remains to be studied.

Regarding lipolysis, there are temporal differences in the mobilization of fats from different depots. Specifically, overnight, upper body fat depots (i.e. visceral and abdominal) are the predominant source of circulating FFA (170, 231). However, there are no studies that compare the effects of pre-sleep PRO consumption on FFA mobilization in different fat depots, and only very few studies that examine the effects of pre-sleep PRO consumption in SCAAT, specifically. In an unpublished study from our laboratory, in a crossover design, young (27 ± 2 yrs), overweight and obese (36.1 ± 1.9 kg/m 2) males ingested either casein PRO (25g micellar PRO, 120 kcals) or a non-nutritive PLA (0 kcal) 30 minutes before going to sleep. Researchers measured overnight lipolysis using a microdialysis technique to measure SCAAT interstitial concentrations of GLY (190). There were no significant differences in overnight SCAAT interstitial GLY concentrations (PRO: 175.0 ± 26.5; PLA: 184.8 ± 20.7 μmol/L; p=0.77), next- morning SCAAT interstitial GLY concentrations (PRO: 171.7 ± 19.1; PLA: 161.5 ± 18.6 μmol/L; p=0.72), RMR (PRO: 2126 ± 111; PLA: 2145 ± 106 kcals/d; p=0.94), or fat oxidation measured by RER (PRO: 0.76 ± 0.01; PLA: 0.76 ± 0.01; p=0.75) between the casein PRO and PLA groups (190).

However, specific to lipolytic rate is the effect of pre-sleep feeding on insulin response. Importantly, although daytime studies suggest that casein ingestion blunts the postprandial insulinemic response compared to whey PRO (158), a pre-sleep casein PRO snack was reported to elicit an increase in next-morning insulin levels in overweight and obese females (193), possibly predisposing them to negative metabolic effects. Specifically, in this population, pre- sleep ingestion of whey, casein and CHO caused significant elevations in next-morning insulin (whey: pre 114.6 ± 7.8, post 144.0 ± 7.8 pmo;/l; casein: pre 154.2 ± 7.8, post 172.2 ± 7.8 pmo;/l; PLA: pre 100.2 ± 7.8, post 113.4 ± 7.8 pmo;/l) with the greatest change occurring with whey PRO consumption (whey: Δ 29.4 pmo;/l; casein: Δ 18.0 pmo;/l; PLA: Δ 13.2 pmo;/l) and no significant differences among groups (193). Additionally, the homeostatic model assessment of insulin resistance (HOMA-IR) was significantly elevated in all groups (whey: pre 3.9 ± 0.3, post 4.9 ± 0.3; casein: pre 6.0 ± 0.3, post 6.8 ± 0.3; PLA: pre 3.6 ± 0.4, post 4.1 ± 0.4), with the greatest change occurring in the whey group (whey: Δ 1.0; casein: Δ 0.8; PLA: Δ 0.5), and no

72 significant differences among groups. Of note, these effects were abolished with the incorporation of just 3x/wk of exercise training (2d of RT, 1d of high intensity interval training), indicating the protective metabolic mechanisms of exercise training (193, 276). Conversely, in overweight males, neither next-morning insulin (PRO: 32.9 ± 6.2 µIU/mL; PLA: 31.6 ± 4.9 µIU/mL; p=0.64) nor HOMA-IR (PRO: 7.3 ± 1.6; PLA: 7.1 ± 1.3; p=0.62) differed between ingestion of casein and a non-nutritive PLA (190). The lack of differences in responsiveness to the PRO versus PLA supplement in overweight males was attributed to hyperinsulinemia (fasting insulin > 30 µIU) and insulin resistance (HOMA-IR > 2.5) both at baseline and every point throughout the study (190). Importantly, because insulin levels at each time point (i.e. baseline and next-morning) were elevated in this population, and levels were not significantly different between baseline and next-morning measures, it was concluded that the supplement did not cause these changes (190). No reported changes in insulin response has been reported in other studies, as well (149, 276).

Although some studies have shown beneficial effects of pre-sleep eating of a small snack, results are not conclusive, and may certainly depend on the macronutrient composition of the snack (148). Hibi et al. (148) had healthy, normal BMI, young women partake in a crossover design, whereby participants consumed a commercially available snack (~200 kcal, mean PRO:fat:CHO of 5:50:45%) for 13 days during the day or at night. Compared to daytime snacking, pre-sleep snacking significantly blunted fat oxidation as measured by direct calorimetry in a whole room respiratory chamber for 23 hours (daytime: 52.0 ± 13.6 vs. pre- sleep: 45.8 ± 14.0 g/d; p=0.02), albeit a very modest difference. This indicated a slightly higher reliance on CHO compared to fat oxidation with PRO consumption at night (148). Notably, it is important to consider the short duration (13 d) of the study. This alone may have transient effects on metabolic outcomes, and likely would not have elicited body composition changes. Also, the macronutrient composition of the snack was very CHO-dense (~90 kcal of the total 200 kcal) with very limited PRO content (~10 kcal of the 200 total kcal), and thus, would preferentially elicit less reliance on fat oxidation, compared to PRO-rich snacks (94).

Although studies of pre-sleep eating have shown promising metabolic and body composition outcomes, the limiting factor in each of the aforementioned studies is the lack of methodologically matching for PRO timing (190, 193, 228, 276, 316, 355). For instance, in a

73 study from our laboratory, overweight and obese men were instructed to consume the same diet throughout the study, consume a standardized diet for 24 hours before laboratory visits (consisting of Ensure drinks), and consume pre-sleep casein PRO or a non-nutritive PLA during separate trials before bed (unpublished study). Thus, during the 24-hour standardization, participants consumed the same amount of PRO at baseline compared to the 24-hour standardization periods (baseline: 102.27 ± 30.5 vs. control, 107.29 ± 5.12 g/day, p=0.636); however, during the PRO trial, participants consumed more PRO with the supplemented drink (casein: 30g PRO vs. PLA: 0g PRO). Importantly, although the PRO was timed at night, there was no daytime PRO consumption trial to make effective comparisons. Likewise, in a study by Res et al. (316), after a RT protocol, participants consumed a bolus of either PRO or CHO PLA before bed. Although both groups consumed a 24-hour standardized diet providing 1.2 g/kg of body weight in PRO, the PRO group consumed 40 g more PRO than the CHO PLA group. To date, no studies have compared a pre-sleep PRO feeding trial to a daytime PRO feeding trial, and therefore, it is unclear whether any reported benefits of the published pre-sleep feeding studies were a consequence of the addition of PRO calories (regardless of the time of day), or the consumption of PRO at night. Thus, to make effective comparisons and conclusions, it is imperative to study the metabolic effects of dietary PRO consumed at night versus during the day.

Additionally, use of the microdialysis technique to measure localized fat metabolism in the SCAAT depot after pre-sleep PRO consumption has only been utilized in one unpublished study from our laboratory (no exercise intervention), where we found no differences in fat metabolism in overweight and obese men with pre-sleep PRO versus non-nutritive PLA consumption (unpublished data). The SCAAT is of particular interest in metabolism research as the upper body subcutaneous tissue (i.e. SCAAT) supplies most of the circulating FFA during the overnight hours in lean individuals (170). Furthermore, dietary PRO intake has been shown to increase RMR (228, 276) and contribute to improvements in body composition (21, 25), and body fat (21, 25), specifically. Therefore, if one can optimize the timing of dietary PRO during a window at which SCAAT is elevated (pre-sleep), theoretically, mobilization and oxidation of fats could potentially be optimized and elevated exponentially. Importantly, the effects of pre- sleep PRO consumption combined with RT on localized fat metabolism using a microdialysis technique has not been explored in RT men or women. Therefore, research is warranted to

74 provide further insight and clarity on the extent to which the practice of pre-sleep versus daytime eating of PRO combined with RT enhances metabolism, and specifically lipolytic rate of the SCAAT in RT men and women.

2.6 Summary

In summary, the concept of pre-sleep eating (after dinner, immediately before bed) is a relatively novel field in metabolism research that in recent research is beginning to show metabolic benefits. This paradigm shift could be promising for future nutrition recommendations, as athletes have been practicing pre-sleep eating of PRO for years with only anecdotal evidence to support the effectiveness of this dietary practice. Thus, not only would further beneficial metabolic findings of pre-sleep PRO consumption justify this common dietary practice, but also it would allow the refining of this practice so that it may be used to enhance body composition and metabolic outcomes when combined with ET.

The literature on pre-sleep PRO consumption thus far has shown positive effects. Specifically, data have shown increases (228, 276) or no influence on next-morning RMR (148, 193, 195), no influence on RQ (190, 193), enhanced muscle recovery through an increase in muscle PRO synthesis and whole body PRO turnover (316, 355), positive effects on markers of appetite (193, 195, 228, 276), improvements in cardiovascular measures such as systolic blood pressure, wave reflection and arterial stiffness (101), and no effect on body weight when used as a chronic practice (276). Importantly, all pre-sleep PRO eating studies report no or limited negative metabolic implications (increases next-morning insulin levels and makers of insulin resistance in overweight and obese women, which are negated with exercise) (118, 151, 193, 221, 276, 415). However, although a popular practice and topic of conversation, research regarding pre- sleep eating, specifically on fat metabolism, is in its infancy, and thus, many gaps in the literature still exist and require elucidation. First, and certainly most importantly, the limited research studies examining the effects of pre-sleep PRO consumption have not methodologically matched for timing of PRO intake (190, 193, 228, 276, 316, 355). Therefore, it is unclear whether any reported benefits of the published pre-sleep PRO studies were a consequence of the addition of PRO calories regardless of the time of day, or the consumption of PRO at night. Thus, to make

75 optimal comparisons and conclusions, it is imperative to PRO match trials by studying the metabolic effects of dietary PRO consumed at night versus other times of day.

Moreover, the ability to measure overnight lipolysis is a recent addition to the scope of literature that exists with pre-sleep feeding. To date, only one unpublished study from our laboratory (in obese men) used the microdialysis technique to analyze localized SCAAT lipolysis after pre-sleep PRO ingestion (independent of a RT regimen). The SCAAT is of particular interest in metabolism research as the upper body subcutaneous tissue (i.e. SCAAT) supplies most of the circulating FFA during the overnight hours in lean individuals (170). Furthermore, dietary PRO intake has been shown to increase RMR (228, 276) and contribute to improvements in body composition (21, 25), and body fat (21, 25), specifically. Therefore, if one can optimize the timing of dietary PRO during a window at which SCAAT is elevated (pre-sleep), theoretically, mobilization and oxidation of fats could potentially be optimized. Overall, there is no current research that examines how SCAAT lipolysis is affected by the combination of pre- sleep PRO consumption and RT in normal-weight and body composition individuals. Therefore, research is needed in this area to elucidate the physiological changes that may occur, so that this dietary practice may be refined and potentially added to clinical and athletic dietary recommendations in the future.

CHAPTER THREE

METHODS

3.1 Participants

A power analysis was performed using JMP PRO 11 (SAS, Cary, NC) with interstitial glycerol concentration as the primary outcome variable. The analysis revealed a need for 12 participants per group with a power of 0.80, α = 0.05, standard deviation = 16 µmol/L (taken from the control group), and difference to detect of 20 µmol/L (treatment-control) (277). Participants were recruited using flyers posted on the university’s campus and Tallahassee businesses and gyms. Prospective participants were pre-screened through an e-mail. Inclusion criteria included healthy, resistance-trained (squat 100% of their body weight, and bench press 70% of their body weight, adapted from ACSM Guidelines (420) and piloted in our 2 laboratory), normal weight (BMI = 18.5-25.0 kg/m ; body fat <33%), normally menstruating (on or off oral contraceptives) females ages 18-35 years. Participants were excluded if they had uncontrolled thyroid dysfunction (not on medication), musculoskeletal disorders, currently smoked, had milk allergies, or if they took medications or supplements known to affect substrate metabolism. A washout period was required (length depending on supplement taken) prior to participation for participants that took dietary or ergogenic supplements other than a multivitamin or protein supplements. Participants were required to refrain from consuming caffeine and performing physical activity for 24 hours prior to each laboratory visit. This study was approved by the Florida State University Human Subjects Institutional Review Board (Appendix A) and written informed consent (Appendix B) and medical history (Appendix C) were completed prior to participation.

3.2 Research Design

This study was a randomized, crossover, double-blind, acute study with two conditions: daytime PRO consumed 30 minutes after RE and pre-sleep PLA consumed 30 minutes’ pre-sleep (PRO-PLA), and daytime PLA and pre-sleep PRO (PLA-PRO) (Friesland Campina and Dymatize® Nutrition, Dallas, TX). There were two primary aims of the study: 1) Aim 1 addressed the effects of RE on fat metabolism, and 2) Aim 2 addressed the effects of PRO timing (PRO-PLA and PLA-PRO) on fat metabolism. Of note, because Aim 1 was focused on RT only

(without influence of nutritional supplementation), the participants’ data from both visits 4 and 5 were averaged and the mean values were reported. However, for Aim 2, participants were randomly assigned to PRO-PLA or PLA-PRO in a randomized, placebo-controlled, cross-over design, (N =13 per group). Participants reported to the Institute of Sports Sciences and Medicine (ISSM) at Florida State University for measurements and assessments on five occasions (Figure 10): pre-testing and familiarization of maximal testing (Visit 1), maximal testing (visit 2), familiarization (Visit 3), and two experimental trials (PRO-PLA and PLA-PRO) (Visits 4 and 5). Visits 1, 2, 3 and 4 were separated by at least 3 days (Figure 10), in order to optimize physical activity normalization through reporting, as well as to avoid a training effect (133). Visits 4 and 5 occurred during menses (days 1-6 of the follicular phase), as research has shown that high levels of E2 during the mid-luteal phase can enhance exercise-induced lipid utilization in women (261). Participants visited the laboratory on Visits 4 and 5 after self-reporting that they were currently in menses. Thus, visits 4 and 5 were separated by approximately four weeks (Figure 10). Visits 3-5 (not Visit 1 or 2) were overnight stays in the laboratory. For these overnight stays, participants were provided with an air mattress, pillow, and clean bedding to sleep. For visits 4 and 5, participants were randomly assigned to their initial treatment condition using a computerized random number generator (416). Before each visit, participants returned to the laboratory under the same pretesting conditions (i.e. consuming the same food and performing the same physical activity). Dietary intake was confirmed by a 24-hour dietary food record (Appendix D) of the day prior to each visit, and dietary intake was controlled on experimental visits (see section 3.3.4.4). Physical activity was reported using an Exercise Log (Appendix E) for three days prior to each visit. Participants recorded the type, duration and intensity (using Borg’s Rating of Perceived Exertion Scale) of the exercise. If the recorded activity was resistance-based, the participant recorded the name of each exercise, as well as sets, repetitions, and weight completed. Prior to beginning collection on each of the visits, participants completed a Visual Analog Scale (VAS) for soreness and fatigue (321) of both the upper (arms, chest, back, abdomen) and the lower body (legs) in order to assess baseline levels of soreness and fatigue using a scale with a range of 0 to 10 cm (Appendix F). Participants performed all RE protocols during experimental visits (Visits 4 and 5) at the same time of day (early afternoon, ~1450-1550 hours) to avoid the potential influence

78 of the circadian cycle. All RE sessions took place under the supervision of research personnel and certified personal trainers.

Figure 10. Timeline of study. VAS: visual analogue scale; PA: physical activity; HR: heart rate; BP: blood pressure; PLA: placebo; PRO: protein; PRO-PLA: daytime PRO and pre-sleep PRO; PLA-PRO: daytime PLA and pre-sleep PRO.

3.3 Procedures

3.3.1 Visit 1, Pre-Testing and Familiarization of Maximal Testing

Participants arrived to the ISSM laboratory for Visit 1 in a fed state, consuming their habitual diet and having eaten within one hour prior to arrival. Participants signed an informed consent (Appendix B) and a verification form indicating that they were not pregnant for the dual energy X-ray absorptiometry (DXA) scan (Appendix H), completed a VAS for soreness and fatigue (Appendix F), and set tentative testing dates based upon their menstrual cycle. Anthropometrics and body composition were measured. Participants then performed a familiarization of the maximal strength test for squat and bench press.

Figure 11. Timeline of Aim 1. : microdialysis sample collection; : blood collection; : dummy microdialysis sample collection; MD: Microdialysis; RMR: resting metabolic rate.

3.3.1.1 Anthropometrics and body composition. Height and body weight were measured using a wall-mounted stadiometer (SECA, Hanburg, Germany) and a digital scale (Detecto®, Brooklyn, NY), respectively. Body composition was measured using DXA (Hologic Discovery W, Bedford, MA) by a certified DXA technician according to the manufacturer’s instructions with the participants assuming the supine position, as described previously (20). 3.3.1.2 Maximal performance testing. After baseline testing, participants were familiarized with a 1RM strength test for squat and bench press. The participant’s 1RM was defined as the highest weight moved one time through the full range of motion using proper form (373). Form requirements for squat and bench press followed the USA Power Lifting (USAPL)

80 rules (419) and repetitions were only accepted if all requirements were satisfied. According to the USAPL rules, proper completion of a squat repetition requires the crease between the hip and abdomen to be lower than the top of the knee cap, while proper completion of a bench press repetition requires the feet to remain on the floor, while hips, shoulders and head remain flat on the bench (bar must touch the chest or abdominal area without bouncing). Baseline estimated 1- RM weight was selected based upon experience of the participant.

Figure 12. Timeline of Aim 2. : microdialysis sample collection; : blood collection; : dummy microdialysis sample collection; MD: Microdialysis; RMR: resting metabolic rate; PLA: placebo; PRO: protein; PRO-PLA: daytime PRO and pre-sleep PRO; PLA-PRO: daytime PLA and pre-sleep PRO.

Prior to maximal attempts, participants followed a warm-up for squat and bench press at 50, 60, 75, 85, and 90% estimated 1RM for 5, 4, 3, 1 and 1 repetitions, respectively, which has

81 been modified from a previous study (201). Thereafter, during maximal attempts, weight was increased by 2.5 to 5.0% until the participant either felt that she reached her maximum or she failed the repetition. If the participant failed the repetition, the participant was permitted to attempt to lift the weight one more time. Each warm-up set was separated by three minutes of rest while each maximal attempt was separated by seven minutes of rest (201). Participants were permitted to use weight-lifting ergogenic aids of their choice (i.e. shoes, belts, wrist wraps).

3.3.2 Visit 2, Maximal Testing Visit

At least three days after Visit 1, participants visited the laboratory a second time for the actual maximal testing of squat and bench press. The protocol for the actual 1RM was the same as the familiarization day (Visit 1).

3.3.3 Visit 3, Familiarization Visit

During the familiarization visit, participants were familiarized with the RE protocol, RMR and sleeping in the laboratory. Exercises for RE targeted the full body using a barbell, and included (in this order): back squat, bench press, Romanian deadlift, bent-over row, shoulder press, and reverse lunges (modified from Ormsbee et al., 2007 (277)). Before beginning each exercise, participants warmed up with one set of 10 repetitions at 40% 1RM of the respective exercise (40% of actual 1RM for back squat and bench press determined from Visit 2, and 40% of predicted 1RM for all other exercises). After the warm up set, exercises were performed in a series of three sets of 10 repetitions (modified from Ormsbee et al., 2007 (277) and NSCA Guidelines (68)). The bench press and back squat exercises were completed with resistance equal to 65% of 1RM determined from the maximal testing day (68). All other exercises began with a weight that could be lifted about 10-12 times, chosen volitionally by the participant. Participants advanced or decreased the weight during these three sets as needed to obtain an optimal intensity (expected to achieve above an 8 on a modified RT RPE scale [Appendix I] by the last repetition of each set). If above an 8 on the modified RT RPE scale was not achieved by the last repetition of the set, the weight was increased accordingly. The weight that elicited greater than an 8 on the RPE scale was the weight that was used for the next two visits (Visits 4 and 5). The participant sat in a designated chair for a rest period of 90 seconds (68) after all exercises and sets. After RE, to familiarize the participant to RMR, the ventilated hood (described below) was placed over the participant’s head and torso for 30 minutes.

3.3.4 Visits 4 and 5, Experimental Trials

Participants arrived to the ISSM laboratory for the pre-testing overnight laboratory visit in afternoon (~1500 hours) in a fed state (with breakfast and lunch meals provided the night before, as described below). The following morning, participants completed a VAS for hunger, satiety, and fullness (Appendix G) of the day of prepared meals that they consumed. 3.3.4.1 Sleep assessment. Participants wore a sleep watch (Fatigue Science Readiband TM , Blaine, WA) (Figure 10) on their non-dominant wrist for experimental visits, as well as three days following either experimental visit to measure sleep quantity and quality in the laboratory and at home. The watch was worn immediately upon arriving to the laboratory and throughout the night. The outcome variables of this measure included total time sleeping, sleep latency (the time in which it took them to fall asleep while in a resting state), sleep efficiency (a measure of sleep quality, or the ratio of the time they spent asleep to the amount of time they spent in bed, including resting), and wake episodes (268). The sleep efficiency measure is based on sleep latency and the average number of wake episodes, with higher scores indicating better sleep quality. 3.3.4.2 Resting metabolism. REE (amount of calories burned at rest) and RER (measure of whole-body substrate utilization) was measured with a dilution technique and open-circuit indirect calorimetry (ParvoMedics TrueOne 2400 metabolic cart, Sandy, UT) using a ventilated hood that covered the participants’ head and torso (212). Participants were asked to remain awake, quiet, and as motionless as possible in a semi-recumbent position in a quiet, dark, climate-controlled, isolated room (20-23°C). Resting metabolism was measured on four occasions: 1) baseline, after microdialysis probe equilibration (~1600 hours); 2) immediately after RT (~1720 hours); 3) after the pre-sleep supplement (~2050 hours), and; 4) the next morning (~0600 hours). Prior to testing, calibrations were made in duplicate and were performed on the flow meter using a 3L syringe and gas analyzers with verified gases of predetermined concentrations. Gas exchange was measured continuously for 30 minutes, with the last 15 minutes of collection being used for data analysis. CHO and fat oxidation were calculated assuming negligible nitrogen excretion, and using RER, VO2, and volume of carbon dioxide exhaled (VCO 2) in the following equations developed by Frayn (106), and adapted by Jeukendrup (173):

CHO oxidation = 4.55 VCO 2 – 3.21 VO 2

Fat oxidation = 1.67 VO 2 – 1.67 VCO 2 3.3.4.3 Blood sampling and biochemical analysis. Blood samples were collected from the antecubital vein on three occasions for Aim 1: 1) before microdialysis probe insertion (1500 hours); 2) mid-RE (~1655 hours); 3) post-RE (~1720 hours), and three occasions for Aim 2: 1) 30 minutes after the daytime supplement (~1800 hours); 2) 30 minutes after the pre-sleep supplement (~2120 hours), and; 3) the next morning immediately upon waking 30 minutes before RMR (~0530 hours). The blood samples were taken 30 minutes after supplementation because as plasma amino acid concentrations are known to significantly increase 30 minutes after pre-sleep consumption of casein PRO (316), and therefore, the most potent potential changes in plasma biomarkers would likely occur around that time. Antecubital blood samples were collected into EDTA-coated plasma vacutainers (Becton, Dickinson & Company, Franklin Lakes, NJ). and samples were centrifuged (SorvallTM ST 16R Centrifuge, Thermo Scientific Inc., Waltham, MA) for 15 minutes at 3500 rpm at 4°C. Aliquots (300 μL) were transferred into microtubes and stored at -80˚C for later batch analysis. Insulin (Cat. No. RE53171, IBL International®, Hamburg, Germany), GLY (Cat. No. MAK117-1KT, Sigma-Aldrich®, St. Louis, MO), NEFAs (Cat. No. RE53171, IBL International®, Hamburg, Germany), CATs (Cat. No. 17-BCTHU-E02.1, Alpco®, Salem, NH), and GH (Cat. No. QC99, R&D Systems®, Minneapolis, MN) concentrations were measured in duplicate using commercially available enzyme linked immunosorbent assays (ELISA) kits, according to the manufacturer’s instructions. Blood was also collected in serum vacutainers (Becton, Dickinson & Company, Franklin Lakes, NJ), and immediately analyzed for concentration of glucose using a YSI (Yellowsprings Instruments, Yellowsprings, OH). 3.3.4.4 Dietary intake standardization. Before each visit to the laboratory, participants were asked to maintain their normal dietary habits, confirmed using a 24-hour dietary food log (Appendix D) for the day prior to each of these visits. Dietary food logs were input and analyzed by two research personnel (The Food Processor SQL, ESHA Research, Salem, OR) to control for changes in dietary intake prior to each visit. During Visits 4 and 5, dietary intake was standardized for the full day. Calories in each meal were provided using 40:30:30 (CHO:PRO:FAT) ratio. Meals were made by a company that prepared customized meals based upon macronutrient and caloric needs of the participants. Calories were determined using the Cunningham Equation (126):

REE = 500 + (22 x FFM) Activity level was taken into account in the previous equation by estimating the thermic effect of exercise (TEE), assuming that participants were moderately active: TEE = RMR x 0.6 Participants chose palatable food items from a standard menu for breakfast, lunch and dinner. Within 24 hours prior to Visits 4 and 5, participants were hand-delivered all of the meals by research personnel. Breakfast and lunch was consumed outside of the laboratory (~0700 and ~1200 hours, respectively). Participants brought the dinner meal (as well as the breakfast and lunch containers from earlier that day to verify compliance) with them when they came to the laboratory that afternoon. During experimental visits, participants were randomized (using Random.org) after RE to consume either daytime PRO and pre-sleep PLA (PRO-PLA), or daytime PLA and pre-sleep PRO (PLA-PRO). Each participant consumed the first supplement 30 minutes after the RE session (~1750 hours). One hour later (~1850 hours), participants consumed dinner. Thirty minutes pre-sleep (~2050), participants consumed the opposite supplement. Participants only consumed water, prepared meals, and the daytime and pre-sleep supplements on experimental visits. 3.3.4.5 Microdialysis. A timeline of microdialysis collection for each experimental trial is depicted in Figure 11. Upon arriving to the laboratory (~1500 hours), microdialysis probes were inserted and equilibrated for one hour while the participant assumed a supine position. Briefly, an area of skin about 10 cm from the umbilicus was cleaned with betadine (Betadine® Solution Swabsticks, Purdue Products L.P., Stamford, CT). The insertion site was then numbed with a topical ethyl chloride spray (Gebauer’s ethyl chloride ®, Gebauer Company, Cleveland, OH) for about 10 seconds to attenuate discomfort. Two previously sterilized microdialysis probes (CMA 20 ELITE: 10×0.5-mm dialysis membrane with 20-kDa pore size; CMA/Microdialysis, Acton, MA) were inserted into the SCAAT of the participant with each location separated by about 10 mm, using techniques previously described (150). Preparation of the probes consisted of soaking the probes in the disinfectant 70% isopropyl alcohol for 20 minutes and then rinsing the probes four times with sterile deionized water (Baxter Healthcare Corporation, Deerfield, IL). To insert the probes, an 18G catheter needle was wrapped with a plastic split catheter and inserted into the SCAAT. The plastic split catheter was held in place

85 while the needle was removed to serve as a conduit for probe insertion. After probe insertion, the plastic split catheter was removed, and the probe was held in place by Steri-Strips TM (3M Health Care, St Paul, MN). The probe was attached to a portable microdialysis pump that continuously perfused (2.0μl/min) a solution (147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl 2, 0.85 mM MgCl 2; order no. P000151, CMA/Microdialysis, Acton, MA) containing 0.9% NaCl and ∼10mM ethanol into the SCAAT. The pumps were secured in a waist belt so that mechanical perturbation to the insertion area was minimized. Residual betadine on the skin was removed using alcohol pads, and the insertion location was protected and held in place with Tegaderm TM film (3M Health Care, St Paul, MN). The pumped perfusate was collected at the exit end of the probe as dialysate with part stored at 4°C for ethanol analysis (index of local blood flow) within 24 hours, and the remainder stored at −20°C for later interstitial glycerol analysis (index of lipolysis). Flow rate of the microdialysis pump remained at 2.0 μl/min for the entirety of the protocol. After 60 minutes of probe equilibration after initial insertion, the dummy vial (D1) was removed, and baseline collection began for 30 minutes (~1600-1630 hours, collection of Vial 1). At ~1630 hours, a new collection vial was attached (Vial 2), and participants began RE. Vials were changed 25 minutes into RE and post-RE (Vials 3-4, ~1655-1720 hours), as well as 30 min after the RE ended (Vial 5, 1755 hours). After RE, participants were randomized to PRO-PLA or PLA-PRO conditions. Participants consumed the daytime supplement, and vials were changed after 30 min (Vial 6, ~1820 hours), and again before their prescribed dinner (Vial 7, ~1850 hours). Participants consumed their prescribed dinner (~1850 hours) and then vials were changed every 60 min (Vials 8-9, ~1950-2050 hours) until the pre-sleep supplement, taken 30 min pre-sleep (~2050 hours). After the pre-sleep supplement and RMR, the perfusate vials were refilled, the pumps were equilibrated for 10 minutes, and the first night vial was attached (Vial 10, ~2130 hours). During sleep, vials were changed every two hours of sleep until waking (Vials 11-13, ~2330-0330 hours). Upon waking, the final vial was attached (Vial 14, ~0530 hours). After 30 minutes of collection (~0600 hours), Vial 14 and the microdialysis probes were removed. Samples were stored at 4˚C overnight. Within 24 hours of the beginning of data collection, ethanol analysis for blood flow determination (described in Section 3.3.4.7) was performed (152). After that, samples were stored at -80˚C for later dialysate GLY analysis using

86 an automated microdialysis analyzer (CMA 600 analyzer, CMA Microdialysis, Solna, Sweden) according to manufacturer’s instructions. The CMA 600 uses colorimetric methods and enzymatic reagents in order to analyze GLY concentration kinetically (417). Calibration was performed with a single multicomponent calibrator (Calibrator A, M Dialysis AB, Solna Sweden) with predetermined concentrations of the analytes. Absorbance change during the first 30 seconds was monitored, and the maximal reaction rate was recorded (417). The measuring principle for the determination of GLY concentration using this technique is as follows: 1) GLY is phosphorylated by ATP and converted to glycerol-3-phosphate and ADP (catalyzed by GLY kinase); 2) glycerol-3-phosphate is consequently oxidized (by adding O 2) to dihydroacetone phosphate and hydrogen peroxide (catalyzed by glutathione peroxidase); and 3) the hydrogen peroxide reacts with 3,5-dichloro-2-hydroxy-benzene sulphonic acid and 4-amino-antipyrine to form the red quinoneimine dye (catalyzed by peroxidase). Averaged duplicate measures of dialysate GLY concentration were used in order to determine interstitial GLY concentrations. The rate of formation of the red quinoneimine dye is proportional to the GLY concentration (417). 3.3.4.7 Blood flow. The ethanol technique was used to estimate local blood flow in the area surrounding the probes, as described previously (152). Ethanol freely diffuses over probe membranes and is not metabolized in adipose tissue to a significant extent (151, 152), and therefore, ethanol removal and consequent collection from the localized area correlates with blood flow. Ethanol concentrations were measured in the perfusate and dialysate using a multimode microplate reader (SpectraMax® M5, Molecular Devices, Sunnyvale, CA) within 24 hours of collection. A portion of the dialysate sample (2μl) was added to a mixture containing an ethanol buffer (pH 8.9; 74 mM Sodium Pyrophosphate, 60 mM Hydrazine Sulfate, 22 mM Glycine) and an NAD solution (100 mM NAD) in black 96-well microplates (ThermoScientificTM Immuno Plates, Nazareth, PA). Alcohol dehydrogenase (10 μl) was added to each well and the plate was read with fluorescence at an excitation and emission of 360 and 415, respectively, following a one-hour incubation (room temperature in the dark). The measured fluorescence is directly proportional to ethanol concentration in the sample (152), and the ethanol outflow/inflow ratio (Ethanol dialysate /Ethanol perfusate ) is inversely related to blood flow in the adipose tissue around the probe.

3.3.4.6 Calculation of interstitial glycerol. Dialysate GLY concentration is only representative of a small fraction of the actual interstitial concentrations. Actual interstitial concentration of GLY can be measured when the in vitro relative recovery of GLY is known by adjusting dialysate concentrations for the in vitro relative recovery. In the present study, we used the same probes and flow rate (2.0 μl/min) in vitro and the recovery rate for GLY was measured to be 73.24%. To determine this value, the probes were inserted into a solution [Dulbecco’s phosphate-buffered saline, 0.1% bovine serum albumin (BSA), 5 mM glucose, 0.2 mM GLY, 5 mM ethanol, and 2 mM of lactate]. In vitro concentrations were used to calculate in vivo concentrations based upon the assumption that the relationship between these two recovery rates were similar. In vitro recovery rates were determined by sampling beaker and dialysate known concentrations every 30 minutes for two hours. The ethanol outflow-to-inflow ratio (O:I) is inversely related to blood flow and was calculated as:

O:I ratio = [ethanol dialysate ]/[ethanol perfusate ] The in vivo GLY recovery rate for each probe was determined using the following step- by-step equation:

GLY recovery in vitro /EtOH recovery in-vitro = GLY recovery in vivo/EtOH recovery in-vivo Or

GLY recovery in vitro /EtOH recovery in-vitro = GLY recovery in vivo/1 - O:I ratio Then:

(GLY recovery in vitro /EtOH recovery in-vitro ) * (1 – O:I ratio) = GLY recovery in vivo

Then, to calculate interstitial glycerol concentration:

Interstitial GLY concentration = GLY concentration in vivo / GLYrecovery in vivo

3.3.4.8 Resistance exercise protocol. The RE protocol for the experimental visits consisted of the same protocol as the familiarization visit (Section 3.3.3). RE began immediately after the equilibration of the probes, and lasted approximately 50 minutes. 3.3.4.9 Supplementation. The micellar casein PRO supplement (PRO, 30 grams of micellar casein PRO, 120 kcals) and non-caloric, sensory-matched PLA (PLA, 0 grams of

88 micellar casein PRO, 0 kcals) specifically formulated by Dymatize® Nutrition and FrieslandCampina® and consumed in a randomized order. Participants consumed the daytime supplement 30 minutes after RE (~1750 hours), and consumed the pre-sleep supplement about 30 minutes before bedtime (~2050 hours). The supplements were premixed by the same research personnel. The supplements were taste-matched, and were provided to the participants in opaque shaker bottles. 3.3.4.10 Compensation. After completion of the study, all participants were given a their results (body composition and metabolism measurements) and $100 for their participation.

3.4 Statistical Analysis

Paired t-tests were used to measure potential differences in baseline measures of the PRO- PLA and PLA-PRO conditions: metabolic rate, serum samples, interstitial GLY concentrations, sleep data, physical activity record measures, 24-hour food log measures (total kcal, PRO, CHO, fat), and VAS for fatigue and soreness (Appendix F). For Aim 1, a one-way ANOVA was used to analyze changes in interstitial GLY concentrations, metabolic rate, and plasma biomarkers around RE compared to baseline (BL) (BL, Mid-RE, post-RE). For Aim 2, a repeated measures ANOVA was used to compare the differences in interstitial GLY concentrations, metabolic rate, and plasma biomarkers between PRO-PLA and PLA-PRO conditions. If a significant finding was noted, a Tukey HSD post-hoc analysis was used to locate where the difference existed. Data were analyzed using SPSS (Version 25) with significance set at p<0.05. Values are presented as mean ± standard error (SE).

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Results

4.1.1 Descriptive Characteristics

Seventy-seven women were interested in participating in the study. Fifty-seven women either did not respond to a follow-up e-mail, said they may participate later, were excluded based on medication, or were self-excluded. Of the 20 women who remained, six of them did not meet the strength requirements, and one of them was injured during the time of the study (outside of the laboratory). Therefore, 13 women participated in the study. Table 2 presents a summary of the descriptive characteristics of participants at baseline. There were no differences between groups. There were no differences between dietary intake and physical activity levels before any of the visits. Average dietary intake for 24 hours before each visit is shown in Table 3.

Table 2. Descriptive characteristics of participants measured at baseline (N=13) Variables Mean ± SE Age (years) 22 ± 1 Height (cm) 163.3 ± 1.5 Weight (kg) 65.1 ± 2.3 BMI (kg/m 2) 19.1 ± 0.6 Body Fat (%) 28.7 ± 1.4 Android Fat (%) 25.6 ± 1.6 Android:Gynoid Ratio 0.75 ± 0.03 Lean Mass (kg) 44.0 ± 51.4 Squat, Percentage of BW (%) 135 ± 6 Bench Press, Percentage of BW (%) 82 ± 4 BMI, body mass index; BW, body weight

4.1.2 VAS Scales of Fatigue and Soreness of the Upper and Lower Body

There were no differences in measures of fatigue and soreness of the upper and lower body before PRO-PLA and PLA-PRO conditions (Table 4).

Table 3. Average dietary intake for 24 hours before each visit Variables Mean ± SE Total C alories (kcal) 1935.7 ± 96.0 Carbohydrate Total Weight (g) 231.9 ± 18.8 Calories (kcal) 913.1 ± 77.0 Grams/kg BW 3.6 ± 0.4 Percent of Total Kcal (%) 46.6 ± 2.2 Protein Total Weight (g) 112.8 ± 6.02 Calories (kcal) 445.4 ± 26.1 Grams/kg BW 1.8 ± 0.1 Percent of Total Kcal (%) 24.0 ± 2.0 Fat Total Weight (g) 70.9 ± 6.0 Calories (kcal) 624.6 ± 52.4 Grams/kg BW 1.1 ± 0.1 Percent of Total Kcal (%) 32.2 ± 2.2 BW: body weight

Table 4. VAS measurements of fatigue and soreness of the upper and lower body before PLA- PRO and PRO-PLA condition visits PLA-PRO PRO-PLA

Fatigue, UB (cm) 21.5 ± 3.3 25.1 ± 5.2 Fatigue, LB (cm) 24.9 ± 5.2 26.2 ± 5.8 Soreness, UB (cm) 22.1 ± 5.5 21.7 ± 5.2 Soreness, LB (cm) 23.4 ± 7.0 24.0 ± 6.5 Values are means ± SE; UB = upper body; LB = lower body; PLA: placebo; PRO: protein; PLA- PRO: daytime placebo and pre-sleep protein; PRO-PLA: daytime protein and pre-sleep placebo.

4.1.3 Sleep Data

There were no significant differences in the measured total time sleeping, sleep latency, sleep efficiency, or wake episodes when comparing PLA-PRO and PRO-PLA conditions or when comparing the home condition (the three days that the participants wore the watch in their own home environment). However, when comparing the average of the PLA-PRO and PRO-PLA experimental conditions and the average of the home setting days, there was a significant difference between wake episodes (Table 5).

Table 5. Sleep quantity and quality measurements Experimental Conditions Home Setting

Total Time Sleeping (hours) 6.7 ± 0.5 7.1 ± 0.6 Sleep Latency (minutes) 21 ± 4 22 ± 4 Sleep Efficiency 82 ± 3 84 ± 3 Wake Episodes 5 ± 1 3 ± 1 * Values are means ± SE; * = significantly different ( P < 0.05) compared to Experimental Conditions.

4.1.4 Dietary Control on Experimental Visits

Participants were provided the same amount of food to consume during both PLA-PRO and PRO-PLA conditions (Table 6). There were no significant differences in feelings of hunger, satiety or fullness the following morning between PLA-PRO and PRO-PLA conditions.

4.1.5 Effect of Acute Resistance Exercise

4.1.5.2 Interstitial glycerol concentrations . Interstital GLY concentrations were higher at mid-RE, and significantly higher at post-RE, compared to BL (Figure 13). Interstital GLY concentrations decreased significantly from post-RE to 30 min post-RE (Figure 13). Individual responses are shown in Figure 14. 4.1.5.1 Metabolism. There were significant differences between baseline and post-RE measures in all measured metabolic variables (Table 7). REE and fat oxidation significantly

92 increased, while RER and CHO oxidation significantly decreased (Table 7). Changes in REE, RER, and FatOx are depicted in Figure 15.

Table 6. Dietary intake during PLA-PRO and PRO-PLA conditions PLA-PRO PRO-PLA

Protein without Supplement Total Weight (g) 85.3 ± 17.9 84.4 ± 16.9 Calories (kcal) 70.1 ± 2.4 69.7 ± 2.7 Grams/kg BW 1.3 ± 0.1 1.3 ± 0.1 Percent of Total Kcal (%) 17.5 ± 0.6 17.4 ± 0.7 Protein with Supplement Total Weight (g) 115.3 ± 17.9 114.4 ± 16.9 Calories (kcal) 95.9 ± 3.6 95.5 ± 4.04 Grams/kg BW 1.8 ± 0.1 1.8 ± 0.04 Percent of Total Kcal (%) 24.0 ± 0.9 23.9 ± 1.0 Carbohydrate Total Weight (g) 211.5 ± 46.2 207.9 ± 46.8 Calories (kcal) 172.5 ± 4.1 169.2 ± 3.8 Grams/kg BW 3.2 ± 0.2 3.2 ± 0.2 Percent of Total Kcal (%) 43.1 ± 1.0 42.3 ± 0.9 Fat Total Weight (g) 72.3 ± 17.3 74.2 ± 17.0 Calories (kcal) 296.3 ± 14.2 304.4 ± 13.7 Grams/kg BW 1.1 ± 0.1 1.1 ± 0.1 Percent of Total Kcal (%) 32.9 ± 1.6 33.8 ± 1.5 Values are means ± SE; BW: body weight; PLA: placebo; PRO: protein; PLA-PRO: daytime placebo and pre-sleep protein; PRO-PLA: daytime protein and pre-sleep placebo.

4.1.5.3 Measurements of plasma markers . Plasma concentrations of insulin, glucose, GH, CATs, NEFAs and GLY are shown in Table 8. There were no significant changes in

93 plasma insulin, glucose or GLY concentrations at mid-RE or post-RE compared to baseline. There was a significant increase in GH, Epi, and NE concentrations at mid-RE and post-RE compared to BL. The inter-assay and intra-assay coefficient of variation for insulin was 10.8% 2.5%, respectively. The inter-assay and intra-assay coefficient of variation for NEFA was 7.3% and 5.1%, respectively. The inter-assay and intra-assay coefficients of variation for GLY was 4.2% and 20.6%, respectively. The inter-assay and intra-assay coefficient of variation for GH was 13.3% and 6.4%, respectively. The intra-assay coefficient of variation for Epi was 18.8%. The intra-assay coefficient of variation for NE was 12.5%.

Figure 13. The effect of resistance exercise on interstitial glycerol concentration. Data from both visits 4 and 5 were averaged and the mean values are reported as means ± SE. * = significantly different from BL, P < 0.05, $ = significantly different from Post-RE, P < 0.05.

4.1.6 Effect of Pre-sleep versus Daytime Protein Consumption

4.1.6.2 Interstitial glycerol concentrations . There were no significant differences in interstitial GLY concentrations during the incremental sleeping measurements in either PLA- PRO or PRO-PLA condition (Table 9). Interstitial GLY concentrations of each time point during PLA-PRO and PRO-PLA conditions (PLA-PRO and PRO-PLA) is depicted in Figure 15.

Figure 14. Individual interstitial glycerol concentration responses to resistance exercise. Data from both visits 4 and 5 were averaged and the mean values are reported as means ± SE.

Table 7. Metabolism measurements at baseline and post resistance exercise BL Post-RE p-value

REE (kcal/day) 1560 ± 49 1756 ± 68 0.02

RER (CO 2/O 2) 0.75 ± 0.01 0.70 ± 0.01 0.004 CHOOx (g/hr) 3.08 ± 0.63 0.44 ± 1.0 0.02 FatOx (g/hr) 5.64 ± 0.24 7.57 ± 0.41 0.0003 Data from both visits 4 and 5 were averaged and the mean values are reported as means ± SE; BL: baseline; Post-RE: post resistance exercise; REE: resting energy expenditure; RER: respiratory exchange ratio; CHOOx: carbohydrate oxidation; FatOx: fat oxidation

4.1.6.1 Metabolism. REE was significantly higher when measured after the evening supplement in the PLA-PRO condition, and the PRO-PLA condition compared to BL (Table 10). FatOx was significantly higher when measured after the evening supplement in the PLA-PRO condition compared to BL (Table 10). There were no other differences in any of the metabolism measurements.

Figure 15. Energy expenditure, respiratory exchange ratio, and fat oxidation before and after resistance exercise. Data from both visits 4 and 5 were averaged and the mean values are Figure 15 continued. reported as means ± SE. Post-RE: post resistance exercise; * significantly different from baseline, P = 0.00.

Table 8. Plasma biomarkers at baseline, mid resistance exercise, and post resistance exercise BL Mid-RE Post-RE

Insulin (µIU/mL) 26.6 ± 3.8 19.8 ± 1.9 20.7 ± 3.1 Glucose (mg/dL) 80.1 ± 2.3 78.9 ± 4.1 82.1 ± 3.0 Growth Hormone (pg/mL) 522.6 ± 131.9 1288.3 ± 83.9 * 1522.8 ± 51.1 * Epinephrine (pg/mL) 23.2 ± 2.7 92.5 ± 16.6 * 84.5 ± 21.4 * Norepinephrine (pg/mL) 139.2 ± 13.6 850.9 ± 155.3 * 695.3 ± 93.5 * NEFA (mEq/L) 0.31 ± 0.02 0.31 ± 0.03 0.30 ± 0.02 Glycerol (mmol/L) 0.040 ± 0.010 0.057 ± 0.007 0.067 ± 0.016 Data from both visits 4 and 5 were averaged and the mean values are reported as means ± SE; BL: baseline; Mid-RE: mid resistance exercise; Post-RE: post resistance exercise; NEFA: non- esterified fatty acids; *: significantly different compared to baseline, P < 0.001.

Table 9. Overnight interstitial glycerol concentrations in PLA-PRO and PRO-PLA conditions PLA-PRO PRO-PLA Baseline 524.3 ± 109.0 669.0 ± 137.0 Supp – Sleep 843.9 ± 338.1 605.1 ± 131.0 Sleep – 2 hours 641.1 ± 127.6 661.6 ± 114.8 2 hours – 4 hours 460.7 ± 71.4 390.7 ± 66.2 4 hours - 6 hours 474.2 ± 63.7 426.9 ± 77.9 6 hours – 8 hours 656.8 ± 257.8 754.5 ± 180.7 Next morning 333.2 ± 68.0 321.3 ± 77.1 Values are means ± SE; All values expressed in units of µM; PLA: placebo; PRO: protein; PLA- PRO: daytime placebo and pre-sleep protein; PRO-PLA: daytime protein and pre-sleep placebo.

4.1.6.3 Plasma biomarkers . Plasma concentrations of insulin, glucose, NEFA, and GL are shown in Table 11 and Figure 16. There were no differences in plasma insulin or GLY concentrations with the pre sleep supplement in either PLA-PRO or PRO-PLA conditions. There was a significant main effect of plasma insulin concentrations measured after the pre-sleep supplement compared to BL in the PLA-PRO condition and not the PRO-PLA condition. The plasma concentration of glucose was significantly lower after PLA-PRO compared to PRO-PLA.

Figure 16. SCAAT lipolytic during PLA-PRO and PRO-PLA conditions. Values are means ± SE. BL: baseline; RE: resistance exercise; Supp = supplement; NM = next morning.

Table 10. Measurements of resting energy expenditure after the pre-sleep pre-sleep supplement and the next morning compared to baseline in PLA-PRO and PRO-PLA conditions PLA-PRO PRO-PLA Baseline REE (kcal/day) 1564 ± 53 1555 ± 47 RER (CO 2/O 2) 0.75 ± 0.01 0.75 ± 0.01 CHOOx (g/min) 3.26 ± 0.92 2.91 ± 0.78 FatOx (g/min) 6.59 ± 0.32 5.70 ± 0.35 Pre-Sleep Supplement REE (kcal/day) 1725 ± 60 * 1693 ± 60 * RER (CO 2/O 2) 0.74 ± 0.01 0.76 ± 0.01 CHOOx (g/min) 2.26 ± 0.77 3.94 ± 0.63 FatOx (g/min) 6.59 ± 0.32 * 5.90 ± 0.35 Next Morning REE (kcal/day) 1544 ± 57 1541 ± 51 RER (CO 2/O 2) 0.76 ± 0.01 0.77 ± 0.01 CHOOx (g/min) 3.66 ± 0.72 4.49 ± 0.52 FatOx (g/min) 5.44 ± 0.27 5.00 ± 0.28 Values are means ± SE; PLA: placebo; PRO: protein; PLA-PRO: daytime placebo and pre-sleep protein; PRO-PLA: daytime protein and pre-sleep placebo; REE: resting energy expenditure; RER: respiratory exchange ratio; CHOOx: carbohydrate oxidation; FatOx: fat oxidation Table 10 continued. *: significantly different from baseline, P < 0.05. 98

Table 11. Plasma markers at baseline, immediately post pre-sleep supplement, and next morning in PLA-PRO and PRO-PLA conditions PLA-PRO PRO-PLA BL Pre-sleep NM BL Pre-sleep NM

Insulin 32.3 ± 6.5 23.2 ± 3.9 14.0 ± 2.1 20.8 ± 3.3 21.9 ± 6.1 18.4 ± 6.8 (µIU/ml) Glucose 81.7 ± 3.5 80.0 ± 2.0 * 77.8 ± 6.0 78.4 ± 2.4 87.6 ± 2.4 81.4 ± 2.2 (mg/dL) NEFA 0.33 ± 0.04 0.33 ± 0.04 0.37 ± 0.02 0.27 ± 0.03 0.33 ± 0.04 0.41 ± 0.03 (mEq/L) Glycerol 0.04 ± 0.01 0.02 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 0.04 ± 0.02 0.07 ± 0.03 (mmol/L) Values are means ± SE; * = significantly different from PRO-PLA, P < 0.05. PLA: placebo; PRO: protein; PLA-PRO: daytime placebo and nighttime protein; PRO-PLA: daytime protein and nighttime placebo; BL: baseline; NM: next morning; NEFA: non-esterified fatty acids.

4.2 Discussion

This is the first study to assess the effects of 1) RE, and 2) pre-sleep versus daytime PRO consumption on SCAAT lipolysis and whole-body substrate oxidation in resistance-trained women. The primary findings of the present study included the following: RE significantly increased SCAAT lipolysis and FatOx with a concomitant increase in plasma GH concentrations, and there were no differences in lipolysis, FatOx, or plasma biomarkers between pre-sleep and daytime PRO consumption. Therefore, our aim 1 hypothesis that an acute bout of RE would increase SCAAT lipolysis (as indicated by increases in concentration of interstitial GLY at mid- RE and post-RE) and whole-body substrate metabolism (to indicate greater reliance on fat as a fuel) was supported. Additionally, our aim 2 hypothesis that there would be no differences in SCAAT lipolysis or whole-body substrate utilization overnight between PLA-PRO and PRO- PLA conditions was also supported. Lastly, our aim 3 hypothesis that: 1) serum GH, GLY and NEFA would increase at mid-RE and post-RE compared to BL, while glucose and insulin would stay the same, and; 2) no plasma biomarkers would be different between PLA-PRO and PRO- PLA conditions was partially supported in that there was a significant increase in CAT and GH

4.2.1 Resistance Exercise and Fat Metabolism

RE has consistently been shown to improve body composition through increases in lean mass and decreases in fat mass (178, 234, 276). Lipolysis is one of the main factors in decreasing fat mass with RE. When discussing the effects of RE on lipolysis, comparisons between women and men must be made because there are no other data on the effects of RE on SCAAT lipolysis in women. Overall, because the data that are available in men and the data from this study in women show similar trends, it indicates that RE affects women to a similar metabolic extent compared to men. Although we did not directly assess the differences between male and females, it is important to discuss. Females tend to store more subcutaneous fat than visceral fat, compared to males who store more in the visceral compartment (210). The men in the Ormsbee et al. study had less body fat compared to the women in the current study (men: 9.0 ± 2.1% body fat; women: 28.7 ± 1.4% body fat). However, regardless of sex and body fat percentage, the data show no significant associations between amount of body fat and either resting or exercise FatOx (159). It has been suggested that the ability to mobilize and utilize fats is more important than the amount of fat, as indicated by the current study, with women having a higher lipolytic rate during and after RE (baseline: 600.6 ± 88.3 µmol/L; Mid-RE: 827.4 ± 111.6 µmol/L, 84% difference from baseline; Post-RE: 955.1 ± 131.2 µmol/L, 136% difference from baseline) compared to men in the study by Ormsbee et al. (baseline: 112.6 ± 21.7 µmol/L; Mid- RE: 200.4 ± 38.6 µmol/L, 78% difference from baseline; Post-RE: 184 ± 41 µmol/L, 75% difference from baseline) (277). In the present study, lipolysis increased during and immediately after RE, however, interstitial GLY concentrations returned to baseline levels within 30 minutes. Others have reported elevations for up to 100 minutes post-RE, however, those data are from men. (277). Therefore, it is unclear whether this acute elevation in mobilization of GLY will result in any direct long-term body composition improvements.

One of the factors of these changes is an increase in resting metabolism after RE. As found in the current study, changes in resting metabolism have also been reported in the literature including increased REE (196, 243, 272, 277, 344), decreased RER (39, 277), and increased FatOx (196). An increase in REE with RE, as was observed in the present study, is consistent with findings from other studies in men (243, 272, 277, 344) and women (196), indicating an increased energy expenditure at rest post-RE compared to BL. In addition, RE has

100 been shown to decrease RER after exercise in men (277) and women (39), indicating elevations in FatOx after exercise. Indeed, RE is fueled primarily by glycolytic energy systems (372), and when glycogen is lowered with a RE bout (97), oxidation of fats is elevated post-RE (for glycogen-sparing purposes) (300). An increase in REE with RE in women is not consistent in all studies. Wingfield et al. (401) demonstrated that a high-intensity RE session in recreationally active women did not significantly change REE or RER compared to baseline measures. The RE session utilized similar movements compared to the current study (leg press, bench press, lunges, shoulder press, biceps curl, and triceps extension), and lower repetitions (three sets of 6RM to 8RM) however, with less rest between sets (20- to 30-s rest) compared to 90 seconds of rest in the present study. These authors mentioned that the intensity (three sets of 6RM to 8RM) they used and duration (25 minutes) may not have been high enough to elicit a significant metabolic response. In the present study, the RE sessions lasted 55 ± 1 minutes. Therefore, it is plausible that with our intensity (1 set of 10 repetitions at 40% 1RM, 3 sets of 10 repetitions at 65% 1RM), and a longer duration of RE, there would be a difference in REE and RER response to RE in our group of women. This may be why we saw increased REE and FatOx, and decreased RER with RE in these women.

Movement of adipocyte constituents into the interstitial space (as indicated by interstitial GLY concentrations), and then into the plasma (as indicated by plasma GLY and NEFA concentrations) is elevated simultaneously during and after RE (272). However, in the current study, although SCAAT lipolysis of GLY increased significantly with RE, neither plasma GLY concentrations (BL: 0.041 ± 0.008 mmol/L; Mid-RE: 0.056 ± 0.008 mmol/L, P=0.10; Post-RE: 0.064 ± 0.016 mmol/L, P=0.10), nor plasma NEFA concentrations (BL: 0.31 ± 0.02 mEq/L; Mid-RE: 0.31 ± 0.03 mEq/L, P=0.47; Post-RE: 0.30 ± 0.02 mEq/L, P=0.45) increased with RE. Despite this non-significant change in plasma GLY concentrations, concentrations may be physiologically significant as they increased by 36.6% from BL to mid-RE, and by 56.1% from BL to post-RE. Ormsbee et al reported that, although plasma GLY concentrations increased during and after RE, plasma NEFA only increased after RE and for a shorter duration than plasma GLY (272). Because there were no significant changes in plasma NEFA concentrations, and there was a significant increase in SCAAT lipolysis of GLY and FatOx, it may be that participants mobilized SCAAT adipocyte constituents into the interstitial space (perhaps with the fate of being re-esterified), and oxidized FA from the intramuscular fat stores. It has been shown

101 that contributions of intramuscular FA to total energy expenditure during exercise are higher in women (241), and thus, that may explain increases in SCAAT lipolysis and FatOx, without increases in plasma concentrations of GLY or NEFA. In total, data from the effect of RE on fat metabolism indicate that an acute RE bout can significantly affect localized fat and whole-body metabolism in resistance-trained women. Further, it may be argued that chronic RE-induced improvements in body composition may be mediated by the accrual of these acute physiological changes with the onset of a RE bout, and the subsequent accumulated effects.

Figure 17. Plasma glucose, insulin, glycerol, and NEFA concentrations before, during and after resistance exercise during PLA-PRO and PRO-PLA conditions. * = significantly different from pre-sleep PL, P < 0.05. Values are means ± SE. Supp = supplement.

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Figure 17 continued

The effect of GH on lipolysis is known to be mediated by RE (272) and fasting (153). Interestingly, GH alone significantly elevates FFA in the blood, reflecting its potent stimulation of lipolysis (255). Ormsbee et al. (272) showed that there was a significant increase in GH concentration with RE (Mid-RE: ~1000% increase from baseline; Post-RE: ~1500% increase from baseline) in men and up to 50 minutes post RE, in addition to increased interstitial and plasma concentrations of GLY, suggesting that GH is likely a driving force in increased fat metabolism with RE. The current study showed similar results in resistance-trained women

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(Mid-RE: +151% from baseline; Post-RE: +197% from baseline). Importantly, because fasting also has an additional effect on lipolysis and GH concentration (153), it may be reasoned that GH and the fasting state have an accumulated effect on lipolytic rate with this intervention. Participants in the current study were also in a fasted state when they had their blood drawn, having eaten the prescribed lunch meal at ~1200 hours, and beginning RE at ~1650 hours (~4.5 hours fasted). In comparison, in the Ormsbee et al. study (272), participants visited the laboratory after an overnight fast, beginning the RE protocol after ~12 hours of fasting. In the present study, participants consumed their first supplement 30 minutes after RE, and food intake is known to suppress GH (153), it would be reasonable to assume that GH concentrations would stay similarly elevated had our participants stayed fasted. Therefore, changes in GH were likely mediated by the combination of RE and being in a fasted state.

There were no changes in glucose or insulin concentrations during or after RE compared to baseline in the present study. However, significantly lower insulin (13, 272) and glucose (13) concentrations after RE have been noted in previous studies. This is logical, as it is known that exercise increases GLUT4 concentration to the cell surface (associated with glucose uptake into skeletal muscle) (47), and increases insulin sensitivity (which would indicate less plasma insulin due to the muscles’ increased insulin affinity) (299). However, it is known that there is a considerable amount of inter-individual differences in blood glucose response to RE (288). It is important to note that perhaps the acute nature of the current study was not conducive to significant changes in plasma insulin and glucose levels, as it has been found that 6 months of RE in young women improves insulin sensitivity, and eight weeks of RE in Type II diabetics has been found to lower insulin and glucose levels (13), eluding to efficiency of glucose uptake into muscle cells and therefore lower plasma concentrations (298).

The current study showed a significant increase in both Epi (BL: 23.2 ± 2.7 pg/mL; mid- RE: 92.5 ± 16.6 pg/mL, +299 from BL; post-RE: 84.5 ± 21.4 pg/mL, +264% from BL) and NE (BL: 23.2 ± 2.7 pg/mL; mid-RE: 92.5 ± 16.6 pg/mL, +511% from BL; post-RE: 84.5 ± 21.4 pg/mL, +399% from BL) at mid-RE and post-RE compared to BL. These findings are supported by the literature, as RE has been shown to stimulate a 180% increase in Epi at post-RE compared to BL and a 350% increase in NE at post-RE compared to BL in lean men, with these effects lasting up to two hours after the RE session (272). Changes in CAT concentration with RE in

104 women have not been observed. These increases in CATs with RE parallel increases in SCAAT lipolysis (272) (as in the present study) indicate the potent effect of the CATs on SCAAT lipolysis. After RE, NE returned to baseline more slowly (110 minutes post-RE) than Epi (50 minutes post-RE). Because it is known that Epi has a greater affinity for the inhibitory α-ARs, which are highly concentrated in the SCAAT (34), it is reasonable to conclude that the impetus of prolonged lipolysis post-RE is due to the longer-lasting NE, rather than Epi. However, we do not know this in the present study, as blood was taken immediately after RE, and not again until after the first supplement.

Overall, this is the first study of its kind to show that RE increases SCAAT lipolysis and FatOx in women. Although there was no direct comparison, these measures increased similarly to males in a previous study (272).

4.2.2 Effect of Pre-sleep versus Daytime Protein Consumption

Our second hypothesis that incremental overnight SCAAT lipolysis and next morning lipolysis and FatOx would not differ between PRO-PLA and PLA-PRO was supported. Mounting evidence in recent literature suggests the metabolic benefits of pre-sleep consumption of PRO Calories in small boluses (~200 kcal) (194). However, previous pre-sleep feeding studies differed from current study in that most of them did not control for PRO timing (day versus night), and they measured metabolism, but did not measure lipolysis. Antonio et al. (17) controlled for PRO timing by having exercise-trained male and female participants consume 54 g of casein PRO either in the morning (before 12:00 pm) or at night (less than 90 minutes before bedtime) for 4 weeks, while maintaining their normal exercise training regimen. After 4 weeks, they reported that the timing of PRO did not significantly affect body composition as measured by the BodPod®. Compared to the present study, the Antonio et al. study used both men and women that were either aerobically or resistance trained, not exclusively women that were resistance-trained. Likewise, the Antonio et al. study did not control for diet or exercise, as in the current study.

In the current study, there were significantly more wake episodes in the laboratory setting compared to the home setting (laboratory setting: 5 ± 3; home setting: 3 ± 2 wake episodes), but because no other sleep quality or quantity measure was different with the exception of wake episodes, it is safe to assume that because the sleep watch uses actigraphy, small movements of

105 the participants during times of vials changes (even if they were still asleep) may have been recorded as a wake episode. Therefore, these data confirm that microdialysis probe insertion into the SCAAT, pre-sleep supplementation, and regular visits during sleep (every 2 hours) by research personnel do not significantly affect sleep quality or quantity.

In addition, although other studies have noted either the benefits or no changes in metabolism resulting from pre-sleep feeding (i.e. REE, RER, FatOx, muscle protein synthesis and recovery) (192, 227, 276), only one other study directly assessed the effects of pre-sleep PRO consumption on SCAAT lipolysis and whole-body substrate utilization (190), and it was performed in overweight and obese men. Although previous work suggests that PRO may be lipolytic, as determined by postpradial plasma GLY concentrations immediately after PRO-rich meals in obese males and females (93), Kinsey et al. (190) found that pre-sleep casein PRO did not affect overnight or next morning SCAAT lipolysis or next-morning whole-body substrate oxidation (after about 8 hours of sleep from the pre-sleep feeding), which is considerably longer than post-prandial analysis of lipolysis. Additionally, like studies reported above, the Kinsey et al. study (190) did not control for PRO timing, and participants consumed either casein PRO or a non-nutritive PLA before going to bed in a crossover design. Further, on the pre-sleep PRO visit, participants consumed more total PRO compared to the PLA visit. In the current study, PRO consumption was the same for both experimental visits (visit 4 and 5), with the only difference being the timing (daytime versus pre-sleep) of the casein PRO supplement. However, in total, there are no significant differences in lipolysis or whole-body substrate metabolism when PRO timing is (as in the current study), or is not (as in the Kinsey et al. study) controlled for. Despite the study design differences, our data agree that pre-sleep PRO is not detrimental to overnight or next-morning fat metabolism.

It is also known that REE is suppressed during sleep (182). With dietary intervention, data indicate that the amount of PRO consumed throughout the day has effects on the pre-sleep attenuation of RMR in women (209). Madzima et al. (227) reported a significant increase in REE with pre-sleep PRO consumption; however, this study utilized active men, who were leaner compared to overweight and obese individuals in other studies with similar findings (191, 192), and it is known that lean mass is the primary driver of REE outcomes (142). Although the females in our study were normal body fatness (28.7 ± 1.4% body fat, healthy/acceptable range:

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25-32%), their percent lean mass (67.0% lean mass) was perhaps closer to the overweight and obese participants in previous pre-sleep PRO studies (191) compared to the lean men in the Madzima et al. study (227). Thus, the next-morning REE difference after pre-sleep PRO consumption between these resistance-trained women and lean men may have been driven by lean mass, regardless of training status, as we were unable to show that REE was significantly increased the morning following a pre-sleep PRO bolus. However, it is important to mention that we measured the participants’ baseline RMR at about 4:00 pm. This may be a limiting factor of the current study, in that it may have been a better option to assess baseline REE upon rising on the morning of the assessment. The Madzima et al. study (355) assessed baseline REE immediately upon waking and coming into the laboratory.

Our data indicate no significant changes in overnight SCAAT lipolysis or FatOx with pre-sleep PRO consumption. Mechanistically, circulating hormones would drive the potential changes in SCAAT lipolysis, measured 30 minutes after the nighttime supplement and upon waking the following morning in the current study. As anticipated, and similar to the present study, in previous pre-sleep feeding studies, generally, there are no significant changes in hormones measured the following morning (i.e. insulin, adiponectin, leptin, HOMA-IR, glucose, GH, cortisol) (148, 191, 192, 275). Only one study has reported significant increases in insulin and HOMA-IR with acute pre-sleep PRO feeding in sedentary obese women (192). These negative effects were attenuated with the addition of 4 weeks of exercise (2 days of RE, 1 day of high-intensity interval training) (275). Thus, any potential consequence of eating PRO before sleep was attenuated by exercise, and no other studies have reported similar issues in healthy populations. The present study agreed with most of the aforementioned studies as we found no significant differences in plasma insulin or GLY between pre-sleep PRO and PLA. No other pre-sleep feeding study measured plasma concentrations of NEFA; however, with no significant differences in SCAAT lipolysis with pre-sleep PRO feeding (191), we would not expect to see differences in these concentrations. Plasma glucose concentrations were significantly higher 30 minutes after consuming the pre-sleep PLA supplement (non-caloric) in the PRO-PLA condition compared to the pre-sleep PRO supplement in the PLA-PRO condition (PRO-PLA: 87.6 ± 2.4 mg/dL; PLA-PRO: 80.0 ± 2.0 mg/dL, P=0.02). Although most pre-sleep feeding studies did not find significant differences in blood glucose concentrations between pre-sleep PRO and PLA (191, 192), those studies measured blood glucose (and all plasma biomarkers) the following

107 morning, and not measure blood glucose concentrations 30 minutes after consumption of the pre- sleep supplement. Further, we found no significant differences in blood glucose concentrations between PRO-PLA and PLA-PRO the next morning, indicating that these differences were temporal and acute. In addition, although blood glucose concentrations were significant different between pre-sleep supplement time points in the PRO-PLA and PLA-PRO conditions, the blood glucose levels were still within the normal post-prandial range (normal range 2-hour postprandial range: below 140 mg/dL; PL: 87.6 ± 2.4 mg/dL; PRO: 80.0 ± 2.0 mg/dL), thus there was likely no health risk concern.

There were several strengths of the current study design that effectively addressed limitations from previous pre-sleep feeding studies. We controlled for the dietary intake of the participants using pre-prepared meals with a 40:30:30 split (CHO, fats, PRO), which has not been done before in other pre-sleep feeding studies, and thus, we can confidently conclude that neither diet during the length of the experimental visits nor diet 24 hours before each of the visits influenced metabolic outcome of our measured variables. However, although the total caloric content of the provided meals was based upon caloric need (from lean mass) and activity level, the provided meals did not have given the same number of calories, but did have the same macronutrient composition compared to what the participants were acclimated to (habitual intake: 1936 ± 96 kcal, 46.6% CHO, 24.0% PRO, 32.3% fat; provided meals: 2410 ± 50 kcal, 43.1% CHO, 24.0% PRO, 32.9% fat). In addition to this strength, we assessed sleep quality and quantity using a sleep watch, because the pre-sleep protocol required us to enter the participants’ room while they were sleeping to attach new microdialysis vials every two hours, and it is known that shortened sleep for extended periods of time can significantly decrease RER (greater reliance of fat as a fuel), and alter hormonal levels (increased CAT, cortisol, and insulin plasma concentrations) (309). Another strength of the current study is that we controlled for the menstrual cycle by having participants visit the laboratory on testing days during menses (on average, day 3 ± 0.3), as it is known that the presence of high E 2 levels during the mid-luteal phase can enhance exercise-induced lipid utilization and REE in women (261). Although we had these strengths, there are a few limitations of the current study that need to be addressed. Only normal weight and BMI, resistance-trained women were included in the present study. Thus, reported results may not be generalizable to other populations. Another limitation was that it is impossible to obtain direct measures of metabolism in human visceral fat.

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Even so, simultaneous measurement of SCAAT lipolysis (microdialysis) and whole-body metabolism (indirect calorimetry) in the current study provided valuable insight on the effects of pre-sleep PRO consumption on fat metabolism. In addition, although participants were instructed to consume only provided meals and supplements on experimental visits, it is possible that compliance to these standards may not have been met. Although there were many comparisons made to other RE and pre-sleep studies using similar designs and supplements (190, 191, 228, 273, 277), direct comparisons are not possible because these studies utilized different populations such as overweight and obese men and women, sedentary lean and obese men, active men, and older individuals. Therefore, this research will continue to add to the growing body of research on the metabolic effectiveness of RE and pre-sleep PRO consumption in resistance- trained women. One possible limitation of the current study may have been the amount of PRO consumed in the supplement. Recently, muscle metabolism has been found to be elevated with 40 grams of pre-sleep PRO in resistance-trained men (316), which is considerably greater than the recommended post-RE 20 gram of PRO required to produce similar effects (402). However, independent of direct dose comparisons, preliminary data from the same authors also found that 30 grams of pre-sleep PRO does not significantly improve next-morning muscle metabolism in resistance-trained men. Further, a more recent study has increased the pre-sleep PRO dose up to 54 grams in men and women (17). Thus, a limitation of the current study may be that the absolute dose provided (30 grams) was too low to elicit a significant response. Another limitation of the current study is that we did not assess each of the plasma biomarkers at each timepoint (glucose, insulin, GLY and NEFA at all timepoints, and CATs and GH just around RE). In addition, we did not have a control group for the effects of RE on lipolysis and FatOx (Aim 1). However, Ormsbee et al. (277) found no significant changes in SCAAT lipolysis during their control trial (remained resting in the supine position during the entirety of the experimental day and did not perform RE). Thus, it is reasonable to assume that there would be no significant differences in SCAAT lipolysis in the current study for a no-RE control condition. In addition, although we collected microdialysis samples every two hours during sleep and found no significant changes, we did not have the participants sleep in a chamber to measure direct calorimetry throughout the evening hours. Therefore, although we found significant increases in FatOx immediately after the pre-sleep supplement in the PLA-PRO condition (measured immediately before sleep and not during sleep), and these measures returned to baseline when

109 measured the next morning, we cannot confidently draw conclusions about the effect of pre-sleep PRO on whole-body substrate oxidation throughout the sleeping hours. Future research in this area should focus on the long-term fat metabolism effects of RE and pre-sleep PRO consumption in women, both independently and together, and also the differences in these outcomes in women and men. Likewise, future work should examine how RE and PRO timing affect the SCAAT at the receptor level by infusing receptor agonists and antagonists to determine mechanisms of change. Lastly, because we cannot ignore the metabolic differences between healthy and clinical populations, future work should be performed acutely and chronically in other female populations, such as those with obesity and metabolic disorders, to elucidate potential mechanisms of improvement with the addition of RE and PRO timing.

In conclusion, RE increases SCAAT lipolysis and FatOx in resistance-trained women, likely mediated by increases in CAT and GH concentrations. Thus, the long-term body composition benefits of RE in women may be mediated by localized and systemic changes in fat metabolism and hormone concentration. In addition, there are no differences in fat metabolism when consuming PRO during the daytime or pre-sleep in resistance-trained women. Therefore, pre-sleep PRO is a viable option for increasing PRO consumption in resistance-trained women, as it does not blunt overnight lipolysis.

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APPENDIX A

INSTITUTIONAL REVIEW BOARD APPROVAL OF STUDY PROTOCOL

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INFORMED CONSENT

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APPENDIX C

HEALTH AND MEDICAL HISTORY QUESTIONNAIRE

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APPENDIX D

FOOD LOG

Twenty-four Hour Dietary Log Participant Number: ______Date: ______Prior to Visit: 1 2 3 4

Directions for 24-Hour Dietary Log 1. Record your 24-hour food record for 1 full day. 2. Please record each food you eat immediately after you eat it. 3. Record only one food item per line. 4. Be as specific as possible when describing a food eaten: how it was cooked and the amount you ate. Don’t forget to include all beverages you drink. For example: Coffee with 1 tsp. Cream, 12 oz. Regular Coke, or 8 oz. Sweetened Tea. 5. Include brand names or labels from food items whenever possible. 6. Record amounts eaten in household measures. For example: one cup nonfat milk, 3 ounces grilled chicken, 2 tablespoons ranch dressing, 1 medium fruit, 2 slices cheese. 7. Include the method used to prepare the food item. For example: fresh, frozen, stewed, fried, baked, canned, broiled, raw, braised. 8. For canned foods, include the liquid in which it was canned. For example: Sliced peaches in heavy syrup or Fruit cocktail in light syrup. 9. If you eat at a restaurant, do your best to estimate portion size and list the restaurant you ate at. List any visible fat, oil, or sauces added to your food. 10. List amount and type of oil or butter you use in the preparation of your food. 11. Do not alter your diet while you are keeping a food record.

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PHYSICAL ACTIVITY RECORD

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VISUAL ANALOGUE SCALE FOR FATIGUE AND SORENESS

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VISUAL ANALOGUE SCALE FOR HUNGER, SATIETY, AND FULLNESS

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APPENDIX H

PREGNANCY CONSENT FORM

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APPENDIX I

MODIFIED RESISTANCE TRAINING RATE OF PERCEIVED EXERTION SCALE

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BIOGRAPHICAL SKETCH

Brittany R. Allman Curriculum Vitae

Professional Preparation

2018 Ph.D. in Exercise Physiology, Minor Concentration Area: Sports Nutrition Florida State University, Tallahassee, FL 2013 M.S. in Exercise Physiology (Summa cum laude) University of Delaware, Newark, DE 2011 B.S. in Exercise Science (Summa cum laude) Indiana University of Pennsylvania, Indiana, PA

Professional Experience 2018 to Present Postdoctoral Scholar Arkansas Children’s Nutrition Center University of Arkansas for Medical Sciences, Little Rock, AR 2013 to 2017 Graduate Course Instructor: -Applied Exercise Physiology Laboratory -Anatomy and Physiology I Lecture -Anatomy and Physiology II Lecture -Anatomy and Physiology I Laboratory -Anatomy and Physiology II Laboratory Department of Nutrition, Food and Exercise Sciences Florida State University, Tallahassee, FL 2013 to 2017 Graduate Teaching Assistant: -Sports Nutrition Online -Introduction to Exercise Science Department of Nutrition, Food and Exercise Sciences Florida State University, Tallahassee, FL

2013 to 2017 Graduate Research Assistant Advisor: Michael J. Ormsbee, Ph.D. Department of Nutrition, Food and Exercise Sciences Florida State University, Tallahassee, FL

2011 to 2013 Graduate Research Assistant Advisor: Shannon Lennon-Edwards, Ph.D., R.D. Department of Kinesiology and Applied Physiology University of Delaware, Newark, DE

Professional Credentials

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2015 to Present Certified Strength and Conditioning Specialist (CSCS) National Strength and Conditioning Association 2015 to 2017 Basic X-Ray Machine Operator The American Registry of Radiologic Technologists 2014 to Present Certified Sports Nutritionist (CISSN) International Society of Sports Nutrition 2009 to Present Professional Rescuer with First Aid American Red Cross 2009 to Present Certified Exercise Physiologist (EP-C) American College of Sports Medicine

Awards, Honors and Scholarships 2017 Florida State University’s Fellows Society 2017 Kappa Omicron Nu’s Honor Society 2017 NSCA Foundation Women’s Scholarship, National Strength and Conditioning Association 2017 Dissertation Award Program, College of Human Sciences, Florida State University, Tallahassee, FL 2017 Pao-Sen Chi Memorial Scholarship Endowment, Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 2017 Dr. Ava D. Rodgers Endowed Scholarship, College of Human Sciences, Florida State University, Tallahassee, FL 2016 Jean A. Reutlinger and Lillian H. Munn Scholarship, College of Human Sciences, Florida State University, Tallahassee, FL 2014 Selected for the Mary Frances Picciano Dietary Supplement Research Practicum, The Office of Dietary Supplements at The National Institutes of Health, Bethesda, MD 2011 Outstanding Presentation in the Undergraduate Scholars Forum, Top 5 of 70, Indiana University of Pennsylvania, Indiana, PA 2010-2011 Ronald E. McNair Post-Baccalaureate Achievement Program 2010 Excellence in Scholarship, Presenting Research at the University at Buffalo’s 16 th Annual McNair Research Conference, Buffalo, NY 2009 Provost Scholar Award for Outstanding Academic Achievement, Indiana University of Pennsylvania, Indiana, PA

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2008 All-American Scholar Athlete (Softball), Indiana University of Pennsylvania, Indiana, PA 2007-2010 Athletic Scholarship, Varsity Softball, Indiana University of Pennsylvania, Indiana, PA 2007-2010 Pennsylvania State Athletic Conference Scholar Athlete Award for Outstanding Academic Achievement, Indiana University of Pennsylvania, Indiana, PA 2007-2010 Honor Roll and Dean’s List, Indiana University of Pennsylvania, Indiana, PA Nominations 2017 2017-2018 Outstanding Teaching Assistant Award, Florida State University, Tallahassee, FL 2015 Student Representative of the Southeast Chapter of the American College of Sports Medicine 2014 Graduate Student Advisory Council of the College of Human Sciences, Florida State University, Tallahassee, FL Membership in Professional Organizations 2014 to Present National Strength and Conditioning Association 2014 to Present Professionals in Nutrition for Exercise & Sport 2013 to Present International Society of Sports Nutrition 2010 to Present American College of Sports Medicine 2015 to 2018 Junior League of Tallahassee

TEACHING Courses Taught As Graduate Student Instructor of Record: Anatomy and Physiology I Lecture Applied Exercise Physiology Laboratory Anatomy and Physiology I Laboratory Anatomy and Physiology II Lecture Anatomy and Physiology II Laboratory As Teaching Assistant:

Introduction to Exercise Science Sports Nutrition Online 172

Graduate and Undergraduate Mentor Graduate Maggie Morrissey Alexa Rodriguez Brett Hanna Undergraduate Shannon Wakeford Jessica McKelvey Alex Shippy Ashley Ferrand Kailee Hernandez, Undergraduate Research Opportunity Program (UROP) Mentor an undergraduate student through research by allowing her to assist in dissertation data collection, help analyze data, and presenting findings.

RESEARCH AND ORIGINAL CREATIVE WORK Publications

Refereed Journal Articles

Kreipke V, Allman BR , Ormsbee MJ, Kinsey AW, Hickner RC, Moffatt RJ. (2015). Impact of four weeks of a multi-ingredient performance supplement on muscular strength, body composition, and anabolic hormones in resistance-trained young men. The Journal of Strength and Conditioning Research, 29(12), 3453-3465.

Lennon-Edwards S, Allman BR , Schellhardt TA, Ferreira CR, Farquhar WB and Edwards DG. (2014). Lower potassium intake is associated with increased wave reflection in young healthy adults. Nutrition Journal, 13(39).

Allman BR , Biwer A, Maitland CG, DiFabio B, Coughlin E, Ormsbee MJ. (2018, In Press). The effects of short term beta-alanine supplementation on physical performance and quality of life in a Parkinson’s Disease: A pilot study. Journal of Dietary Supplements .

Invited Journal Articles

Ormsbee MJ, Carzoli JP, Klemp A, Allman BR , Zourdos MC, Kim JS, Panton LB. (2017, In Press). Efficacy of the Repetitions in Reserve-Based Rating of Perceived Exertion for the Bench Press in Experienced and Novice Benchers. The Journal of Strength and Conditioning Research .

Kinsey AW, Cappadona SR, Panton LB, Allman BR , Contreras RJ, Hickner RC, Ormsbee MJ. (2016). The Effect of Casein Protein Prior to Sleep on Fat Metabolism in Obese Men. Nutrients, 8(452), 1-15.

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Allman BR , Kreipke V, and Ormsbee MJ. (2015). What Else is in Your Supplement? A Review of the Effectiveness of the Supportive Ingredients in Multi-Ingredient Performance Supplements to Improve Strength, Power and Recovery. Strength and Conditioning Journal, 37(3), 54-69.

Nonrefereed Journal Articles

Allman BR. What’s the Harm in a Midnight Snack? Tallahassee Democrat, December 16, 2016.

Allman BR. Assess What to Eat Before a Workout. Tallahassee Democrat, December 5, 2016.

Allman BR. Does a Pound of Weight Loss Really Equal 3,500 Calories? Tallahassee Democrat, October, 2016.

Interviews and Media Coverage

Allman BR. Eating Before Bedtime? Jiu-Jitsu Magazine, May-June, 2017.

Allman BR. What’s the Harm in a Midnight Snack? Tallahassee Democrat, December 16, 2016.

Angle S. “Sweat or Skip? When to Crush Your Workout and When to Pass: Your Stomach Is Empty, or You Just Ate a Huge Meal.” SHAPE Magazine, September, 2016, p. 80.

FSU: Parkinson's and MS Patients May Find Help from Sports Supplement . WCTV.TV, August 9, 2013. Grants and Fellowships Grants and Fellowships Funded Allman BR . (2017). Fat Metabolism in Resistance-Trained Women in Response to Exercise and Protein Timing. Dissertation Research Grant funded by the Congress of Graduate Students (COGS), the Office of the Provost, and the Office of Research, Florida State University. Total Award $1,000. Allman BR . (2017). Fat Metabolism in Resistance-Trained Women in Response to Exercise and Protein Timing . Undergraduate Research Opportunity Program Materials Grant funded by the Center for Undergraduate Research and Academic Engagement, Florida State University. Total Award $250. Role: Co-Primary Investigator. Ormsbee MJ and Allman BR . (October 2016 – August 2016). Fat Metabolism in Resistance- Trained Women in Response to Exercise and Protein Timing. Funded by Fatigue Science. Total Award $7,000. Gift-in-kind; Role: Co-Primary Investigator.

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Ormsbee MJ and Allman BR . (October 2016 – August 2016). Fat Metabolism in Resistance- Trained Women in Response to Exercise and Protein Timing . Funded by FrieslandCampina. Total Award $50,000. Monetary and Gift-in-kind, Role: Co-Primary Investigator.

Grants and Fellowships Denied

Allman BR and Ormsbee MJ. (2016). Fat Metabolism in Resistance-Trained Women in Response to Exercise and Protein Timing. National Strength and Conditioning Association Dissertation Grant. Total Award up to $5,000 Presentations Refereed Presentations at Conferences For refereed presentations at conferences, 50.0%were national and 50.0% were local in scope. Allman, BR . (Presented 2018). The Effect of Protein Timing and Resistance Exercise on Lipolysis and Fat Oxidation in Resistance-Trained Women. The American College of Sports Medicine Annual Meeting 2018 . Conducted in Minneapolis, MN. Carzoli JP, Klemp A, Allman BR , Zourdos MC, Kim J, Panton LB, Ormsbee MJ. (Presented 2017). Efficiency of the Repetitions in Reserve-Based Rating of Perceived Exertion for the Bench Press in Experienced and Novice Benchers. Southeast American College of Sports Medicine’s Annual Meeting. Conducted in Greenville, SC. Allman BR , Maitland CG, Hagberg J, Ost EC, Ormsbee MJ. (Presented 2015). Blood lactate concentrations following isometric squats in multiple sclerosis patients. Southeast American College of Sports Medicine’s Annual Meeting . Conducted in Jacksonville, Florida. Kreipke VC, Allman BR , Kinsey AK, Hickner RC, Dubis GS, Tanner CJ, Moffatt RJ, Ormsbee MJ. (Presented 2015). The Impact of T+ TM Supplementation on Strength Performance in Power Athletes. National Strength and Conditioning Association’s Annual Meeting . Conducted in Las Vegas, NV. Allman, BR . (Presented 2013). The Relationship between Habitual Sodium and Potassium Intake on Vascular Function in Healthy, Older Adults. Thesis Defense . Conducted from University of Delaware.

Refereed Presentations at Symposia and Forum For refereed presentations at symposia and forum, 100.0% were local in scope. Allman, BR . (Presented 2018). The Effect of Protein Timing and Resistance Exercise on Lipolysis and Fat Oxidation in Resistance-Trained Women. College of Human Sciences Research Showcase . Presentation conducted at Florida State University, Tallahassee, Florida.

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Nonrefereed Presentations at Conferences For nonrefereed presentations at conferences, 66.7% were national and 33.3% were regional in scope. Allman, BR . (Presented 2011). The Physiological and Psychological Differences between an Aerobic Based Program and a Yoga Based Program on Breast Cancer Patients. 2011 McNair National Research Conference . Presentation conducted at the University of Maryland, College Park, Maryland. Allman, BR . (Presented 2010). The Physiological and Psychological Differences between an Aerobic Based Program and a Yoga Based Program on Breast Cancer Patients. The University at Buffalo’s 16 th Annual McNair Research Conference . Presentation conducted from The University at Buffalo, Buffalo, New York.

Nonrefereed Presentations at Symposia and Forum For nonrefereed presentations at symposia and forum, 100.0% were local in scope. Allman, BR . (Presented 2018). The Effect of Protein Timing and Resistance Exercise on Lipolysis and Fat Oxidation in Resistance-Trained Women. Ingestive Behavior Group Seminar . Presentation conducted at Florida State University, Tallahassee, Florida. Allman, BR . (Presented 2011). The Physiological and Psychological Differences between an Aerobic Based Program and a Yoga Based Program on Breast Cancer Patients. Undergraduate Scholars Forum . Presentation conducted at Indiana University of Pennsylvania, Indiana, Pennsylvania. Allman, BR . (Presented 2010). The Physiological and Psychological Differences between an Aerobic Based Program and a Yoga Based Program on Breast Cancer Patients. IUP Ronald E. McNair Post-Baccalaureate Achievement Program’s End of the Year Symposium . Presentation conducted from Indiana University of Pennsylvania, Indiana, Pennsylvania.

Nonrefereed Invited Presentations to Lay Audiences For nonrefereed invited presentations to lay audiences, 100.0% were local in scope. Allman, BR. (Presented 2017). Nutrition Basics. Orange Theory Fitness, North Location – Tallahassee, FL Allman, BR. (Presented 2017). Sports Nutrition Seminar Series: Nutrition for Athletes and Active Individuals. Zicro Academy – Tallahassee, FL. Allman, BR. (Presented 2016). Sport Nutrition Seminar Series: Nutrition for Weight Loss and Weight Cuts. Zicro Academy – Tallahassee, FL. Allman, BR. (Presented 2015). Element3 Church E3Fit Ministry: Supplements. Tallahassee, FL.

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Allman, BR. (Presented 2015). Element3 Church E3Fit Ministry: Fitness Nutrition. Tallahassee, FL. Allman, BR. (Presented 2015). Element3 Church E3Fit Ministry: Healthy Summer Recipes. Tallahassee, FL. Allman, BR. (Presented 2015). Nutrition Basics for Athletes. Seminar presentation to young swimmers. Tallahassee, FL. Allman, BR. (Presented 2015). Nutrition Basics for Athletes. Seminar presentation to young cheerleaders. Cheer Nation Athletics – Tallahassee, FL. Allman, BR. (Presented 2015). Fit for You Nutrition Seminar Series: Nutrition Myths. Success Athletic Training – Tallahassee, FL. Allman, BR. (Presented 2015). Fit for You Nutrition Seminar Series: Basic Nutrition. Success Athletic Training – Tallahassee, FL. Allman, BR. (Presented 2015). Fit for You Nutrition Seminar Series: Cooking, Baking and Eating Out Alternatives. Success Athletic Training – Tallahassee, FL. Allman, BR. (Presented 2015). Fit for You Nutrition Seminar Series: Fitness Nutrition. Success Athletic Training – Tallahassee, FL. Allman, BR. (Presented 2015). Fit for You Nutrition Seminar Series: Supplements. Success Athletic Training – Tallahassee, FL. Allman, BR. (Presented 2015). Fit for You Nutrition Seminar Series: Eating for Weight Loss. Success Athletic Training – Tallahassee, FL. Allman, BR. (Presented 2015). Nutrition Basics for Athletes. Seminar presentation to young swimmers. Tallahassee, FL.

ACADEMIC SERVICE AND LEADERSHIP

2017 to 2018 Reviewer, CHS Dissertation Award Program (DAP) Committee Florida State University, Tallahassee, FL 2017 to 2019 Student Representative, Nutrition, Metabolism, and Body Composition Special Interest Group National Strength and Conditioning Association 2016 to 2017 Undergraduate Research Opportunity Program (UROP) mentor, Kailee Hernandez Florida State University, Tallahassee, FL 2016 Grievance Committee Florida State University, Tallahassee, FL

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OTHER Health and Fitness Professional Experience 2016 to 2017 Group Fitness Instructor (Kickboxing) Zicro Academy, Tallahassee, FL 2015 to Present Group Fitness Instructor (High Intensity Interval) OrangeTheory Fitness,Tallahassee, FL 2013 to 2015 Personal Trainer, Group Exercise Instructor (High and Low Impact) Success Athletic Training, Tallahassee, FL 2013 Personal Trainer, Group Fitness Instructor (Low Impact, Sports Training) RAW Tennis and Fitness, Newark, DE 2012 to 2013 Group Fitness Instructor (Bootcamp, Heavy Bag Kickboxing, TRX) Wholistic Fitness, Lansdale, PA 2011 to 2012 Wellness Desk, Group Fitness Instructor (Les Mills Body Pump) North Penn YMCA, Lansdale, PA 2011 Personal Training and Boot Camp Intern Action Personal Training, Doyelstown, PA 2010 to 2011 Corporate Fitness Center Associate, Group Fitness Instructor Gorell Windows and Doors, Indiana, PA 2009 to 2011 Personal Trainer YMCA of Indiana County, Indiana, PA

Community Service and Leadership 2017 to 2018 Co-Chair, Teen Board Committee Junior League of Tallahassee, Tallahassee, FL 2016 to Present Big sister, Big Brothers Big Sisters of the Big Bend, Community-Based Program Tallahassee, FL 2016 to 2017 Co-Chair, PACE Committee Junior League of Tallahassee, Tallahassee, FL 2015 to Present Nutrition Seminar Speaker, Element3 Fit Ministry Element3 Church, Tallahassee, FL 2015 to Present Member, Junior League of Tallahassee Tallahassee, FL 2015 Coach, Girls on the Run Tallahassee, Desoto Trail Elementary Tallahassee, FL 2013 to Present Big sister, Big Brothers Big Sisters of the Big Bend, Mentor 2.0 Program Tallahassee, FL

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