THE RELATIONSHIP OF TESTOSTERONE, APPETITE, FOOD INTAKE ... · 2.3.1.3 Physiology of Testosterone...

THE RELATIONSHIP OF TESTOSTERONE, APPETITE, FOOD INTAKE AND EXERCISE IN MALE

ADOLESCENTS

by

Alexander Schwartz

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Nutritional Sciences

University of Toronto

© Copyright by Alexander Schwartz 2019

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THE RELATIONSHIP OF TESTOSTERONE, APPETITE, FOOD

INTAKE AND EXERCISE IN MALE ADOLESCENTS

Alexander Schwartz

Doctor of Philosophy

Graduate Department of Nutritional Sciences


2019

ABSTRACT

The hypothesis that testosterone has an interactive relationship with food intake and high

intensity exercise in adolescent males was explored in three experiments. Combined (Experiment

1) and separate (Experiment 2) effects of acute glucose and protein ingestion on testosterone

were observed. In Experiment 1, testosterone decreased by 18.6 ± 3.1% (p < 0.01) after one hour

of ingesting the combined glucose/protein beverage. In Experiment 2, testosterone decreased

acutely 20 min after both protein and glucose ingestion with the decrease continuing after protein

but not glucose after 65 minutes (p = 0.0382). No associations between testosterone and appetite

or food intake were found. Experiment 3 aimed to define the effect of sustained bouts of high

intensity exercise on testosterone. The effect of high intensity aerobic exercise (HIEX) on

testosterone in adolescent males was measured through two sessions of either: 1) three 10-minute

bouts of HIEX cycling at 75% VO2peak or 2) rest. Plasma testosterone concentrations increased

by 21.5 ± 13.6% after three 10-minute bouts of HIEX when compared to rest (p = 0.0215) but

testosterone was not correlated with appetite or appetite related hormones. Thus, the results of

this research show the novel effects of acute nutrient intake and high intensity aerobic exercise

on testosterone levels in adolescent males but no relationship with appetite and food intake

regulation was established. Therefore, this research does not support the hypothesis that

testosterone has a short-term interactive relationship with food intake.

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ACKNOWLEDGMENTS

I dedicate this thesis to my late parents who sacrificed their lives and left our birthplace and

home to seek a better future for my brother and I. I am forever grateful for the unconditional love

of both of them. My mother never lived to see me grow into the person I am today but would be

proud. My father, in spite of losing his own battle with cancer during the preparation of this

dissertation, has shown me strength, support and love throughout my graduate studies and made

me appreciate life more every day. Loss would never be painful if weren’t for love and

happiness.

I also dedicate this to my life partner Dr. Shannon Vettor who has been there for me during the

most difficult time in my life and given me strength when I felt my weakest. I feel fortunate

every day to be waking up and knowing your love, intelligence, support and compassion can get

me through anything. I’m excited to raise our soon to be born son Simon with you and begin this

new chapter in our lives together.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS ............................................................................................................. iii

TABLE OF CONTENTS ............................................................................................................... iv

LIST OF TABLES ....................................................................................................................... viii

LIST OF FIGURES ....................................................................................................................... ix

LIST OF ABBREVIATIONS ......................................................................................................... x

LIST OF PUBLICATIONS .......................................................................................................... xii

1 INTRODUCTION .................................................................................................................... 1

2 REVIEW OF LITERATURE ................................................................................................. 4

2.1 Introduction ......................................................................................................................... 5

2.2 Overweight and Obesity in Adolescence ............................................................................ 5

2.3 Puberty and Growth in Males and Normal Development ................................................... 6

2.3.1 Sex Hormones ....................................................................................................... 10

2.3.1.1 Physiology of Luteinizing Hormone and Role in Male Puberty ............ 10

2.3.1.2 Physiology of Follicle Stimulating Hormone and Role in Male

Puberty .................................................................................................... 11

2.3.1.3 Physiology of Testosterone and Role in Male Puberty .......................... 11

2.3.1.4 Physiology of Estradiol and Role in Male Puberty ................................ 16

2.3.1.5 Physiology of Sex Hormone Binding Globulin and Role in Male

Puberty .................................................................................................... 18

2.3.2 Measurement of Puberty in Males ........................................................................ 19

2.3.2.1 Clinical Staging ...................................................................................... 19

2.3.2.2 Biochemical Measures ............................................................................ 20

2.4 Hormonal Appetite and Food Intake Regulation .............................................................. 25

2.4.1 Central Appetite and Food Intake ......................................................................... 25

2.4.2 Ghrelin .................................................................................................................. 25

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2.4.3 Insulin.................................................................................................................... 26

2.4.4 Leptin .................................................................................................................... 26

2.4.5 Peptide YY ............................................................................................................ 27

2.4.6 Glucagon-like-peptide-1 ....................................................................................... 28

2.5 Appetite and Puberty ......................................................................................................... 28

2.5.1 Clinical Feeding Trials in Adolescents ................................................................. 28

2.5.2 Appetite Hormones and Puberty ........................................................................... 28

2.5.3 Appetite Hormones and Overweight/Obesity ....................................................... 30

2.5.4 Testosterone and Overweight/Obesity .................................................................. 31

2.5.5 Testosterone and Food Intake/Appetite ................................................................ 32

2.6 Testosterone and Exercise ................................................................................................. 35

2.6.1 Studies of testosterone change from exercise in males ......................................... 35

2.6.2 Physiology of testosterone in exercise .................................................................. 37

2.7 Summary and Research Rationale .................................................................................... 37

3 HYPOTHESES AND OBJECTIVES ................................................................................... 40

3.1 Overall Hypothesis and Objective .................................................................................... 40

3.2 Specific Hypotheses and Objectives ................................................................................. 40

4 ACUTE DECREASE IN SERUM TESTOSTERONE AFTER A MIXED GLUCOSE

AND PROTEIN BEVERAGE IN OBESE PERIPUBERTAL BOYS .............................. 42

4.1 Abstract ............................................................................................................................. 44

4.2 Introduction ....................................................................................................................... 45

4.3 Materials and Methods ...................................................................................................... 46

4.3.1 Subjects ................................................................................................................. 46

4.3.2 Protocol ................................................................................................................. 46

4.3.3 Biochemical Assays .............................................................................................. 48

4.3.4 Statistical Analyses ............................................................................................... 48

vi

4.4 Results ............................................................................................................................... 49

4.5 Discussion ......................................................................................................................... 50

4.6 Conclusions ....................................................................................................................... 52

5 ACUTE DECREASE IN PLASMA TESTOSTERONE AND APPETITE AFTER

EITHER GLUCOSE OR PROTEIN BEVERAGES IN ADOLESCENT MALES ........ 57

5.1 Abstract ............................................................................................................................. 59

5.2 Introduction ....................................................................................................................... 59


5.3.1 Subjects ................................................................................................................. 61

5.3.2 Protocol ................................................................................................................. 61



5.4 Results ............................................................................................................................... 66

5.5 Discussion ......................................................................................................................... 69

5.6 Conclusions ....................................................................................................................... 71

6 ACUTE DECREASE IN APPETITE IS UNRELATED TO INCREASE IN

PLASMA TESTOSTERONE DURING HIGH INTENSITY CYCLING IN

ADOLESCENT MALES ....................................................................................................... 78

6.1 Abstract ............................................................................................................................. 80

6.2 Introduction ....................................................................................................................... 81


6.3.1 Subjects ................................................................................................................. 82

6.3.2 Protocol ................................................................................................................. 82



6.4 Results ............................................................................................................................... 86

6.5 Discussion ......................................................................................................................... 87

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6.6 Conclusions ....................................................................................................................... 89

7 GENERAL DISCUSSION..................................................................................................... 93

7.1 Significance and Implications ........................................................................................... 97

7.2 Conclusion ........................................................................................................................ 98

8 FUTURE DIRECTIONS ....................................................................................................... 99

9 REFERENCES ..................................................................................................................... 102

10 APPENDICES ..................................................................................................................... 133

APPENDIX 1. Pubertal Assessment Flow Chart........................................................................ 134

APPENDIX 2. GCMS Method Development and Validation .................................................... 135

APPENDIX 3. Study 2 Sub-Analysis of Pre-Early Pubertal Subjects ....................................... 138

APPENDIX 4. Consent Forms.................................................................................................... 145

APPENDIX 5. Screening Questionnaires ................................................................................... 171

5.1. Telephone Screening Questionnaire ............................................................................... 172

5.2. Recruitment Screening Information Questionnaire......................................................... 174

5.3. Eating Habit Questionnaire ............................................................................................. 177

5.4. Physical Activity Questionnaire ...................................................................................... 179

5.5. Puberty Questionnaire ..................................................................................................... 180

APPENDIX 6. Study Day Questionnaires .................................................................................. 184

6.1. Baseline/Recent Food Intake Questionnaire ................................................................... 185

6.2. Motivation to Eat VAS.................................................................................................... 186

6.3. Physical Comfort VAS .................................................................................................... 187

6.4. Treatment and Test Palatability ...................................................................................... 188

6.5 Test Meal Record ............................................................................................................. 189

viii

LIST OF TABLES

Table 4.1. Subject Characteristics and change in hormone levels following the

glucose/protein beverage........................................................................................................53

Table 5.1. Participant characteristics ..................................................................................72

Table 5.2. Baseline levels of appetite- and sex-related hormones ......................................72

Table 5.3. Relationships between testosterone and luteinizing hormone with

dependent measures (Δ from baseline means)1 .....................................................................73

Table 6.1 Change in plasma testosterone, GLP-1, PYY, ghrelin, glucose and

insulin in response to rest and HIEX1 ...................................................................................90

Table 6.2 Effect of HIEX and rest on plasma testosterone levels between 0 and

65 minutes on each individual subject ..................................................................................91

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LIST OF FIGURES

Fig. 2.1. Onset of normal puberty in boys ...........................................................................7

Fig 2.2. Leydig cell testosterone synthesis ..........................................................................14

Fig 2.3. Proposed relationship between testosterone and food intake during

puberty ...................................................................................................................................34

Fig. 4.1. Association of fasting active ghrelin and fasting testosterone in males at

all pubertal stages ...................................................................................................................54

Fig 4.2. Testosterone level changes from baseline to 60 minutes after ingesting

the glucose/protein beverage in pre-early puberty and mid-late puberty...............................55

Fig 4.3. Association of changes in testosterone and luteinizing hormone levels in

boys following a glucose/protein beverage ............................................................................56

Fig. 5.1. Experimental protocol. ..........................................................................................73

Fig. 5.2. Effect of glucose and protein on plasma testosterone over time ...........................74

Fig. 5.3. Effect of glucose and protein on plasma luteinizing hormone ..............................74

Fig. 5.4. Effect of glucose and protein on plasma glucose ..................................................75

Fig. 5.5. Effect of glucose and protein on plasma insulin ....................................................75

Fig. 5.6. Effect of glucose and protein on plasma GLP-1 ....................................................76

Fig. 5.7. Effect of glucose and protein on plasma ghrelin ...................................................76

Fig. 5.8. Effect of glucose and protein on appetite score .....................................................77

Fig. 6.1. Experimental protocol ..........................................................................................92

Fig. 7.1. Summary of experimental results .........................................................................94

x

LIST OF ABBREVIATIONS

α-MSH α- Melanocyte Stimulating Hormone

5α-DHT 5α-Dihydrotestosterone

AgRP Agouti-Related Peptide

ANOVA Analysis of Variance

ARC Arcuate Nucleus

AUC Area Under the Curve

BMI Body Mass Index

CART Cocaine-and-Amphetamine-Regulated-Transcript

CLIA Chemiluminescent Assay

DHEA Dehydroepiandrosterone

DMH Dorsomedial Hypothalamus

ELISA Enzyme-Linked Immunosorbent Assay

FFM Fat-Free Mass

FI Food Intake

FSH Follicle Stimulating Hormone

GCMS Gas Chromatography Mass Spectrometry

GLP-1 Glucagon-Like-Peptide-1

GnRH Gonadotropin-Releasing Hormone

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HPGA Hypothalamic-Pituitary-Gonadal Axis

IA Immunoassay

IGF-1 Insulin-Like Growth Factor-1

Kcal Kilocalories

LatH Lateral Hypothalamus

LCMS Liquid Chromatography Mass Spectrometry

LDL Lipoprotein Cholesterol

LH Luteinizing Hormone

LHCGR Luteinizing Hormone/Chorionic Gonadotropic Receptor

MS Mass Spectrometry

NPY Neuropeptide Y

POMC Pro-Opiomelanocortin

PYY Peptide Tyrosine Tyrosine

RIA Radioimmunoassay

SEM Standard Error of the Mean

SHBG Sex Hormone Binding Globulin

VAS Visual Analogue Scale

VMH Ventromedial Hypothalamus

xii

List of Publications

Peer Reviewed Articles:

Chapter 4

Schwartz, A., Anderson, G.H., Patel, B.P., Vien, S., McCrindle, B.W., Hamilton, J. Acute

decrease in serum testosterone after a mixed glucose and protein beverage in obese peripubertal

boys. Clinical Endocrinology 2015; 83(3); 332-338.

Chapter 5

Schwartz, A., Hunschede, S., Lacombe, R.J. S., Chatterjee, D., Sánchez-Hernández, D., Kubant,

R., Bazinet, R., Hamilton, J.K., Anderson, G.H. Acute decrease in plasma testosterone and

appetite after either glucose or protein beverages in adolescent males. Clinical Endocrinology

2019; 91(2); 295-303

Publications Related to the Thesis:

Lockwood, J., Jeffery, A., Schwartz, A., Manlhiot, C., Schneiderman, J., McCrindle, B.,

Hamilton, J. Comparison of a Physical Activity Recall Questionnaire with Accelerometry in

Children and Adolescents with Obesity: a pilot study. Pediatric Obesity 2016; 12; E41-E45

Hunschede, S., Schwartz, A., Kubant, R., Thomas, S.G., Anderson, G.H. The role of IL-6 in

exercise-induced anorexia in normal-weight boys. Applied Physiology, Nutrition and

Metabolism 2018; 43; 979-987

Poirier, K.L., Totosy de Zepetnek, J.O., Bennett, L.J., Boateng, T., Brett, N.R., Schwartz, A.,

Luhovyy, B.L., Bellissimo, N. Effect of commercially available sugar-sweetened beverages on

subjective appetite and short-term food intake in boys. Nutrients. 2019; 11(2); 270.

Kucab, M., Boateng, T., Brett, N.R., Schwartz, A., Totosy de Zepetnek, J.O., Bellissimo, N.

Effects of eggs and egg components on cognitive performance, glycemic response, and

subjective appetite in children aged 9-14 years. Current Developments in Nutrition. 2019; 3(S1);

P14-017-19.

Published Abstracts:

Schwartz, A., Manlhiot, C., Malkin, A., Rafii, M., Pencharz, P., McCrindle, B., Hamilton, J.

The Relationship of Physical Activity Intensity on Plasma Glucose and Adiposity in

Adolescents. Canadian Journal of Diabetes (2013); 37: S261

Jeffery, A., Schwartz, A., Manlhiot, C., Schneiderman, J.E., McCrindle, B., Hamilton, J. The

Clinical Challenge of Accurate Physical Activity Measurment in Overweight Adolescents.

Canadian Journal of Diabetes (2013); 37: S261

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Jamil, M., Schwartz, A., Manlhiot, C., McCrindle, B., Hamilton, J. Evaluation of Body

Composition Assessment Techniques to Measure Adiposity in Adolescents. Canadian Journal

of Diabetes (2013); 37: S271

Hunschede, S., Schwartz, A., Kubant, R., Akilen, R., Thomas, S., Anderson, G.H. Mechanisms

of Appetite Suppression After High Intensity Exercise in Lean and Obese Boys. The FASEB

Journal (2016); 30; S1

Hammond, L.R., Plastina, S., Villaume, V., Husson, F., Brett, N.R., Al-Shammaa, M., Paterakis,

S., Schwartz, A., Lee, J.L., Rousseau, D., Bellissimo, N. Assessing the reproducibility and

validity of the Dedicated Ryerson University In-Vitro Digester to estimate glycemic response.

Applied Physiology, Nutrition, and Metabolism, 2019, 44(4): S1-S54

Hammond, L.R., Villaume, V., Brett, N.R., Brans, R., Plastina, S., Schwartz, A., Rousseau, D.,

Bellissimo, N. Effect of low carbohydrate-containing foods on in-vitro glucose release in the

Dedicated Ryerson University In-Vitro Digester. Applied Physiology, Nutrition, and

Metabolism, 2019, 44(4): S1-S54

Lee, J.L., Villaume, V., Amalraj, R., Brett, N.R, Schwartz, A. Rousseau, D., Bellissimo, N. A

comparison of glycemic response to white potato products in-vitro and in healthy older adults.

Applied Physiology, Nutrition, and Metabolism, 2019, 44(4): S1-S54

1

Chapter 1

INTRODUCTION

2

INTRODUCTION

During adolescent pubertal development, obtaining the necessary nutrients required for

healthy growth into adulthood is critical. Though most Canadian adolescents meet their daily

nutritional recommendations and ingest acceptable portions, 30% of Canadian children and

adolescents are overweight or obese [1]. Vast epidemiological evidence suggests that excess

adiposity in adolescence persists into adulthood; excess BMI and obesity during adolescence

are connected to mortality [2].

There is contradictory evidence in the literature with regards to excess adiposity and the onset

of male puberty. A Danish study indicated that lower vocal timbre was reached earlier in

obese adolescent males than normal-weight ones [3]. However, other population studies have

demonstrated that the ‘relative risk’ to stay in pre-pubertal status in males as defined by

pubertal staging is higher in those with a higher body mass index (BMI) [4]. Further studies

following monozygotic and dizygotic twin pairs from birth using peak growth velocity and

peak height concluded that growth during puberty is strictly regulated by genetics [5]. These

conflicting findings highlight the difficulty of measuring when puberty begins in males due to

inconsistencies in defining the parameters of pubertal onset.

Given the known inverse relationship of testosterone and excess body fat in adult males,

focusing on testosterone and obesity/growth in this population may be beneficial, [6, 7].

Testosterone is a steroid hormone that increases during onset of male puberty and shows a

strong correlation to growth and velocity during male puberty [8]. A recent paper

demonstrated that obesity and lower testosterone in adolescent males led to comorbidities in

adulthood such as insulin resistance [9].

Considering the importance of testosterone in adolescent male development and the intimate

relationship between obesity and food intake, there is a need for understanding how food

intake modifies testosterone levels in teenage males, as this interaction could play a role in

male development. There is scarcity of well-controlled research examining the acute effect of

food intake on testosterone, and none to date that examines the effect of foods on testosterone

in adolescent males. In adults, a decrease of postprandial testosterone was demonstrated

following an oral glucose tolerance test (OGTT) challenge [10], but there are no studies

utilizing protein in a similar manner to glucose and no studies exist to determine the effects of

food intake on testosterone in adolescent males.

3

In contrast to the effect of food intake on acute testosterone levels, there is compelling

evidence to show that testosterone levels increase after both acute bouts and habitual

engagement of physical activity in adult males [11-13]. However, limited evidence exists on

how these levels are affected in the adolescent population due to a myriad of methodological

issues such as the need to reach an intensity threshold in these activities in order to stimulate

peripheral testosterone production [14]. Teenagers in Canada consistently fall short of

obtaining the minimal requirement of physical activity. Approximately 45% of boys aged 12-

17 years fail to meet the intensity levels necessary for physiological benefits [15]. Higher

intensity levels of objectively measured physical activity have been shown to be inversely

related to adiposity in adolescents; those who engaged in at least 60 minutes of moderate-to-

vigorous physical activity per day were leaner in comparison to those who did not [16].

In view of the suspected interplay between testosterone, nutrition and physical activity in

teenage males, it is necessary to have research defining how they may interact to increase our

understanding of how nutrition and exercise affect development. Therefore, this dissertation

will investigate the effects of nutrients and exercise on testosterone and examine the

relationship testosterone has with appetite regulation in adolescent males.

4

Chapter 2

REVIEW OF LITERATURE

5

REVIEW OF LITERATURE

2.1 Introduction

The purpose of the review is to provide rationale for the research presented in this

dissertation. This review begins by addressing the prevalence and issues surrounding

adolescent obesity. The following section reviews pubertal development in males since this is

the specific population studied throughout this thesis. Hormonal regulation during puberty

will be discussed and, furthermore, specific pubertal abnormalities pertinent to this research

will be addressed. Section four deals specifically with hormonal regulation of appetite and

food intake and section five synthesizes the previous two sections by providing insight into

appetite during puberty. Section six discusses the relationship of physical activity/exercise

and testosterone and finally, a summary and research rationale will be provided in section

seven.

2.2 Overweight and Obesity in Adolescence

Obesity is a medical condition where excess body fat leads to negative health consequences

and reduced lifespan. It affects 1.9 billion adults [17] and approximately 10% of children

worldwide [18]. It is defined by body mass index (BMI), which is calculated by weight

divided by the square of the height. This measure has been shown to be a reasonable estimate

of adiposity in the general population, though begins to break down in active individuals with

higher levels of lean body mass [19]. In adults, the World Health organization defines a BMI

of >25kg/m2 as overweight and a BMI of >30kg/m2 as obese [20].

In children and adolescence, healthy weight and height varies with age and sex and is defined

using BMI percentile. For boys 2-20 years of age, a BMI percentile of 85th to less than the

95th percentile is defined as overweight, whereas anything above the 95th percentile is

considered to be obese [21]. In Canada it is estimated that nearly a third of 5 to 17-year olds

are overweight and 19.5% of boys are obese [22, 23]. Childhood and adolescent obesity often

persists into adulthood and is associated with chronic illnesses such as hypertension,

hyperlipidemia, cardiovascular disease, diabetes and fatty liver disease in addition to

premature mortality [24, 25].

6

In addition to metabolic disorders, excess adiposity has an impact on growth. Though

adolescent males with overweight or obesity show advanced bone age in comparison to

normal weight boys, their bone mineral content is still lower than what would be expected for

their body weight [26]. Furthermore, excess adiposity in boys with obesity is associated with

a delay in pubertal development [27], though paradoxically, the opposite holds true for

females [28]. This pubertal delay may be a consequence of decreased testosterone levels in

obese adolescent boys when compared to lean adolescents [29, 30]. The differences of

testosterone between boys who are obese vs. lean persists throughout puberty into adulthood

[9] and may result from diminished testicular function due to excess adiposity [31].

Understanding how excess body fat can affect normal pubertal development in males during

a critical period in male growth is an important and often overlooked facet of obesity

prevention.

2.3 Puberty and Growth in Males and Normal Development

Pubertal Onset

The onset of ‘true’ male puberty begins with the activation of the hypothalamic-pituitary-

gonadal axis (HPGA), when gonadotropin-releasing hormone (GnRH) pulsatility increases

after a quiescent period and stimulates the production of follicle-stimulating hormone (FSH)

and luteinizing hormone (LH) from the pituitary gland [32, 33] (Figure 2.1). These

gonadotropins signal spermatogenesis (FSH) and the production of testosterone (LH) in the

testes, resulting in secondary sexual characteristics associated with puberty in boys. Though

normal puberty is said to begin between nine and 14 years of age in boys [34], average age of

pubertal onset in males is difficult to define and varies as a result of a multitude of factors

including ethnicity, genetics, and nutritional status.

7

Figure 2.1- Onset of normal puberty in boys. Puberty begins with activation of the hypothalamic-pituitary-

gonadal axis (HPGA) stimulating gonadotropins (FSH & LH) to bind to target cells in the testicles resulting in

spermatogenesis and development on secondary sexual characteristics.

Though physiological signals from the HPGA indicate that puberty has initiated, the exact

mechanism responsible for pubertal onset remains to be elucidated. Puberty is dependent on

an interplay between genetic and environmental factors, and various studies indicate that in

well-nourished populations, genetics influences approximately 40-80% of the variation in the

timing of puberty in boys [35-38]. The summation of research for the genetic influence on

pubertal timing has been drawn primarily from single-gene disorders and indicates that

though there are many genes involved, the gene which encodes the kisspeptin protein

(KISS1) as well as its corresponding G-protein coupled receptor 54 (GPR54) and

gonadotropin-releasing hormone receptor (GnRH) play pivotal roles in pubertal timing.

KISS1/GPR54 signaling acts in the initiation of GnRH secretion at puberty by acting on the

GnRH neuron which expresses GPR54, resulting in the release of gonadotropins[39, 40].

Evidence from animal models provide support in that KISS1/GPR54 mRNA levels in

primates increase during puberty [41] and additionally, mice with GPR54 and KISS1

knockouts fail to enter puberty [42, 43].

8

Given that genetics do not explain all of the variation seen with pubertal timing,

environmental influences, therefore, must also play an important role in pubertal onset and

potentially mediating the genetic regulation. One of the strongest and most influential factors

from the environment may be nutrition. The association of overall diet quality with the timing

of puberty in both boys and girls has only been analyzed in the DONALD study [44]. Using

dietary records and a nutritional quality index, a multivariate analysis revealed that, after

adjustment for sex, maternal overweight, energy intake at baseline, and respective

anthropometry at baseline, a lower nutritional quality index in prepuberty was still associated

with an earlier age of pubertal onset; children with a lower diet quality according to their

nutritional quality index score entered puberty ∼0.4 y earlier than children with a higher diet

quality. Though there are several limitations with using dietary records, it does demonstrate

that diet can have an effect on pubertal timing. This is further evidenced when looking at

protein intake and protein quality. When compared to animal protein, a higher vegetable

protein intake at 5-6 years of age has been shown to delay puberty in both males and females

[45]. This is not surprising given that animal protein, in particular dairy, stimulates secretion

of insulin thereby decreasing IGF binding protein 1 and increasing availability of IGF-1 [46],

the significance of which with puberty is discussed further in section 2.5.2. Energy imbalance

(caloric surplus) resulting in obesity is an additional important nutritional consideration and

the relationship between obesity and puberty will be discussed in depth in section 2.5.4.

A variety of other physical and psychosocial stressors may also influence the timing of

puberty. Physical stressors such as high levels of exercise have been demonstrated to

attenuate HPGA activity in adult male runners [47, 48], but whether this translates to pre-

pubertal males remains unknown. Psychosocial factors such as parental absence, income

status and ethnicity have also been shown to affect entry into puberty for girls, but there is a

gap in evidence for similar findings in males [49, 50]. Some evidence shows similar trends in

boys entering puberty at different rates based on race/ethnicity, but the socioeconomic and

environmental factors surrounding different ethnicities remain to be addressed [51].

Pubertal Characteristics and Development

Measurement of puberty using the Tanner Stages is discussed in detail in section 2.3.3.1. The

first manifestation of puberty in boys is typically testicular enlargement (gonadarche).

9

Testicular volume, correlated with Tanner Stage, is one of the critical measures for

pubertal development of boys in the Tanner Scale and ranges between ≤ 3ml (stage I) to ≥

20ml (stage V). A testicular volume of > 3.0ml indicates entry into puberty and is an

important element of genital Tanner Stage 2 [52]. After approximately one year of testicular

enlargement, the size of the penis gradually begins increasing and developing adult

proportions [53], and this coincides with the development of varying levels of pubic hair

around the genitals, torso, legs, face and axillary regions. Pubic hair development (pubarche)

results from increased production of adrenal and gonadal dehydroepiandrosterone (DHEA) in

males which converts to dihydrotestosterone in hair follicles and dermal exocrine glands

stimulating hair growth [54, 55]. Additionally, the increased levels of DHEA play a major

role in adolescent acne [56, 57].

The increased levels of androgens in males affect other organs such as the larynx, muscles,

and bones. Abrupt changes in voice characteristics (deepening) have been noted to occur

between Tanner Stage 4 and 5 [58]. Under the influence of testosterone, males have

significant increases of bone and muscle size and strength concomitant with decreased

adiposity; 50% of adult body weight is gained during puberty and peak weight velocity

(9kg/y) occurs at the same time as peak height velocity, which ceases with epiphyseal closure

at approximately 17 years of age [59]. The bone growth of males is reflected by temporary

increases seen in the levels of alkaline phosphatase (corresponding to osteoblast activity) due

to the rapid bone growth which peaks at approximately Tanner Stage 3 [60, 61]. In addition

to increased bone mass, adult males have approximately 150% of the lean body mass of

females and nearly twice the number of myocytes [62].

Though puberty is associated with the ‘classical’ physiological changes mentioned above,

other important systems also go through modifications during adolescence. Hemoglobin

concentrations increase in adult males more so than in females as a result of increased

erythropoietin/red blood cell production from elevated testosterone production [63]. Total

cholesterol also increases, peaking during early puberty along with triglycerides and blood

pressure. However, low-density lipoprotein cholesterol (LDL) peaks during late puberty and

high-density lipoprotein cholesterol concentration remain somewhat constant, though there

may be a small decrease from early to mid-puberty [64].

Sequentially, male puberty usually begins with gonadarche [52] followed by the pubarche.

Though some ethnicities may not show pubarche this early, Tanner stage 3 pubarche occurs

10

approximately 1 to 1.5 years after initial testicular enlargement [59]. A pubertal growth

spurt occurs at approximately genital stage 3 and 4 at the same time as sperm development

(spermarche) [65]. Masculinization such as facial hair appearance and larynx enlargement

occurs at around genital stage 4 and male puberty is typically completed or near completion

by 16-18 years of age.

2.3.1 Sex Hormones

2.3.1.1 Physiology of Luteinizing Hormone and Role in Male Puberty

Luteinizing hormone (LH) is a gonadotropin and glycoprotein which is produced within the

gonadotropic cells of the anterior pituitary gland in the hypothalamus. The hypothalamus

releases GnRH, which controls the release of LH [33]. Throughout fetal development,

fluctuations in circulating LH can be observed with peak values occurring before 20 weeks

and decline again before birth [66-69]. Immediately after birth, neonates experience a surge

in LH due to the withdrawal of maternal derived estrogen, and this surge is larger in male

than female infants [70]. In male infants, LH peaks at approximately 1 to 3 months of age,

declining rapidly and reaching a nadir at 4-9 months [71, 72].The steroidogenic actions of LH

are exerted primarily through cAMP-mediated events in the gonads via the enzymes of the

Leydig cells [73, 74]. LH controls both the amount of cholesterol entering the Leydig cell and

the production of testosterone from cholesterol within the cell itself [75].

The GnRH pulse generator begins to mature approximately 1 to 2 years prior to the onset of

puberty and testosterone production, which leads to a gradual increase in frequency and

amplitude of LH pulsatile secretion that occurs nocturnally [76, 77]. As puberty progresses,

these secretions occur with increased frequency throughout the day and as part of the

hypothalamic-pituitary-gonadal axis (HPGA) [78], and in turn stimulate steroidogenesis in

the testes (see 2.3.1.3) [79]. In pubertal boys, daytime plasma LH values were above 0.3

IU/L, with periods of values of 0.1-0.3 IU/L in addition to short periods of undetectable

levels. Nocturnally, up to 4.7 IU/L were found in all boys with a higher frequency of pulses at

night [80].

11

2.3.1.2 Physiology of Follicle Stimulating Hormone and Role in

Male Puberty

Follicle-stimulating hormone (FSH), a gonadotropin and glycoprotein similar to LH, is

produced within the gonadotropic cells of the anterior pituitary gland in the hypothalamus.

The hypothalamus releases GnRH which controls the release of FSH [33]. The primary

function of FSH is gamete production during the fertile phase of life [81], and in males this is

manifested through spermatogenesis in the Sertoli cells of the gonads [82]. Mutations in the

FSH receptor have resulted in delayed sexual maturity and diminished testosterone

production and reductions in fertility [83-85]. In addition to fertility, FSH plays a role in

thyroid regulation [86]. FSH is important to normal pubertal development; deficiency of FSH

or the loss FSH receptor signaling decreases the number of Sertoli cells and diminishes

testicular size [87] in addition to reduced adult body size [88] and delayed puberty due to

hypogonadism [89].

2.3.1.3 Physiology of Testosterone and Role in Male Puberty

Testosterone is a steroid hormone and plays a pivotal role in male pubertal development.

Production of testosterone is initiated by secretion of LH from the pituitary gland, which

binds to the G protein-coupled LH/chorionic gonadotropic receptor (LHCGR) on the surface

of the Leydig cells situated on the testes [90]. The activation of the LHCGR stimulates

intracellular concentrations of cAMP in the Leydig cell resulting in the production of proteins

necessary for steroidgenesis. This activation subsequently leads to the conversion of

intracellular cholesterol into testosterone [74]. LH acts as a central regulatory factor within

the Leydig cells as it regulates both the amount of cholesterol entering the cell and the

production from cholesterol within the cell itself [75].

Cholesterol is incorporated into the cell from either receptor mediated endocytosis via LDL’s

or synthesized de-novo starting from acetyl CO-A and stored in cytoplasmic lipid droplets

[75]. Testosterone synthesis requires conversion of cholesterol into testosterone through five

different enzymatic steps which can take approximately 30 minutes [91] (Figure 2.2). The

cholesterol molecule’s side chains are shortened through C22 and C20 hydroxylases

(Cytochrome P450Scc) followed by cleavage of the bond between C20 and C22, leading to

production of pregnenolone within the mitochondria of the Leydig cell [92, 93].

12

Once pregnenolone is formed, it enters the endoplasmic reticulum of the Leydig cell and

follows either the Δ4 or Δ5 pathway depending on the location of the double bond in the

pregnenolone steroid. The Δ4 pathway involves dehydration and conversion to progesterone

and then into 17-hydroxy-progesterone whereas the Δ5 pathway is more common in humans

[94] and is converted to the intermediate 17-hydroxy-pregnenolone which in turn is converted

into dehydroepiandrosterone (DHEA) [95]. Both 17-hydroxy-progesterone from the Δ4

pathway and DHEA from the Δ5 pathway are used to produce the intermediate

androstenedione, which is then converted to testosterone [96].

During transport, testosterone is mainly bound to albumin or to sex hormone binding globulin

(SHBG) which is produced by hepatocytes. In normal men, only 2% of total testosterone

circulates freely (unbound), while 44% and 54% are bound to the binding proteins SHBG and

albumin, respectively [97, 98]. Though the binding affinity of testosterone to SHBG is much

higher (approximately 100 times greater), the greater concentration of albumin results in a

fairly close binding capacity between the two proteins [99]. It is important to note that the

movement of testosterone from the blood into cellular compartments such as the brain or

muscle is reduced by SHBG but not by albumin [100, 101]. Thus, unlike SHBG, albumin

allows for biological action of testosterone with the intra-cellular nuclear androgen receptor

(bioavailability) [99]. Since binding proteins cannot move across cellular membranes,

testosterone must enter target cells in the following ways, though the most relevant pathway

of these remains to be elucidated:

i) Diffusion- disassociation

Diffusion- disassociation of testosterone from binding proteins occurs primarily in the

capillaries near target cells as a product of the interaction of endothelial glycocalyx resulting

in structural modifications that allow testosterone to be set free from the extracellular space

into the intracellular space [102]. From here, testosterone can enter the cell freely across the

membrane through diffusion. It then binds to an intracellular receptor and enters the nucleus

to regulate gene expression [103, 104].

ii) Receptor-mediated endocytosis of steroid

While still bound to SHBG, testosterone binds to a cell importer protein called megalin [104,

105]. The entire hormone carrier is degraded within the cell, and the ligand testosterone

13

hormone is released into the free cytoplasm, where testosterone is broken apart from the

SHBG and can enter the nucleus [106].

iii) Non-genomic mechanisms of testosterone action

The established model of genomic testosterone transport and transcription dictates that

testosterone, like other steroid hormones, freely crosses the plasma membrane of the cell and

binds to specific receptor proteins within the cytoplasm. The bound steroid receptors bind to

homodimers and heterodimers in target gene promoters and activates/de-activates

transcription leading to protein synthesis [107-111]. This process may take up to several

hours after steroid exposure and takes additional time for messenger RNA to be translated

into proteins which then illicit signaled responses [112]. Steroid hormones can indeed affect

cellular processes in a non-genomic fashion. Evidence has shown that androgens are capable

of binding to receptors in and around plasma membranes which activate cell signaling

pathways that in turn elicit responses within seconds to minutes. Though much of this

research has been demonstrated in estrogens [113, 114], there is growing evidence for similar

effects from androgens [115].

A documented non-genomic effect of testosterone has been demonstrated with intracellular

calcium regulatory mechanisms. Testosterone has been shown to affect calcium

concentrations within neuroblastoma [116] and male osteoblast cells [117]. Furthermore,

testosterone has been demonstrated to induce both vasoconstriction and vasodilation within

various cardiovascular system structures, including the aorta and coronary arteries [118-123].

The relationship between testosterone and calcium regulation has also been proposed as a

beneficial mechanism for improvements in sport performance; testosterone induced skeletal

muscle sarcoplasmic reticulum calcium release may enhance the ability of explosive muscle

contraction [124]. Additionally, androgen receptors have also been found to activate second

messenger pathways independent of their transcriptional action within the cytoplasm [125]

though it remains unclear as to whether these secondary pathways ultimately lead to

transcriptional changes [126]. Regardless, the non-genomic action of testosterone provides

insight into the possibility of more precipitous action on target cells.

Within the reproductive system, testosterone provides neuroendocrine regulation of GnRH

through feedback. Specifically, high levels of testosterone inhibit LH secretion and vice-

versa, a function that results from changes of pituitary sensitivity to GnRH [127]. However,

14

some findings have suggested that there is a neuronal component for androgen

regulation of LH secretion [128-130] and though the specific sites are still unknown, this

does open the possibility that testosterone may exert its effects on other neuronal regions such

as the hypothalamus which, amongst other functions, regulates appetite and food intake

[131].

Figure 2.2- Leydig cell testosterone synthesis. Synthesis requires conversion from cholesterol through a

sequence of enzymatic steps. Cholesterol is incorporated into the cell by de novo synthesis from acetate or

through extracellular supply via the LDL receptor. Cholesterol molecule’s side chains are shortened leading to

production of pregnenolone within the mitochondria of the Leydig cell. Once formed, pregnenolone enters the

endoplasmic reticulum of the Leydig cell and follows either the Δ4 or Δ5 pathway dependent on the location of

the double bond in the pregnenolone steroid. The Δ5 pathway is more common in humans and is converted to

the intermediate 17-hydroxy-pregnenolone which in turn is converted into dehydroepiandrosterone (DHEA).

Both 17-hydroxy-progesterone from the Δ4 pathway and DHEA from the Δ5 pathway are used to produce the

intermediate androstenedione, which is then reduced to testosterone by 17β-hydroxysteroid dehydrogenase.

Testosterone and male puberty

Testosterone is crucial in male pubertal development and production increases during normal

male pubertal progression, exerting its effects on target organs critical for development.

15

Testosterone concentration correlates strongly with growth and growth velocity during

pubertal onset. A 24-hour steroid profile of 41 pubertal males showed that growth velocity

was significantly associated with morning testosterone levels (r = 0.88, p < 0.001) and when

dividing the males into pubertal groups based on testicular volume, those with volumes

indicating early puberty (3-6ml) showed the strongest association [8]. Given that peak growth

hormone secretion is pareallel to peak height velocity in males during mid-late puberty [132],

it could be hypothesized that testosterone is critical for initial pubertal growth but is less

important during later stages. The greater role of testosterone in early pubertal growth is

supported by evidence showing weak correlations between testosterone and growth velocity

during mid-late puberty [8]. Growth is primarily seen through the musculoskeletal system,

and though testosterone does not seem to elicit a direct effect on bone growth, aromatization

of testosterone into estradiol may be a critical step in bone development as estradiol plays an

important role in bone formation [133] further discussed in 2.3.1.4.

Muscular growth during puberty is likely mediated through androgen receptors, which are

located within both the muscle and the mesenchymal pluripotent muscle cells [134].

Testosterone binds to these receptors, translocating to the nucleus and binding to specific

DNA sequences within the muscle [135]. From there, testosterone induces muscle fibre

hypertrophy by acting at multiple steps in the pathways that regulate muscle growth.

Testosterone stimulates pluripotent mesenchymal stem cells and commits them to muscle

satellite cells, which increase in number [136, 137]. In addition, testosterone stimulates cell

replication and increases myoblast activity while simultaneously down-regulating muscle

protein degradation, which results in net gains of muscle tissue [138, 139]. Taken together, it

appears that testosterone increases muscle volume as a result of myoblasts fusing to existing

multinucleated muscle fibers called myotubes which then go through myogenic

differentiation [134]. Along with this increase in muscular size, male puberty is also

synonymous with secondary sexual characteristic development such as increased body hair

and deepening of the voice.

During puberty, sexual maturation includes the production of axillary and pubic hair, which

can be used as part of subjective measures of pubertal milestones [140]. Hair is produced by

hair follicles which go through regular growth cycles that shed and grow new hair; a cycle

hormonally regulated based on age/stage of development [141]. Androgens stimulate tiny

fine colourless hairs (vellus follicles) forming longer and thicker pigmented hairs, particularly

16

in the axilla and pubis [140, 142]. These follicles must pass through a full hair cycle so

that they may be shed and regenerate the lower follicle that will have more prominent

changes associated with pubic hair growth development such as that on the upper pubic

diamond, chest, and face [143]. During the latter stages of puberty, the male larynx goes

through extensive changes as a result of increased testosterone, lowering the frequency of

sound production; average adult male vocal frequency is 100 Hertz (Hz) compared to females

who average 213 Hz [58, 144]. The growth of the larynx is primarily seen through

enlargement of both the thyroid cartilage and vocal folds in addition to the lengthening of the

vocal tract from androgen stimulation [145, 146].

Other manifestations of puberty include changes to the skin and acne. The appearance of

substantial acne are noted during pubertal development in some males, and this is directly

related to enhanced sebaceous gland activity, which have all the necessary enzymes for

conversion of testosterone to 5α-dihydrotestosterone (5α-DHT) [147, 148]. Increased levels

of testosterone are converted to 5α-DHT and bind to the nuclear androgen receptors of the

sebaceous gland, where differentiation, proliferation and lipid synthesis occur [149].

2.3.1.4 Physiology of Estradiol and Role in Male Puberty

Estrogens are steroid hormones that are responsible for the development and regulation of

female reproductive system in addition to female secondary sex characteristics development.

There are three naturally occurring estrogens in human physiology; estrone, estradiol, and

estriol. Of these, estradiol is the predominant hormone in terms of absolute levels and

estrogenic activity. Estradiol in males is synthesized and secreted by testes and peripherally

in the adrenal gland, where estradiol is converted from prehormones [150]. Additionally,

desulphuration of conjugated estrogens in the liver provides added estradiol in males; estrone

sulphate circulates in the blood and is converted by the liver into estrogen [151].

Synthesis of estradiol results from a side chain of cholesterol being cleaved from

pregnenolone, which ultimately converts to androstenedione and testosterone. From there,

androstenedione and testosterone are converted by specific isoenzymes and aromatase in both

the gonads and peripheral tissues to convert to estradiol [152, 153].

17

Similarly to testosterone, 98% of plasma estradiol is bound to SHBG or albumin, and the

remaining 2% is unbound [154]. Estradiol binds to an estrogen receptor, which changes

shape, dimerizes, and interacts with DNA [155]. Though estrogen-receptor-α is the more

common receptor and is expressed in the breast, uterus, cervix, and vagina as well as other

organs, estrogen-receptor-β is more pertinent to males and can be primarily expressed in the

prostate, testis, spleen, hypothalamus, and thymus [156]. Furthermore, sites such as the brain,

bone, and vascular system have direct non-genomic estrogen action in the membrane [157].

There are four main mechanisms of estrogen signaling as described by Hall et al.[155]:

i) Classical ligand-dependent

Estradiol/estrogen receptor complexes (E2-ER) bind to DNA response elements (EREs) in

target promoters, which results in an up or down-regulation of gene transcription followed by

a response from the tissue.

ii) Ligand-independent

In the absence of estradiol, estrogen receptor function can be modulated by extracellular

signals. Growth factors of cyclic adenosine monophosphate activate intracellular kinase

pathways, which lead to phosphorylation and activation of the estrogen receptor at ERE-

containing promoters without ligand/complex as described in the aforementioned classical

signal.

iii) ERE Independent

E2-ER complexes alter the transcription of genes without ERE through association with other

DNA-bound transcription factors.

iv) Cell-Surface Signaling

Unlike the other three mechanisms, this is a non-genomic signaling pathway. The three

genomic pathways lead to mRNA that contain polysomes which allow for synthesis of

proteins that give way to tissue responses. In contrast, cell-surface signaling involves

estradiol activating a membrane-associated binding site that generates rapid tissue responses

such as those seen in the brain, bone and vascular system [157].

Estradiol and male puberty

18

Morning values of 17β-estradiol were positively associated with both morning

testosterone values and growth velocity in pubertal males [8]. Aromatase, an enzyme

responsible for conversion of androgens into estrogens, was found to be deficient in some

males and in addition to nearly undetectable estrogen levels, the individuals had low bone

mass, unfused epiphyses, increased gonadotropins, and a poor lipid profile. These males also

showed abnormal growth patterns during puberty and a larger than average body length due

to unfused epiphyses [133, 158, 159]. This role of estradiol with epiphyseal fusion may in

part also explain the rapid and earlier maturation of epiphyseal closure in girls [160].

One of the more critical functions of estradiol in males is bone growth and development.

Previous work has shown that idiopathic osteoporosis in males is related to low peripheral

estradiol levels [161] and bone acquisition in younger men is related to total and bioavailable

estrogen levels, but not testosterone [162]. An interventional study in elderly men attempted

to determine the effects of estradiol and testosterone on male bone formation. The males were

rendered hypogonadal through administration of a GnRH agonist and an aromatase inhibitor

which was followed by supplementation of testosterone, estrogen or a combination of both.

Bone resorption was measured by urinary excretion of deoxypridinoline (Dpd) and N-

telopeptide (NTx). Estradiol decreased Dpd and NTx excretion and, in comparison to

testosterone, was more effective in increasing bone formation markers [163]. The authors

concluded that although both sex hormones are important to bone formation, estrogen is the

dominant sex hormone regulating bone resorption.

Estradiol may also play a role in reproduction and spermatogenesis as it aids in regulating the

reabsorption of luminal fluid in the epididymis [164]. Research in animal models has

demonstrated that there was excessive fluid accumulation in the seminiferous tubules of male

mice with estrogen-receptor-β knockout, which consequently led to tubular atrophy and

ultimately infertility [165].

2.3.1.5 Physiology of Sex Hormone Binding Globulin and Role in Male

Puberty

Sex hormone binding globulin (SHBG) is a liver-derived glycoprotein that binds to sex

hormones, specifically testosterone, estradiol and DHT [166]. SHBG is increased by thyroid

hormone and estrogen, and it is decreased by androgens, growth hormone, insulin and

glucocorticoids [154]. SHBG levels are unrelated to meals or the time of day [167]. Once

19

testosterone enters circulation into the blood, nearly half is tightly bound to SHBG and

is considered to be unavailable for biological action on target cells [168], whereas albumin-

bound and free testosterone can enter target cells through passive diffusion (2.3.1.3). Plasma

SHBG is an important determinant in the amount of testosterone that can freely enter cells;

small increases in plasma SHBG reduce the amount of circulating free testosterone [98].

There is a substantial rise in SHBG from birth to early childhood in both males and females,

and during sexual maturation, a decline that is much more pronounced in males occurs due to

high androgen levels [169]. Peripubertal obesity is associated with insulin-induced reductions

in SHBG, which increases bioavailability of sex steroids including E2 [170, 171]. This

reduction in SHBG leads to increase proportions of free testosterone resulting in increased

clearance of testosterone from the body and a fall in production of testosterone and LH via

the negative feedback mechanism [172].

2.3.2 Measurement of Puberty in Males

2.3.2.1 Clinical Staging

In a clinical setting, the Tanner Scale is used to assess pubertal maturity. The scale

determines physical development using primary and secondary sex characteristics such as the

testicular volume and pubic hair development in males and is based on a scale of 1 to 5 [140].

For testicular volume measurement, a Prader orchidometer is used to compare with palpated

volume of the testicles, and this method is a valid and reliable measure of puberty [173, 174].

Tanner et al. [175] initially defined that the onset of male puberty is associated with a bone

age (taken with the mean of several small bones of the hand and wrist) of approximately 13

years in boys. Though there is a fair amount of individual variation in addition to variation

with different ethnicities, the approximate age of pubertal onset in boys is between 9 and 14

years of age [176]. An assessment using additional hormonal measures provides greater

accuracy in determining pubertal stage. Hormonal measurements indicative of male pubertal

progression (testosterone and LH), levels are measured fasting and in the morning since a

diurnal and seasonal variation in the circulating testosterone levels exists [177].

20

2.3.2.2 Biochemical Measures

Testosterone

Due to the limitations of clinical staging, assessment of puberty can be further evaluated with

the measurement of sex hormones pertinent to pubertal development. In adolescent males, the

primary pubertal/sex hormones of interest are testosterone, SHBG, LH, and FSH. Of these,

testosterone presents the greatest challenge in measurement due to methodological

discrepancies and the lack of a recognized reference standard, particularly for low

concentrations of testosterone seen in early adolescent males as well as high concentrations

of interfering cross-reacting steroids [178]. These irregularities have ultimately led to a

consensus statement [179] made by the Endocrine Society and the Center for Disease Control

providing recommendations to improve consistency with testosterone measures in a clinical

setting.

The most common methods of measuring testosterone are mass spectrometry (MS), and

immunoassays (IA), including radioimmunoassay (RIA). MS works under the principle of a

mass-to-charge ratio which is a dimensionless quantity formed by dividing the mass number

of an ion by its charge number [180]. There are two forms of MS; gas chromatography

(GCMS) and liquid chromatography (LCMS). To measure testosterone, a deuterium-labeled

internal standard must be added to the sample since the stable isotope allows for precise

measurement given the multiple steps involved in MS. Testosterone is then isolated and

dried, and the sample is charged with electrons so that the molecules of testosterone are

ionized. These ions are separated/deflected according to their mass-to-charge ratio by an

electric or magnetic field; ions with the same mass-to-charge ratio will undergo the same

amount of deflection, therefore, all the testosterone ions will aggregate together. A detector

looking at charged particles of testosterone then identifies these ions [181].

An alternative to MS is measuring serum testosterone using IA, one form of which is RIA.

This method has been in practice since the late sixties, with very few methodological changes

since that time [182]. A known quantity of testosterone (antigen) is radioactively labeled and

mixed with a known amount of antibody. The serum sample to be measured is then added

and the antigen from this serum and competes with the radiolabeled antigen for binding sites.

The more unlabeled antigen from the serum, the more it binds to the antibody and displaces

21

the radiolabeled antigen. The bound antigens are separated from the unbound ones, and a

gamma counter is used to measure the radioactivity of the unbound antigens [183, 184].

Due to the complicated process of RIA, the most popular alternatives are non-radioactive

labeled tracer IA’s such as chemiluminescent assay (CLIA) and enzyme-linked

immunosorbent assay (ELISA). Much like the RIA, the known antigen is labeled and

competes with the unknown serum antigen. In the ELISA, the tracer is represented by the

antigen coupled by an enzyme that initiates a colorimetric reaction [185]. Specific to the

testosterone antigen, colour density is inversely proportional to the amount of testosterone

present and is read by a spectrophotometer. Similarly, the CLIA assay uses a competitive

antigen labeled with a tag that emits light when bound. The sensitivity and reliability of this

method is similar to RIA and much simpler to execute [186].

One of the fundamental challenges for researchers has been finding an accurate, sensitive and

reliable measure of testosterone. Quite a few studies have compared IA vs. MS using

relatively large sample sizes. Wang et. al. [187] compared both automated platform IA and

RIA against LC/MS as the gold standard using samples from both healthy and hypogonadal

adult males. Though there was a tendency to either overestimate or underestimate

testosterone in the various IA and RIA analyses, they were generally acceptable for assessing

testosterone values for adult males and identifying hypogonadism. Due to the lack of

precision and accuracy of IA and RIA at lower serum concentrations of testosterone, they

may not be as effective in measuring children and females. Similarly, other papers comparing

MS to IA have concluded that IA tends to overestimate serum testosterone values in women

and children [188, 189] where low values are expected. This overestimation may be due to

the cross-reactivity with DHEA sulfate [190], though the exact cause remains to be

determined. An analysis of first and second-generation testosterone IA’s compared against

LC/MS indicated that all IA platforms showed good correlation with LC/MS when the

testosterone concentration was >4.0nmol/l, but performed poorly when concentrations were

below this value [191]. Tandem MS has consistently shown improved accuracy, requires low

sample volume and is far more rapid and robust when compared to all other methods [192].

LH & FSH

22

Since an important marker of pubertal onset is pituitary gonadotropin activity, it is

necessary to measure LH and FSH in conjunction with testosterone in males. Based on the

biological profile of these glycoproteins, IA’s are the most common ways to measure LH

[193-195]. Much like testosterone, various methods of IA’s have been used to measure

gonadotropins since the 60’s. RIA’s for LH and FSH function similarly to that of testosterone

where a radiolabeled LH or FSH preparation and serum LH or FSH compete for binding of

the gonadotropin specific antibody [60]. Some of the limitations of RIA’s include the

potential health hazards of dealing with radioactive tracers in addition to a short shelf life and

high cost of equipment. Furthermore, there may indeed be issues with RIA capturing an

accurate measure of gonadotropin molecules in low serum levels [196].

A non-competitive IA based on the sandwich technique is the most common form of

measuring gonadotropins. This type of technique involves the use of two antibodies which

bind to different sites on the antigen (gonadotropin). Two antibodies are added before and

after the antigen (capture antibody and detection antibody respectively), and they bind to the

antigen at different parts of the antigen (epitopes). The antigen is therefore ‘sandwiched’

between two antibodies, and as the antigen concentration increases, the amount of detection

antibody increases, leading to a higher measured response [197]. In Immunofluorometric

(IFIA) and CLIA’s, antibodies are labeled by linking to another chemical; a fluorescent label

or a luminescent label allow for ease of identifying the quantity of the gonadotropins [194].

Similar to IFIA and CLIA’s, ELISA’s use a sandwich principle that was developed for the

gonadotropins and was reproducible, highly specific and sensitive though showed limitations

of measurement in prepubertal children [198]. Compared to RIA, ELISA techniques have

several advantages, which include eliminating the problems associated with the use of

radioisotopes, higher sensitivity, low inter‐assay variation and simplicity/less working time

per sample [194]. Since their initial development, the kits have improved with sensitivities of

measuring serum gonadotropins [199], FSH and LH ten times higher than previous kits

making it possible to obtain measurements at concentrations as low as 0.02 IU/L [200].

Pubertal Assessment

Some of the challenges of accurately assessing boys using self-staging may include

variability of pubic hair growth in different ethnicities, over or underestimating genital size,

and discordance between physical characteristics and gonadotropin production. Currently,

23

part of an accepted method of pubertal assessment in males is utilizing measurements of

testicular volume [59], performed by a clinical expert in this technique. However, this may

not be an accurate proxy in some males, including those that are obese who have shown

lower testosterone values for similar volumes to that of normal weight teenagers [31].

Similarly, in many pediatric studies, genital exams may reduce participant willingness to

consent and raises some concerns regarding feasibility. The main limitation with boys self-

staging is that it is problematic when compared to both trained pediatric endocrinologists

[31], and to actual hormonal derived pubertal assessment methods [201]; 39% of boys

incorrectly rated their pubic stage when compared to a trained endocrinologist [202].

Creating a Flow Chart for Pubertal Assessment

To evaluate pubertal stages based on a combination of hormonal measures and Tanner

staging in this study, various lab values have been estimated from multiple sources of

pubertal assessment literature [203-211]. A flow chart synthesizing this information for

guiding pubertal assessment using the hormonal measures of interest in conjunction with

testicular volume has been developed and presented in Appendix Figure A2.1. One of the

issues relevant when reviewing the literature relates to the sensitivity and cross-reactivity of

hormonal assays performed. For example, typical analyses that utilize direct assay

measurements (ELISA or RIA) have limited accuracy at both low and high testosterone

concentrations and lack standardization. Assays using a tandem MS or multiple-step IA

analyzers show improved sensitivity and specificity and are preferred for these measures in

boys, especially those with lower circulating levels of testosterone [212, 213]. Similarly,

gonadotropin assays have become much more sensitive with lower levels of detection able to

discriminate prepubertal from early pubertal boys. Thus, the results derived from these

studies were chosen based on use of sensitive assay measurements and MS methods were

weighted favourably due to the greater precision needed to detect low pre-pubertal hormonal

levels. In addition, the number of subjects measured, and method of Tanner staging for

comparison (i.e. self-staging versus clinician conducted testicular exam) was also taken into

consideration. Using the cumulative data to develop three tiers of analysis (LH, testosterone

& testicular volume), three proposed stages of puberty have been developed: i) Pre-Early

Puberty, ii) Early to Mid-Puberty and iii) Mid-Late/Post Puberty.

In a study by Chada et al. [204], 78 boys in various stages of puberty and pre-puberty were

assessed through genital Tanner staging while venous blood samples were taken for

24

measurement of testosterone, inhibin B, FSH and LH using ELISA. Based on this study,

all boys with a LH value of ≤ 0.12 IU/L were pre-pubertal (Tanner stage 1). However, there

was a considerable overlap of LH values between Tanner stage 1 and 2. LH values that fall

between 0.12 and 0.44 IU/L are not specific enough and would indicate that additional

measures such as testosterone and testicular volume need to be considered. There is an

overlap between LH values of 1.05 and 2.39 IU/L during Tanner stage 3 (0.45-2.39 IU/L) and

that of 4 and 5 (1.05-5.54 IU/L). Thus, incorporating the work of Resende et al. [210] where

59 pre-pubertal and pubertal males were divided by Tanner stage using a clinician to assess

testicular volume and immunochemiluminometric and immunofluorometric assays, a Tanner

stage 4 LH range was reported as 0.3-1.6 IU/L. Using the upper end of both Tanner stage 3

from Chada et al. and stage 4 from Resende et al, boys who have a LH value between 1.6 to

2.4 IU/L may be in either Tanner 3 or 4 stage.

Testosterone cannot be used as the primary surrogate of Tanner staging due to its high

variability throughout puberty but can be used in combination with LH values (falling within

the range of 0.12 - < 0.44 IU/L). Based on the work of Mouritsen et al. [214] and the

established lower range of late pubertal males ages 14-16 and 16-19 from Konforte et al.

[215], testosterone values that were shown to be ≤ 32-36 ng/dl would indicate the subject is

pre-pubertal, thus ≤ 35 ng/dl is proposed as the cut off. If testosterone values are above this

threshold (35-150 ng/dl), then a tertiary measure of testicular volume of < 3.0 ml could be

included to define pre-early pubertal status and 3.0-11.0 ml to define early-mid puberty.

However, if the testosterone level is 410 ng/dl as determined by Kulle et al. [208] using

LCMS, this will indicate that the boy is indeed in late puberty (Tanner 4 or 5). More recent

data [214] utilizing a combination of pubic hair growth, testicular size and LCMS

testosterone measures have indicated that the median testosterone concentration of

testosterone increased twofold every 6 months from 12 months before onset of testicular

growth (≥ 3ml) and that 6 months after the onset, testosterone ranged from 140-261 ng/dl.

Based on the summation of this data, Mid-Late/post-puberty (Tanner stages 4 & 5) can be

defined as an LH value of ≥ 2.4 IU/L, a testosterone value >150ng/dl and testicular volume >

12ml, as this seems to be both the lower end of adult testicular volume range and, in addition,

indicative of maximum growth velocity in pubertal males [216].

25

2.4 Hormonal Appetite and Food Intake Regulation

The control of food intake (FI) and appetite comprises both short-term mechanisms and long-

term signals of energy balance that involves central and peripheral systems working in

concert with dietary components and FI [217]. An example of this includes the reduction of

subsequent FI. Short-term control of FI occurs after a coordinated post-prandial response

involving mechanoreceptors in the gut, nutrient concentration changes in the blood and

release of post-prandial anorectic gut hormones [218]. This section will briefly discuss the

physiology and mechanisms of some of the important central and peripheral hormones and

systems involved in regulating appetite and FI.

2.4.1 Central Appetite and Food Intake

The arcuate nucleus (ARC) within the hypothalamus is critical in appetite regulation. The

ARC is situated close to the median eminence where an incomplete blood-brain barrier may

allow for peripheral signals to enter the central nervous system [219]. Situated within the

ARC are a group of neurons responsible for control of FI whose axons project from the ARC

to other hypothalamic nuclei. Neuropeptide Y (NPY) and agouti-related peptide (AgRP)

enhance FI when activated (orexigenic). In contrast to these, pro-opiomelanocortin (POMC)

and cocaine-and amphetamine-regulated transcript (CART) act to coordinate a decrease in

appetite and FI. POMC is the precursor to α- melanocyte stimulating hormone (α-MSH)

which signals to decrease FI via the melanocortin receptors [131, 220].

The brainstem dorsal vagal complex (DVC) also plays an important role in appetite/FI

regulation. The DVC plays a critical role in communication between the peripheral signals of

FI (see 2.4.2 – 2.4.11) and the aforementioned hypothalamic nuclei [221]. This

communication may be possible due to both an absence of a complete blood-brain barrier

within the area postrema of the DVC in addition to neural projections from the brainstem to

the hypothalamus [222]. Furthermore, vagal nerve afferents have been shown to carry

information from the gut directly to the nucleus of the tractus solitarius within the DVC

[223].

2.4.2 Ghrelin

Ghrelin is a 28 amino-acid-long gut peptide derived primarily from the stomach in addition to

the proximal small intestine and pituitary gland. It activates NPY neurons in the ARC

26

stimulating appetite in addition to being a growth hormone secretagogue [224]. Ghrelin

has been shown to significantly increase appetite and FI; it increases before a meal and

decreases after a meal [225-227]. During fasting, ghrelin mRNA expression increases, and

peptide levels of ghrelin return to baseline after feeding [228]. In rodents, central ghrelin

administration induces FI [229] and peripheral ghrelin administration in healthy lean and

obese individuals increases FI, induces the sensation of hunger and increases neural

activation of specific regions of the brain associated with reward [227]. Furthermore, ghrelin

decreases insulin secretion and increases gastric acid secretion [230] in addition to

stimulating gastric motility and increases feeding frequency with no effect on meal size [225,

229, 231, 232].

2.4.3 Insulin

Insulin is a peptide hormone produced by the pancreatic beta cells, which regulates

metabolism of carbohydrates and fats. Animal research has provided much insight into the

effects of insulin on appetite. In rat models, central infusion of insulin resulted in decreased

intake of food [233, 234], and conversely, infused antisense oligodeoxynucleotide (generated

to prevent insulin receptor proteins from developing in the cerebral ventricle of the rat brain)

resulted in hyperphagia and subsequent fat mass [235]. In adult humans, a meta-analysis

performed with 92 normal weight and 44 overweight subjects showed that blood glucose

might be an important satiety signal of short-term appetite regulation in normal-weight but

not overweight subjects [236].

2.4.4 Leptin

Leptin, a product of the human leptin gene, is composed of 167-amino acids and is secreted

and stored primarily in white adipose tissue, showing a positive correlation with white

adipose tissue volume [237, 238]. Leptin acts on specific (ObRs) receptors in the brain

located in nuclei of the arcuate nucleus (ARC), ventromedial (VMH), dorsomedial (DMH),

and lateral (LatH) hypothalamus [239]. Several signal transduction pathways critical for

regulation of energy homeostasis, FI, and glucose homeostasis are activated which includes

Janus Kinase-Signal Transducer and Activator of Transcription-3 (JAK-STAT3) and

Phosphatidylinositol 3-Kinase (PI3K) [240, 241].

27

In humans, the most significant roles of leptin are regulation of energy homeostasis and

metabolism [242]. Leptin circulates throughout the body and serves as a measure of energy

reserves in adipose tissue to guide central nervous system regulation of FI. Within the

hypothalamus, leptin binds to ObRb receptors and inhibits a neural circuit that inhibits the

appetite-suppressing POMC while activating the appetite-inducing NPY neuropeptides [131].

In addition to hypothalamic regulation of FI and appetite, leptin is also involved in the

mesolimbic dopamine system, critical for motivation and reward of feeding [243].

2.4.5 Peptide YY

Peptide YY (PYY) is an anorexigenic 36 amino-acid peptide that is secreted by the L-cells of

the distal gastrointestinal tract primarily from the ileum in addition to the colon and rectum

[244]. The two endogenous forms, PYY1-36, and PYY 3-36 are released postprandially from L-

cells after FI in humans and decrease FI [245, 246]. PYY 3-36 is further produced by cleavage

of the Tyr-Pro amino terminal residues of PYY1-36 by the dipeptidyl peptidase IV enzyme

(DPP-IV). This allows for hypothalamic Y2 receptor selectivity [247]. PYY1-36 is

predominantly circulated in the fasted state whereas PYY 3-36 largely circulates post-

prandially [248].

Following FI, there is a marked increase of PYY 3-36 within 15 minutes, reaching a post-

prandial peak at approximately 90 minutes. This peak may be elevated for up to 6 hours [245,

249]. The rapid post-meal increase of PYY 3-36 indicates that initially, the release may be

under neural control as the meal has not likely yet reached the ileum of the distal small

intestine. More PYY 3-36 is released thereafter when nutrients appear in the distal small

intestine [250].

High circulating levels of PYY 3-36 have been demonstrated in individuals with anorexia

nervosa, in contrast to obese patients who have frequently shown low levels and a blunted

postprandial response [251, 252]. Furthermore, in humans, peripheral administration of PYY

3-36 has been shown to reduce FI by diminishing rewarding aspects of food in the brain [253,

254].

28

2.4.6 Glucagon-like-peptide-1

GLP-1 is a peptide produced and released postprandially by the intestinal L-cells (like PYY)

[255] and acts on the hypothalamus [256] to decrease FI in both normal and overweight

humans in addition to acting as an incretin and stimulating approximately 60% of the

postprandial insulin secretion [257]. When stimulated, GLP-1 receptors, found in the central

nervous system, have been shown to directly reduce both hunger and FI [258, 259], though

the effect of GLP-1 on the expression of appetite mediating peptides AgRP, POMC, CART,

and NPY is not completely known [260]. Secretion of GLP-1 is biphasic; an early peak

occurs within minutes of FI as a result of neurohormonal reflex and direct nutrient exposure

of proximal L-cells, a later peak from nutrient stimulation of distal L-cells [244]. In addition

to central mediation of appetite and FI, GLP-1 plays an inhibitory role in gastric emptying

through the ileal break which regulates the passage of nutrients [261-263].

2.5 Appetite and Puberty

2.5.1 Clinical Feeding Trials in Adolescents

Anecdotally it has been reported that boys ingest a high number of calories during puberty. In

a study exploring short-term FI, Shomaker et al. [264] tested 103 males and 101 females

between 8 and 17 across the weight spectrum by providing two identical lunch buffet meals

following a standardized breakfast. Males ate more than females at all stages of puberty

(including pre-pubertal) and FI increased across puberty. However, when the researchers

adjusted for body composition (fat mass and fat-free mass), height, overweight status, and

meal instruction, the main effect of sex remained significant. Since sex was shown to be an

important contributor to larger FI in males, the male sex hormone testosterone may contribute

to differences in FI compared to females.

2.5.2 Appetite Hormones and Puberty

A paucity of data exists demonstrating the relationships of appetite hormones with sex

hormones and male puberty. Pubertal status affects appetite and appetite hormone secretion

[265-267]. During puberty, plasma ghrelin decreases throughout developmental stages

assessed by Tanner staging [268]. It remains to be seen if ghrelin is indeed a determining

29

factor in FI behaviour. Though fasting total and deacylated ghrelin were lower in

pubertal boys who are overweight in comparison to boys who are normal-weight in similar

pubertal stages, meal induced suppression of ghrelin was blunted in this population [269].

Furthermore, mean ghrelin concentrations of boys in later stages of puberty were lower in

comparison to those in Tanner stage 1, and fasting blood concentrations of acylated and non-

acylated ghrelin are higher in pre-pubertal boys when compared to those in puberty [270,

271].

Anorexigenic peptides have also shown relationships with male pubertal development. In

adolescent boys, those who were overweight tended to have lower fasting and postprandial

GLP-1 in comparison to normal-weight boys [272]. In addition, PYY was inversely related to

growth hormone secretion during varying stages of puberty which could be beneficial during

growth (lower postprandial satiety resulting in more nutrients ingested for growth) and it was

shown to be lowest in mid to late pubertal boys [273]. The increased growth hormone

secretion is synonymous with puberty and is in direct relation to pubertal insulin resistance.

Insulin resistance in boys increases significantly between early puberty to mid puberty

(Tanner 2 to Tanner 4) and decreases to near prepubertal levels in Tanner stage 5 independent

of fatness and body mass index (BMI) [274]. It has been proposed that this increase in insulin

resistance may result from increased production of growth hormone and subsequent

peripheral increases of IGF-1, as resistance has shown a positive correlation with serum IGF-

1 levels and mean serum GH levels [275, 276].

The aforementioned hormones are all implicated in short-term appetite signaling with

puberty, but leptin, a chronic mediator of appetite has indeed shown relationships with

pubertal development. Leptin is an important signalling molecule involved in the timing of

onset of pubertal development [277]. In boys, rising serum leptin levels precede the onset of

puberty and decrease immediately after initiation of puberty even after adjusting for body

composition [278, 279]. The sexual dimorphism of leptin throughout pubertal progression

(decreasing in boys and increasing in girls) implicates a role of gonadal steroids as mediators

in circulating leptin [280].

30

2.5.3 Appetite Hormones and Overweight/Obesity

In contrast to pubertal development, a great deal of work aiming to determine the relationship

of appetite hormones in overweight/obese individuals has been performed. In obese boys,

short-term FI is affected by body fatness [281]. Obese individuals also demonstrate ghrelin

levels which are lower than normal weight subjects during periods of weight stability, though

ghrelin does increase during diet-induced weight loss [266, 282]. Furthermore, there does not

seem to be a resistance to ghrelin as previously thought [283]. Though it seems that ghrelin

signaling follows what would be expected for adiposity/energy availability, less is clear about

anorexigens.

Circulating postprandial levels of PYY are lower in obese individuals [246], and postprandial

stimulation of PYY may be blunted in obese adolescents [269]. However, results from our

laboratory [266] comparing normal weight and obese adolescent boys contradict the findings

of lower circulating PYY in obese adolescents and showed that PYY AUC is significantly

higher in obese boys. Boys with obesity within our prior study protocol had a lower whole-

body insulin sensitivity index (WBISI) in comparison to normal weight boys (5.5 ± 1.7 and

11.4 ± 1.8) respectively as well as greater fasting insulin resistance (HOMA-IR) values (3.2 ±

1.0 vs. 0.9 ± 0.2 respectively) implicating insulin resistance within the cohort of obese boys.

Previous literature has indicated that there is in fact a high level of serum PYY in obesity

associated insulin resistance and type 2 diabetes [284]. Since insulin resistance is related to

incretins, it would be expected that the anorexigenic incretin GLP-1 would be affected by

obesity. Obesity has been shown to be associated with a decreased postprandial GLP-1

response to feeding [285, 286]. Administration of GLP-1 analogs to obese patients decreased

bodyweight and improved blood glucose regulation and insulin sensitivity [287, 288].

It is well established that obesity is associated with insulin resistance, though there is still

debate with regards to the primary underlying mechanisms [289]. In adult humans, a meta-

analysis performed with 92 normal weight and 44 overweight subjects showed that blood

glucose may be an important satiety signal for short-term appetite regulation in normal-

weight but not overweight subjects [236]. This discrepancy in appetite signaling may be a

function of increased insulin levels leading to increased satiety levels in normal-weight

subjects which could also be impaired in overweight subjects due to central insulin resistance.

In children with overweight, insulin resistance and hyperinsulinemia has been associated with

increased energy intake during an ad libitum buffet after an overnight fast a 10% increase in

31

HOMA-IR was associated with 29 kcal greater energy intake [290]. During pubertal

development, obese children had larger increases of insulin resistance (measured by HOMA-

IR) than normal-weight children and, additionally unlike the normal-weight children, did not

return to normal values after puberty [291].

Much like insulin, individuals with higher levels of adiposity have greater circulating leptin

levels when compared to normal-weight subjects [292]. However, the satiety promoting

properties of leptin seem to be attenuated as a result of leptin resistance in overweight and

obese patients, though it remains to be seen whether the resistance is a function of the excess

body fat or, whether leptin resistance predisposes an individual to greater FI resulting in

increased adiposity [293]. Given that leptin and insulin act upon the same hypothalamic areas

to suppress FI [294, 295], central resistance to both hormones in the hypothalamus may be

related and an important factor in obesity and appetite regulation.

2.5.4 Testosterone and Overweight/Obesity

The inverse relationship between body weight and testosterone levels in males has been well

documented [296-299]. Testosterone may also be predictive of weight regain after weight

loss in males [300], and severe childhood obesity is associated with impaired Leydig cell

function in young men [301]. Though some controversy exists, pubertal boys with obesity

have lower SHBG and lower total testosterone in comparison to normal-weight boys [302],

with increased aromatization due to high adiposity advancing skeletal age in

overweight/obese boys [303]. The relationship between body weight and testosterone seems

to be specific to adipose tissue and body fat as demonstrated in comparisons of younger and

older men [304]. Further studies looking at both healthy and hypogonadal men showed that

the latter had increased fat mass in addition to increased abdominal/central obesity [305, 306].

Similar findings in pubertal males indicated that adolescents with obesity had lower free

testosterone levels and signs of Leydig cell impairment in spite of testicular volume being

equivalent between boys with obesity and and their normal weight peers [31].

Testosterone has been demonstrated to have direct physiological effects on adipose tissue

deposition. Aceyl-CoA synthetase is a fatty acid (FA) activator which signals enhanced FA

uptake in adipose tissue [307]. Testosterone may decrease expression of aceyl-CoA

synthetase. A study of hypogonadal men showed that in comparison to eugonadal men,

hypogonadal males had a propensity to store FA’s and FFA’s in lower subcutaneous fat

32

which was associated with increased expression of aceyl-CoA synthetase [308].

Lipoprotein lipase (LPL) is an extracellular enzyme which hydrolyzes circulating blood

lipoproteins into FA’s which are taken up into the adipocyte and esterified into triglycerides

that are then stored in the adipocyte [309]. In sedentary obese men, abdominal adipose tissue

LPL activity was inversely correlated with bioavailable plasma testosterone [310].

A hypogonadal obesity cycle has been proposed by Cohen in male patients with obesity

[311]. High aromatase activity in adipose cells converts testosterone to oestradiol and

decreases tissue testosterone. This decrease in testosterone results in greater LPL activity

which increases triglyceride uptake into the adipocyte. As well as adipocyte volume

increasing, low testosterone stimulates pluripotent stem cells to be differentiated into

adipocytes thus increasing both the size and number of these cells. The increased levels of

oestradiol, TNFα and leptin from increased adipocyte cell mass prevent the hypothalamic-

pituitary-testicular axis response to decreased testosterone levels [312]. This is a result of

kisspeptin being both inhibited by the inflammatory cytokines as well as oestradiol and being

resistant to leptin, thus preventing downstream signaling of GnRH and LH [313, 314].

Finally, in addition to partitioning greater fat oxidation within the mitochondria [315],

testosterone enhances norandrenaline stimulated lipolysis which may be a result of

testosterone increasing the number of β-adrenoceptors within fat cells [316, 317].

Collectively, testosterone helps mitigate adiposity in males and decreased levels of

testosterone result in increased adiposity. This relationship is cyclical as an increase of

adiposity decreases testosterone through both aromatization and greater clearance of SHBG

[311, 318].

2.5.5 Testosterone and Food Intake/Appetite

Testosterone may influence hormones involved in appetite and vice-versa (Figure 2.3).

During pubertal progression, increased testosterone and subsequent aromatization to estrogen

has been shown to exert increases of pulsatile GH release, which in turn increases insulin-like

growth factor 1 (IGF-1) [319]. Insulin-like growth factor 1 (IGF-1) is known to suppress

PYY and there is an observed decrease of PYY during pubertal progression [273]. Thus,

increased testosterone during pubertal progression is presumed to depress PYY indirectly by

its stimulatory action on the somatotropic axis. Since PYY decreases during pubertal

33

progression in boys [273], it can be posited that this decrease of the anorexigen could in

part play a role in the observed increases of FI in boys.

Another important link with appetite and sex hormones is one between testosterone and

ghrelin. In boys, there is a marked decrease in ghrelin with increased pubertal stage [268].

Ghrelin has been detected in Leydig cells of the testes and is inversely correlated to

testosterone in adult males [320]. In boys and adolescents, short peripubertal boys given

therapeutic injections of testosterone had significant decreases in ghrelin [268]. Since ghrelin

is considered a short-term regulator of feeding initiation, it is unknown whether this hormone

plays a key role in overall FI of adolescent boys. Indeed, the inverse relationship between

testosterone and ghrelin, a known GHRH stimulant, observed during pubertal progression,

may simply result from a negative feedback loop due to increased GH/IGF-1.

Insulin promotes satiety by inhibiting arcuate nucleus expression in the neuropeptide-Y

region of the hypothalamus, thereby decreasing FI [321, 322]. Insulin resistance increases

during puberty by approximately 30% as a result of increases in GH/IGF-1 [275, 276].

Estrogen increases, in part due to aromatization of testosterone, also promote GH/pituitary

secretion and insulin resistance [319]. Testosterone could therefore indirectly increase FI

through pubertal insulin resistance mediated by GH/IGF-1. This is similar to what has been

proposed with PYY.

Leptin is an essential hormone in energy homeostasis through its effects on inducing satiety

[131]. Leptin is a critical hormone for normal pubertal development; it increases prior to

pubertal onset and stimulates the release of GnRH [323]. Unlike girls, leptin in boys begins to

rapidly decrease after the onset of puberty, irrespective of body composition [278]. This sex

difference could relate to testosterone lowering plasma leptin, as shown in a study of

hypogonadal adult men [324]. In a multiple regression analysis of factors related to puberty,

leptin was inversely related to testosterone levels but not fat mass, fat-free mass or estrogen,

LH and FSH levels [278, 279]. Thus, lower leptin may be another mechanism leading to an

increased FI during puberty in boys. Leptin is important in both energy homeostasis and

pubertal onset. Taken together, lower leptin could lead to an increased FI during puberty.

Furthermore, leptin and insulin signaling pathways share an overlap in the ventromedial

hypothalamic neuron and insulin resistance could interfere with leptin signal transduction

[325], leading to an increase of FI.

34

Figure 2.3- Proposed relationship between testosterone and food intake during puberty. Testosterone may

indirectly increase food intake through aromatization resulting in a decrease of postprandial PYY1-36, and PYY 3-

36 (PYY) in addition to increased insulin and leptin resistance with a concomitant decrease of circulating leptin

resulting in increased food intake during puberty.

Much like the research available in puberty and GLP-1, limited work has been performed on

determining relationships between GLP-1 and testosterone. Previous research in animals has

implicated GLP-1 in LH release by showing that cerebroventricular injections of GLP-1

increased plasma LH [326]. In addition, GLP-1 may play a role in decreasing testosterone

pulsatility in humans through direct GLP-1 infusion. However unlike in animals, this seems

to be independent of LH [327]. Animal studies suggest that the down-regulatory effect of

GLP-1 on testosterone may be explained by a neural pathway between the hypothalamus and

testes which regulates secretion of testosterone independent of the pituitary [328]. The

relationship between GLP-1, appetite, and testosterone during puberty in humans remains

unclear as testosterone pulsatility increases during pubertal growth.

Testosterone’s relationship with appetite may not be limited to indirect effects via appetite

hormones. As puberty advances, so too does FFM and FI [329] but even after adjusting for

body composition, height, and weight status, short-term FI in 8-17-year-old males remained

greater than in females [264]. Testosterone may have a direct effect on appetite and FI as

35

androgen receptors are located in the hypothalamic-pituitary-gonadal axis [330]. In

animal studies, orchiectomy decreased FI [331, 332] and this effect was reversed through

testosterone administration [331, 332] which has been previously shown to stimulate FI in

adult rats with intact testicles [333].

Though testosterone does have a relationship with appetite and appetite-related hormones,

there is also evidence to show that FI can affect testosterone levels. Chronic high protein

intake in a group of males have been shown to reduce fasting testosterone concentrations

[334, 335]. Furthermore, 6 weeks of meals containing animal fats attenuated the reduction of

testosterone in comparison to meals that were either high in plant-based fats or low fat in

adult males [336]. Less is known about acute FI and testosterone, though previous research

has shown a decrease in serum testosterone in adult males immediately after glucose

ingestion [10].

2.6 Testosterone and Exercise

The aforementioned literature focuses on the relationship between testosterone and

energy intake, one half of basic human energy balance. Thus, it would be imperative to also

discuss the relationship between testosterone and energy output in adolescent males.

2.6.1 Studies of testosterone change from exercise in males

In adult males, an increase of plasma testosterone levels has been shown as a result of both

anaerobic and aerobic modes of exercise such as weight lifting [337] or cycling [338] and

running [339] respectively. In contrast, studies of the effect of exercise on testosterone levels

in adolescent males are far less abundant. Longitudinal studies in boys who are in Tanner

stages 1 and 2 showed no significant change in testosterone during cardiorespiratory exercise

(20 min cycle ergometer, 60% of VO2peak) and later when they were more mature, still

showed no differences. However, testosterone in these boys was measured with

radioimmunoassay which may not be sensitive enough to detect low levels in this cohort.

Furthermore, no control/non-exercise sessions were performed to determine whether there

were differences between exercise and regular diurnal rhythms [340].

36

Research examining the effect of exercise in boys has focused primarily on longitudinal

interventions and aerobic style exercise programs and it has been observed that training in

young individuals may modify sex hormone responses at rest and during exercise. Cacciari et

al. [341] reported that in 175 trained male soccer and untrained males aged 10 to 16 years,

the plasma level of DHEA-S was significantly higher in trained children. This enhanced

plasma DHEA-S concentration was related to higher levels of testosterone. Mero et al. [342]

described serum testosterone levels under conditions of rest before, during and after a year of

long distance running, sprint running, weightlifting and tennis training in boys aged 10 to 12

years. Compared with untrained boys, the initial plasma testosterone concentration was

almost 3 times higher in these individuals. Furthermore, the mean testosterone level was

approximately doubled after a year of training. These results suggest that endurance training

may alter gonadal hormone production in young athletes.

Fewer studies have examined the acute effects of exercise on testosterone in adolescent

males. Klentrou et al.[343] studied the effects of anaerobic style exercise training on male

athletes ages 12-14. The subjects were asked to perform 30 minutes of resistance or

plyometric exercise compared with a 30-minute control session. The results of this study

showed that testosterone increased from pre-exercise to post exercise in both the resistance

and plyometric modalities. Other studies looking at acute testosterone levels in males found

similar results with anaerobic protocols such as weightlifting [344] and sprinting [345]. In

one of the few aerobic studies observing testosterone change, Hackney et al. presented the

effect of aerobic style exercise training in males who were in Tanner stages 4 and 5. The

participants performed 20 minutes of incremental exercise to exhaustion using a cycle

ergometer. Results indicated that there were no significant differences between pre- and post-

exercise testosterone levels. The lack of control session was a major limitation of the

Hackney study . Additionally, the males were only fasted for three hours prior to testing

which could impact the baseline testosterone levels. Finally, because testosterone was

measured using immuno-chemoluminescence, sensitivity to detect small changes may be an

issue [346]. Given that there is little research in comparison to anaerobic protocols, further

work on the effects of aerobic exercise on acute testosterone levels in adolescent males using

appropriate controls should be performed.

37

2.6.2 Physiology of testosterone in exercise

Though there is indeed a marked increase of testosterone from exercise in adult males and

possibly adolescent males, the physiological mechanisms behind these phenomena are less

clear. Several studies using different forms of exercise have repeatedly shown that the level

of LH in plasma does not change after exercise [11-13] indicating that the increase of

testosterone from exercise is not centrally mediated and exercise itself may stimulate

peripheral changes of testosterone production.

With both anaerobic and aerobic physical exertion, there is an increased production of lactate

as a result of glycolysis and glycogenolysis within skeletal muscle tissue [347]. Lin et al.

discovered that in male rats, infusion of 5-20mM of lactate resulted in a dose-dependent

increase of testosterone production by 25-OH-choesterol in the Leydig cells. It is thus

hypothesized that lactate directly affects the Leydig cell cAMP production of testosterone and

that P450scc is a target of lactate [14]. Further studies have confirmed that exercise-induced

lactate production resulted in dose-dependent increases in testosterone and testicular cAMP

in rats [348]. Thus, it seems that there is a concomitant increase of testosterone and lactate

production during exercise, but this has yet to be validated in humans.

2.7 Summary and Research Rationale

It is clear that puberty is a time of adjustment of FI regulatory mechanisms and presents the

risks of setting a course for increased FI and obesity. However, unlike obesity, the changes in

pubertal FI may be a consequence of the increases of sex hormones rather than the

homeostatic imbalances seen with obesity. For boys, testosterone plays a major role in

growth and development. What is currently unknown is (a) whether the relataionship between

testosterone and FI differs in boys with obesity vs. pubertal boys of normal weight, (b)

whether acute changes of testosterone correlate with FI in pubertal boys across the weight

spectrum, (c) how food and macronutrients affect acute testosterone changes, and (d) how

testosterone affects the postprandial response of appetite hormones. Furthermore, it remains

unclear what the response of testosterone is after aerobic exercise in this population and

whether this response is related to appetite and FI.

38

Therefore, the objective of this thesis is to determine how testosterone is affected by

food and exercise and describe the relationship between testosterone, FI and appetite in

adolescent males. It is expected the results of this thesis will add to current knowledge of

testosterone by providing new insights into the role of this hormone in appetite and FI.

39

Chapter 3

HYPOTHESIS AND OBJECTIVES

40

HYPOTHESES AND OBJECTIVES

3.1 Overall Hypothesis and Objective

Objective

• To determine the interactions between testosterone responses to food intake and

exercise with appetite and food intake in adolescent males.

Hypothesis

• Testosterone responds to food intake and high intensity exercise and has an interactive

relationship with the short-term regulation of appetite and food intake in adolescent

males.

3.2 Specific Hypotheses and Objectives

Chapter 4: ACUTE DECREASE IN SERUM TESTOSTERONE AFTER A MIXED

GLUCOSE AND PROTEIN BEVERAGE IN OBESE PERIPUBERTAL BOYS (published

in Clinical Endocrinology 2015, 83: 332-338).

Objective: • Explore the effect of a mixed glucose/protein beverage on acute endogenous

testosterone levels in overweight adolescent males.

Hypothesis: • A mixed glucose/protein beverage will decrease endogenous testosterone levels an

hour after ingestion in adolescent overweight males.

Chapter 5: ACUTE DECREASE IN PLASMA TESTOSTERONE AND APPETITE

AFTER EITHER GLUCOSE OR PROTEIN BEVERAGES IN ADOLESCENT MALES

(published in Clinical Endocrinology 2019, 83: May 4th)

Objective: • Compare the effect of glucose or protein provided at 1g/kg body weight in a beverage

form on acute responses in plasma testosterone and appetite hormones, appetite, and

food intake in adolescent males 12 to 18 years of age.

41

Hypothesis: • Postprandial testosterone levels play a role in the regulation of short-term appetite and

food intake in teenage boys.

Chapter 6: ACUTE DECREASE IN APPETITE IS UNRELATED TO INCREASE IN

PLASMA TESTOSTERONE DURING HIGH INTENSITY CYCLING IN ADOLESCENT

MALES

Objective: • Determine whether high intensity aerobic exercise will increase testosterone and

decrease appetite in adolescent males and define whether the responses are related.

Hypothesis: • Three 10-minute bouts of high intensity recombinant cycle exercise at 75% VO2peak

will increase testosterone levels and decrease appetite in adolescent males but the

responses will be related.

42

Chapter 4

ACUTE DECREASE IN SERUM TESTOSTERONE AFTER A

MIXED GLUCOSE AND PROTEIN BEVERAGE IN OBESE

PERIPUBERTAL BOYS

The following chapter is a reproduction of a manuscript that has been published in Clinical

Endocrinology 2015 Sept; 83 (3): 332-338

43

ACUTE DECREASE IN SERUM TESTOSTERONE AFTER A MIXED GLUCOSE AND PROTEIN BEVERAGE IN OBESE PERIPUBERTAL BOYS

Alexander Schwartz1, 2, Barkha P. Patel1, Shirley Vien1, Brian W. McCrindle3, G. Harvey

Anderson1, Jill Hamilton1, 2 *

1 Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto,

M5S 3E2, Canada

2 Division of Endocrinology, Department of Paediatrics, The Hospital for Sick Children,

University of Toronto, Toronto, M5G 1X8, Canada

3 Division of Cardiology, Department of Paediatrics, The Hospital for Sick Children,


Corresponding author and person to whom reprint requests should be addressed:

Jill Hamilton, MD, FRCPC

Division of Endocrinology

The Hospital for Sick Children

555 University Ave., Toronto (On), Canada, M5G 1X8

Phone: 416-813-5115; Fax: 416-813-6304

E-mail: [email protected]

Disclosure Statement: The authors have nothing to disclose

mailto:[email protected]

44

4.1 Abstract

Background and Objectives Delayed puberty and lower levels of testosterone (T) have been

observed in adult obese males and some adolescent males. In adult men, enteral glucose

ingestion results in acute lowering of serum testosterone levels, however this has not been

studied in adolescents. We aimed to examine the acute effect of a glucose/protein beverage

on serum T concentration changes in obese peripubertal males. A second objective was to

determine whether change in T concentration was related to appetite hormone levels.

Patients and Methods 23 overweight and obese males age 8-17 in pre-early (Tanner stage 1-

2) and mid-late (Tanner stage 3-5) puberty were included in this cross-sectional study at the

Clinical investigative unit at the Hospital for Sick Children. Participants consumed a

beverage containing glucose and protein and blood samples measuring pubertal hormones,

ghrelin and glucagon-like-peptide-1 (GLP-1) were taken over 60 minutes.

Results Across pubertal stages, there was a significant decrease in T levels in adolescent

boys (-18.6 ± 3.1%, p < 0.01) with no proportional differences between pre-early and mid-

late puberty (p =0.09). Decrease in T was associated with a decrease in LH (r = 0.52, p =

0.02) and fasting T was inversely correlated to fasting ghrelin (r = -0.51, p = 0.03) with no

correlation to GLP-1.

Conclusions Intake of a mixed glucose/protein beverage acutely decreases T levels in

overweight and obese peripubertal boys. A potential mechanism for this decrease may be

secondary to an acute decrease in LH but this requires further evaluation.

45

4.2 Introduction

Obesity is associated with a reduction in serum total testosterone levels in adult men [349].

Testosterone, a steroid hormone which plays a key role in male pubertal development and

secondary sexual characteristics, is 40-50% lower in obese boys when compared with normal

weight boys [9] and may be associated with pubertal delay in overweight and obese boys

[27].

Several mechanisms have been proposed to account for the inverse association between

obesity and testosterone levels. Androgens are converted to estrogens via an aromatase

enzyme present in adipocytes, lowering the testosterone: estrogen ratio [311]. Obesity is also

associated with chronic hyperinsulinemia and insulin resistance [350] which in turn, may

lower sex hormone binding globulin (SHBG) resulting in lower levels of circulating

testosterone as well as an increased clearance of testosterone [351]. Furthermore, excess

adiposity results in a greater production of the energy homeostasis-regulating hormone leptin

leading to central leptin resistance at the level of the hypothalamus, which may decrease the

production of gonadotropins and testicular testosterone production [293, 314, 352]. Finally, a

small number of studies in adult males suggest that testosterone may also be affected by acute

glucose intake. In these studies, direct parenteral insulin and glucose infusion led to

increased testosterone levels [353, 354]; however, enteral ingestion of a glucose beverage

acutely lowered testosterone levels [10]. Reasons for this discordance are unclear yet may

relate to the effect of gut hormones released with enteral intake, such as glucagon-like

peptide-1 (GLP-1) and ghrelin which have been shown to decrease testosterone levels and

pulsatility in animals and healthy adult males [327].

GLP-1 is secreted by the L cells of the intestines in response to feeding and is responsible for

increasing insulin release and inducing satiety centrally [355]. In one study performed in

adult males, GLP-1 infusion during an intravenous glucose challenge resulted in decreased

testosterone pulsatility implicating a down-regulatory effect of GLP-1 on testosterone [327].

Ghrelin is a 28 amino-acid-long gut peptide derived from the stomach in addition to the

proximal small intestine and pituitary gland. Ghrelin activates neuropeptide-Y neurons in the

arcuate nucleus to stimulate appetite [224] and has been shown to rise acutely prior to feeding

initiation and decrease following food intake [227]. In boys, fasting ghrelin levels decrease

with increased pubertal stage [356]. Ghrelin is also inversely correlated to testosterone in

46

adult males and has been detected in Leydig cells of the testes, which are responsible for

testosterone production, although its role in the testes remain unclear [320].

The interplay between testosterone, glucose, and other hormones related to acute food intake

has not been assessed in peripubertal obese boys. Accordingly, the primary objective of this

study was to determine the acute effect of a glucose/protein beverage on testosterone levels in

obese adolescent males across different pubertal stages. A secondary objective was to explore

the relationship of testosterone following the beverage to degree of adiposity, insulin

sensitivity, GLP-1 and ghrelin levels.

4.3 Materials and Methods

4.3.1 Subjects

Overweight and obese but otherwise healthy adolescent boys age 8 to 17 were recruited as

part of a larger study of girls and boys across the weight spectrum exploring childhood and

adolescent obesity (HISTORY: High Impact Strategies Towards Overweight Reduction in

Youth) and the appetite hormone response related to clinical pubertal status [357].

Recruitment occurred via flyers posted in the hospital, and a print ad in the local newspaper.

All eligible overweight and obese boys were included in the analysis. Obesity was defined

using the Center for Disease Control BMI charts where overweight is categorized as 85th-95th

percentile and obesity is categorized as ≥95th percentile [21]. Subjects were excluded if they

had a history of prematurity, chronic illness or were taking any medications known to affect

glucose homeostasis, appetite or pubertal development. The results presented in this paper are

based on a secondary analysis of a previous study to examine appetite hormones in response

to a mixed glucose-whey drink [357]. The study was approved by the University of Toronto

Research and Ethics Board for Humans and The Hospital for Sick Children Research Ethics

Board.

4.3.2 Protocol

After a 10-12 h fast, participants arrived at 0800 and were administered a motivation-to-eat

VAS scale, which measure dimensions of subjective appetite and have been previously

validated for use in boys after glucose preloads [281]. The scale was composed of four

questions: (1) How strong is your desire to eat? (“very weak” to “very strong”), (2) How

47

hungry do you feel? (“not hungry at all” to “as hungry as I’ve ever felt”), (3) How full

do you feel? (“not full at all” to “very full”), and (4) How much food do you think you can

eat? (prospective food consumption, PFC) (“nothing at all” to “a large amount”). Participants

were instructed to read each question and place an “x” along the 100 mm line depending on

their current feelings. An intravenous catheter was placed for blood sampling.

Participants were then administered a mixed beverage containing 30 g glucose monohydrate

(Grain Process Enterprises, Toronto, ON Canada) and 30 g of whey protein isolate (plain

whey-protein isolate; Interactive Nutrition International Inc., Ottawa, Ontario, Canada) plus

aspartame-sweetened, orange-flavored crystals (1.1 g, Sugar Free Kool-Aid, Kraft Canada

Inc., Don Mills, ON Canada) to standardize flavor. The combination of a standardized

glucose load and protein has been shown to generate a greater insulin increase and ghrelin

decrease in comparison to a glucose load only potentially through protein-stimulated GLP-1

secretion [358, 359]. Thus, this combination was chosen to promote a greater change in

hormone flux. The beverage was consumed within 5 min, followed by 50 mL of water to

minimize aftertaste.

Blood samples were taken of testosterone, LH, FSH, GLP-1 and ghrelin at fasting and at 15,

30 and 60 minutes after completely ingesting the mixed beverage. 15 and 30 minutes were

chosen in order to capture the rapid postprandial rise (GLP-1) and fall (ghrelin) of these

peptides in the circulation [355, 360]. The 60 minute time point was chosen as a previous

study in adults has indicated post-glucose load nadir testosterone values at this time [354].

In a separate visit conducted within 4 weeks of the visit described above, body composition

was analyzed via BOD POD (Life Measurement Incorporated, Concord, CA) air-

displacement plethysmography (ADP) [361]. A standard, calibrated scale and wall-mounted

stadiometer were used to measure weight and height, and body mass index (BMI) calculated

as weight (kg)/height (m). Three trials of these measurements were completed and the mean

was taken for analyses. Puberty was assessed through a validated Tanner staging

questionnaire and via examination by a paediatric endocrinologist [362]. A multiple-sampled

(0. 30, 60, 90 and 120 minutes) oral glucose tolerance test (7 1.75g/kg to a max of 75g

glucose) was performed to assess insulin sensitivity [363]. Insulin sensitivity was calculated

by the Matsuda Model whole body insulin sensitivity index (WBISI) where WBISI =

(10,000/square root of [fasting glucose x fasting insulin] x [mean glucose x mean insulin

during OGTT]). Assessment of normal glucose tolerance, impaired fasting or 2-hour glucose

48

and type 2 diabetes was completed by measurement of the 0 and 120-minute glucose

levels using the Canadian Diabetes Association Clinical Practice Guidelines [364].

4.3.3 Biochemical Assays

Glucose was analyzed using Glu Microslides, Vitros 950 chemical system (Ortho Clinical

Diagnostics). Insulin was measured using a chemiluminescence immunoassay (Siemens

Immulite 2500 platform; range 15–2165 pmol/l, intra- and inter-assay coefficient of variation

[CV] <7.6%). Human active GLP-1 (intra-CV: <8%; inter-CV: <5%; #EGLP-35K), total and

acylated ghrelin (intra-CV: <2%; inter-CV: <8%; #EZGRT-89K) were measured with ELISA

kits (Millipore, Billerica, MA, USA). Samples were stored at -80ºC until analysis, and all

samples were run in duplicate.

FSH was assayed to ensure that none of the males had evidence of primary testicular

insufficiency (indicated by an elevated FSH) using a chemiluminescent assay platform

(Abbott ARCHITECT ci8200) used for clinical and research purposes at the Hospital for Sick

Children. The same platform was also used to measure testosterone and LH (testosterone

sensitivity 0.3 nmol/L and cross reactivity of 0.01-2.11% for various metabolites; LH

sensitivity of 0.07 IU/L and cross reactivity of 0.84% for TSH with no cross-reaction to FSH

or hCG).

4.3.4 Statistical Analyses

Sample size was assessed using PS Power and Sample Size Calculations (Version 3.0.43,

2009, Vanderbilt University). The mean of three adult studies showed decreases of

testosterone of 24% following an OGTT [10, 365, 366]. Based on this estimated relative

decrease, and using mean and standard deviation values from studies measuring testosterone

in boys from T1 to T5 puberty, 13 boys were required to achieve power of 0.8 and an α =

0.05 [208].

A two-way repeated measured ANOVA was used to analyze the effects of puberty and time

and their interactions on testosterone before and after the glucose and protein beverage. A

Tukey’s test post-hoc analysis was then performed. Pearson correlation was performed to

evaluate testosterone (baseline) and change in testosterone to measures of adiposity, insulin

49

sensitivity (WBISI), and change in LH, ghrelin and GLP-1. All analyses were performed

with Statistical Product and Service Solutions software, version 17.0 (SPSS Inc., Chicago,

IL). Effects were considered significant at p < 0.05 and data are presented as mean ±(SEM).

4.4 Results

Subject characteristics are presented in Table 4.1. A total of 23 boys with a mean age of 13.3

± 0.6 years and mean BMI of 33.1 ± 2.2kg/m² participated. 10 boys were classified as being

in the pre-early stages of puberty (Tanner stage 1-2) while the remainder (n = 13) were in the

mid-late stages of puberty (Tanner 3-5) mean WBISI values of 3.3 ± 1.1 and 3.4 ± 0.8

respectively. All were normoglycemic with the exception of two boys in mid-late puberty

who exhibited impaired glucose tolerance on the 2-hour OGTT.

Fasting testosterone levels significantly differed between pre-early and mid-late puberty (2.3

± 0.7 vs. 9.9 ± 1.6 nmol/L respectively, p =0.007). Fasting testosterone levels were inversely

correlated to body fat percentage (r= -.49, p = 0.02) but not to BMI Z-score (r= -0.21, p =

0.34) or WBISI (r= .21, p = 0.34). There was a significant inverse relationship with fasting

testosterone levels and fasting active ghrelin prior to ingestion of the beverage (Fig. 4.1).

After controlling for percentage body fat, there remained a trend in this relationship;

however, it was no longer statistically significant (r = -.46, p = 0.06).

For the group as a whole, testosterone levels decreased significantly by 1.9 ± 0.4 nmol/L 30

minutes after ingestion of the glucose and protein beverage (p < 0.01). At 60 minutes after

ingestion of the beverage, testosterone levels remained significantly lower than baseline at

1.6 ± 0.3 nmol/L (p < 0.01), however there was no difference between testosterone levels at

30 and 60 minutes (p = 1.0). The entire cohort had a decrease of approximately 18.6 ± 3.1%,

60 minutes after ingestion of the glucose and protein beverage, independent of the pubertal

stage (Fig. 4.2). A decrease in LH of 0.4 ± 0.1 IU/L 60 minutes after the ingestion of the

beverage was also seen (p < 0.01). The decrease in LH level was significantly correlated to

the decrease seen in testosterone level (Fig. 4.3) (r = 0.52; p = 0.02).

For all subjects, consumption of the beverage resulted in higher insulin and GLP-1, and lower

LH, FSH, testosterone, and ghrelin (Table 4.1). The change in testosterone was not

significantly correlated with either the AUC for ghrelin (r = .412, p = 0.06) or GLP-1 (r =

.17, p = 0.49). Changes in testosterone level had no significant relationship with percentage

50

body fat (r = 0.64, p = 0.08), WBISI (r = 0.005. p = 0.98), or the mean of the combined

appetite scores (r = -.16, p = 0.48).

4.5 Discussion

This is the first study to demonstrate that enteral glucose/protein intake acutely lowers

testosterone levels in overweight and obese adolescent boys across puberty. Furthermore, it

shows that the postprandial decreases of testosterone levels may be centrally mediated as

indicated by lowering of LH levels after the beverage. The ~19% decrease of testosterone

from glucose/protein intake in these boys is consistent with a 25% lower testosterone levels

in adults males after a glucose beverage [10]. Not surprisingly, mid-late pubertal boys had

greater absolute decreases in testosterone after the beverage in comparison to early pubertal

boys however the relative change in testosterone was similar between the two. Fasting

testosterone was inversely related to adiposity and ghrelin but not to insulin sensitivity.

Change in testosterone in response to the beverage was significantly related to change in LH,

but not correlated to adiposity, insulin sensitivity nor to changes in ghrelin or GLP-1.

The decrease of both LH and testosterone levels following enteral glucose/protein intake may

be age-related and more pronounced in children. A study of older men (mean age 51 ± 1.4

years) undergoing an OGTT found a lowering of testosterone [10] but not LH, however, a

study of adult men across a wider age range found that the testosterone decrease was related

to lowered LH pulsatility, and that these effects were more pronounced in younger men

[367]. Therefore, the decrease in testosterone in peripubertal children after glucose/protein

intake may be centrally mediated via lowering of LH levels. Consistent with this possibility,

a previous study in adult males demonstrated that changes in LH concentrations correlate

with changes in serum testosterone levels within 40-120 minutes, in line with our

measurements [368]. Although it would be expected that the decrease in LH would occur

prior to testosterone if the testosterone decrease was centrally mediated, our study showed a

concomitant decrease of LH and testosterone at 15 minutes. It may be possible that the LH

decrease preceded the decrease in testosterone between baseline and 15 minutes, however

earlier samples were not available for analysis.

While we show that the mixed beverage reduced testosterone, it is not clear if the protein or

the glucose component was the primary stimulant for this change. The response observed

may be primarily due to the glucose content of the beverage. Previous work has shown that

51

increased glucose uptake into the central nervous system via glial GLUT-1 receptors in

the hypothalamus [369] and glucose stimulation of the intestinal-mucosal vagal pathway

feeding back to the central nervous system results in lower LH [327, 370].

As postprandial GLP-1 increases have been proposed to explain the decrease of testosterone

after glucose intake [327], this study explored the association between GLP-1 and

testosterone. GLP-1 response to the beverage was unrelated to either fasting testosterone or

its response and contrasts with the effect of GLP-1 infusion during a glucose challenge which

decreased testosterone pulsatility in adult males, implicating a down-regulatory effect of

GLP-1 on testosterone [327]. Because GLP-1 is more strongly stimulated by whey protein

than glucose beverages [371] it is interesting that, while the protein/glucose beverage

increased both GLP-1 and insulin, we did not detect a relationship between GLP-1 and

testosterone change. It may be possible that this is a consequence of methodological

differences as GLP-1 stimulation in our study occurred via enteral beverage intake rather than

intravenous delivery as used in the previous study. Furthermore, the study by Jeibmann et al.

[327] found decreases of testosterone pulsatility but not mean testosterone levels after the

GLP-1 infusion.

Although fasting ghrelin and fasting testosterone were inversely correlated, there was no

association between the change in ghrelin and testosterone after ingestion of the beverage.

These results in the boys after an overnight fast are consistent reports in animals [320] and

peripubertal boys [372]. In addition, higher ghrelin is associated with reduced

spermatogenesis and smaller Leydig cell size in animals and humans [373]. Furthermore,

ghrelin is typically lower in overweight/obese adolescents than normal weight adolescents

[356] and in overweight subjects, post-meal ghrelin declines more slowly than in lean

subjects [374], though our study did not immediately establish a direct association with post-

prandial ghrelin and testosterone.

The interpretation of these results has several limitations. Although a statistically significant

decrease was seen in testosterone levels, it does not necessarily imply a biological

significance. Both adult males and pubertal boys exhibit a diurnal rhythm in testosterone,

with highest levels early morning, declining gradually to a nadir in the evening, so it is not

possible to determine how much of the decrease in testosterone seen was related to this

phenomenon, nor how long the effect may have lasted. Studies in adult males, however, do

indicate a decline in testosterone with a subsequent rise at 120-150 minutes following a

52

glucose drink, indicating that the testosterone changes were at least, in part, due to

nutrient intake, and not due to diurnal rhythm alone [327, 365]. It was not possible to

separate the role of glucose and protein contained within the beverage on the depression of

testosterone in this study, and unfortunately, blood samples were not available to test for

changes in testosterone from the oral glucose tolerance test conducted earlier. We did not

assess other macronutrients, in particular fat, which has been examined in adults studies that

demonstrate plant-based, but not animal-based fat in mixed meals results in lowering of

testosterone [375].

4.6 Conclusions

Our findings suggest that acute glucose-protein intake lowers testosterone levels in

overweight and obese males across pubertal stages and that this effect may be mediated by a

reduction in LH. However, it is not known if these acute changes in testosterone may

themselves impact on appetite and short-term feeding behaviours. Further studies to explore

the acute and chronic ingestion of glucose, protein and fat, and their individual effects on LH

and testosterone in obese boys and adolescents are warranted.

53

Table 4.1- Subject Characteristics and change in hormone levels following the glucose/protein beverage

(n= 23). Subjects are also presented in pre-early puberty (n= 10) and mid-late puberty (n= 13).

All Subjects Pre-Early Puberty Mid-Late Puberty

Baseline 60 min. post-drink Baseline

60 min. post-

drink Baseline

60 min. post-

drink

Age 13.3 ± 2.6 11.2 ± 0.6 14.8 ± 0.7

Height (cm) 161.4 ± 12.0 154.3 ± 3.4 166.8 ± 3.3

Weight (kg) 89.2 ± 31.8 69.3 ± 5.0

104.4 ±

10.5

FM (kg) 37.6 ± 19.9 29.7 ± 3.2 42.4 ± 7.4

FFM (kg) 54.6 ± 16.1 42.5 ± 3.4 62.0 ± 4.7

Body Fat (%) 39.5 ± 9.2 40.7 ± 2.6 38.8 ± 3.3

BMI (kg/m²) 33.1 ± 10.7 27.6 ± 1.4 37.3 ± 3.9

BMI Z Score 2.4 ± 0.4 2.3 ± 0.1 2.5 ± 0.2

Glucose (mmol/L) 4.7 ± 0.3 4.7 ± 0.7 4.8 ± 0.1 4.7 ± 0.2 4.7 ± 0.1 4.7 ± 0.3

Insulin (pmol/L) 121.4 ± 93.5 477.8 ± 312.0 ** 123.8 ± 34.4 447.9 ± 93.3**

119.2 ±

33.7 504.4 ± 137.3**

WBISI 3.4 ± 2.5 3.3 ± 1.0 3.4 ± 0.8

LH (IU/L) 1.7 ± 1.1 1.3 ± 0.9 ** 0.9 ± 0.3 0.6 ± 0.2** 2.2 ± 0.3 1.7 ± 0.3*

FSH (IU/L) 2.1 ± 0.9 1.8 ± 0.8 ** 1.7 ± 0.3 1.5 ± 0.2** 2.3 ± 0.3 2.1 ± 0.3**

Testosterone

(nmol/L) 6.8 ± 6.0 5.5 ± 4.8 ** 2.3 ± 0.7 1.8 ± 0.5* 9.9 ± 1.9 7.7 ± 1.5**

GLP-1 (pM/ml) 3.7 ± 2.7 5.3 ± 3.9 * 4.6 ± 1.7 7.5 ± 2.2* 4.7 ± 1.1 5.0 ± 1.1

* denotes significant difference from baseline at p < 0.05 and ** denotes significant difference from baseline at

p < 0.01.

54

Figure 4.1- Association of fasting active ghrelin and fasting testosterone in males at all pubertal stages (n= 19).

55

Figure 4.2- Testosterone level changes from baseline to 60 minutes after ingesting the glucose/protein beverage

in pre-early puberty (n = 8) and mid-late puberty (n = 13). Data are means ± (SEM); N = 10-13/group.

Treatment (beverage): p<0.0001; pubertal stage: p<0.001; treatment x pubertal stage: p < 0.005. * p<0.05, post-

beverage at 60 minutes vs. baseline. ‡ p<0.05 between pre-early puberty and mid-late puberty.

56

Figure 4.3- Association of changes in testosterone and luteinizing hormone levels in boys following a

glucose/protein beverage (n = 21).

57

Chapter 5

ACUTE DECREASE IN SERUM TESTOSTERONE AND

APPETITE AFTER GLUCOSE AND PROTEIN BEVERAGES IN

ADOLESCENT MALES

The following chapter is a reproduction of a manuscript that has been published in Clinical

Endocrinology 2019 Aug; 91 (2): 295-303

58

ACUTE DECREASE IN PLASMA TESTOSTERONE AND APPETITE AFTER EITHER GLUCOSE OR PROTEIN BEVERAGES IN ADOLESCENT MALES

Alexander Schwartz1, 2, Sascha Hunschede1, R.J. Scott Lacombe1, Diptendu Chatterjee1,

Diana Sánchez-Hernández1Ruslan Kubant1, Richard P. Bazinet1, Jill K. Hamilton1, 2, G.

Harvey Anderson1,2


M5S 3K1, Canada

Department of Physiology




G. Harvey Anderson, PhD.

Department of Nutritional Sciences


150 College St., Toronto (On), Canada, M5S 3E2

Phone: 1-416-9781832


59

5.1 Abstract

Objective: Chronic testosterone blood concentrations associate with food intake (FI), but

acute effects of testosterone on appetite and effect of protein and glucose consumption on

testosterone response has had little examination.

Methods: In a randomized, crossover study, twenty-three adolescent (12-18 y old) males

were given beverages containing either: 1) whey protein (1g/kg bodyweight), 2) glucose

(1g/kg bodyweight) or 3) a calorie-free control (C). Plasma testosterone, luteinizing hormone

(LH), GLP-1 (active), ghrelin (acylated), glucose, insulin and subjective appetite were

measured prior (0) and at 20, 35 and 65 minutes after the consumption of the beverage. FI at

an ad libitum pizza meal was assessed at 85 min.

Results: Testosterone decreased acutely to 20 min after both protein and glucose with the

decrease continuing after protein but not glucose to 65 minutes (p = 0.0382). LH was also

decreased by both protein and glucose but glucose had no effect at 20 min in contrast to

protein (p<0.001). Plasma testosterone concentration correlated positively with LH (r =

0.58762, p < 0.0001) and negatively with GLP-1(r = -0.50656, p = 0.0003). No associations

with appetite, ghrelin or glycaemic markers were found. Food intake was not affected by

treatments.

Conclusion: Protein or glucose ingestion results in acute decreases in both plasma

testosterone and LH in adolescent males. The physiological significance of this response

remains to be determined as no support for testosterone’s role in acute regulation of food

intake was found.

5.2 Introduction

Chronic blood testosterone concentrations have been associated with FI. The relationships

between testosterone and growth velocity [8], fat-free mass (FFM), fat oxidation [376] and,

indirectly, bone growth [133] in pubertal males have been well established. In adults,

elevated circulating testosterone levels are associated with reduced risk of eating disorders

during and after puberty [377] and salivary testosterone with greater taste preference for chili

peppers [378] implying a relationship between testosterone and FI. Furthermore, advances in

60

pubertal status associate with increases in FFM and FI in boys [329]; short-term FI in 8-

17-year-old males is higher than in females of similar body composition, height and weight

status [264].

Animal models provide more direct evidence that testosterone affects FI. Androgen receptors

are located in FI regulatory pathways in the hypothalamic-pituitary-gonadal axis [330].

Testosterone injections to pregnant rats affects development of the hypothalamic-pituitary

orexinergic system, specifically the G protein-coupled orexin receptors 1 and 2 during the

neonatal period [379]. Orchiectomy decreases FI in rodents [331, 332], an effect which is

reversed by providing doses of testosterone to match normal physiological levels [331, 332].

Testosterone injections also have a stimulatory effect on FI in intact adult rats [333].

However, there has been little exploration of the effects of FI ingestion on acute responses in

testosterone concentrations or the acute effects of testosterone on FI, appetite or appetite

regulatory hormones in humans. Ingestion of glucose by adult men [10] or a mixed drink of

glucose and protein by adolescent males [380], decrease testosterone concentrations. In adult

males, a rapid 25% decrease and suppression of testosterone levels over two hours was found

after a 75g oral glucose tolerance test [10]. Similarly, ingestion of a mixed glucose-protein

(60 g) beverage resulted in an 18% decrease of plasma testosterone levels after only one hour

in overweight/obese adolescent males [372]. Neither of these studies measured the

relationship of plasma testosterone with subjective appetite and food intake. However, the

possibility is suggested by the observation that testosterone administration to peri-pubertal

boys aged 8-12.5 years lowered circulating levels of the appetite-related hormones ghrelin

and leptin [372].

Therefore, the hypothesis of this study was that the macronutrient composition of food affects

acute post-ingestion responses in testosterone which in turn associates with post-ingestion

responses in FI, appetite and appetite hormones in adolescent boys. The objective of this

study was to compare the effect of glucose or protein provided at 1g/kg body weight in a

beverage form on acute responses of plasma testosterone, LH, glucose, insulin, GLP-1,

ghrelin, appetite, and FI.

61


5.3.1 Subjects

Normal weight and overweight/obese adolescent males (n = 23) 12 to 18 y of age were

recruited via print advertisement in a local Toronto newspaper. After an initial phone contact,

participants were scheduled for a screening session to determine their eligibility

for participation in a study. Overweight was categorized as in the 85th-95th percentile and

obesity in ≥95th percentile [21]. Participants’ height (m) and weight (kg) were measured

using a stadiometer and a digital scale, respectively. Age-specific BMI percentiles were

calculated using WHO growth charts [17]. Body composition was estimated with

bioelectrical impedance analysis (RJL Systems BIA, 101Q) using the Horlick equation [381].

During the first experimental visit, a nurse trained by a pediatric endocrinologist examined all

participants to assess their pubertal status using the Tanner method [59]. Participants were

excluded from study if they had a history of prematurity, chronic illness or were taking any

medications known to affect glucose homeostasis, appetite or pubertal development. Twenty-

four 12-18 y old males completed the study, but only adolescent males in mid-late puberty

with testosterone values above 150 ng/dl (which is the lower range value for what is expected

of males in mid-late puberty [215]), were included in this analysis (n=23). All study

participants were familiarized with the visual analog scales (VAS) used in the experimental

sessions, and informed written consent was obtained from them and their legal guardians.

The study was approved by the University of Toronto Research and Ethics Board for

Humans.

5.3.2 Protocol

After a 12 h fast, participants arrived at 08:30 am and were administered a motivation-to-eat

VAS, which measures dimensions of subjective appetite and has been previously validated

for use in boys after glucose and whey protein preloads [281]. Protein and glucose beverages

were chosen as previous research indicates that a mixed beverage in adolescent males results

in decreased testosterone [380], but the independent contribution of each remains unclear.

The 100 mm scale was composed of four questions anchored at each end with contrasting

62

terms: (1) How strong is your desire to eat? (desire to eat, “very weak” to “very strong”),

(2) How hungry do you feel? (hunger, “not hungry at all” to “as hungry as I’ve ever felt”), (3)

How full do you feel? (fullness, “not full at all” to “very full”), and (4) How much food do

you think you can eat? (prospective food consumption or PFC, “nothing at all” to “a large

amount”). Participants were instructed to read each question and place an “x” along the 100

mm line depending on their current feelings. An aggregate score of these four questions was

determined as the mean subjective appetite score as previously described in our laboratory

[382-384].

Following the completion of the questionnaires, an intravenous catheter was placed for blood

sampling. The first blood draw was taken prior to ingesting the experimental beverage at

baseline (0 minutes). Participants were then given 5 minutes to ingest the beverage, and blood

was later obtained 20, 35 and 65 minutes after baseline blood draw. Plasma was separated

and stored at -80ºC for later measurement of testosterone, glucose, insulin, glucagon-like

peptide-1(GLP-1, active), active ghrelin, and luteinizing hormone (LH). The pizza meal was

provided at 65 minute as it was 60 minutes after completion of the drink (Fig. 5.1), when a

post-glucose load nadir for testosterone is reached in adults [332] and following a mixed

protein and glucose beverage in adolescent males [380].

The experimental beverages contained either 1g of glucose monohydrate (BioShop Canada

Inc., Burlington, Ontario, Canada) or 1g of protein (plain whey-protein isolate; BiPro USA.,

Eden Prairie, Minnesota, U.S.A) per kg of bodyweight. Independent third-party analysis

(Maxxam Analytics, Mississauga, Ontario, Canada) of the protein demonstrated 90.4% of the

powder was protein, with 5.7% moisture, 2.2% ash, 1.18% fat, and 0.6% carbohydrates.

Given these percentages, the protein beverage was 3.74 kcal/kg bodyweight while the glucose

monohydrate beverage was 4 kcal/kg bodyweight. A non-calorie drink was used as control.

All beverages were flavoured with 1.5ml of chocolate extract (Vanilla Food Company,

Markham, Ontario, Canada) to account for the flavour differences and mixed with 500ml of

water, similar to previous protocols [281]. The whey protein and control beverages were

sweetened with 0.2g sucralose (Tate & Lyle, Stoney Creek, Ontario, Canada) in order to

match sweetness with the glucose beverage. Sucralose was chosen as it has been shown to

have no effect on postprandial plasma glucose or insulin [385]. Test beverages were prepared

the evening before the study and refrigerated in order to be served chilled the following

morning. Participants were served the drink in a large covered opaque cup through a straw.

63

The beverage was consumed within 5 min, followed by 50 mL of water to minimize

aftertaste.

After the final blood draw, an ad libitum pizza meal was provided. The participants were

instructed to eat during the next 20 min until they were comfortably full. Based on the

participant preferences determined during screening, two varieties of Deep ‘N Delicious 5-

inch-diameter pizza were provided for consumption; pepperoni or three-cheese pizzas

(McCain Canada Ltd., Florenceville, Ontario, Canada). Pepperoni pizza (87g) contained 9g

of protein, 6g of fat and 23g of carbohydrates for a total energy content of 180 kcal. Each

three-cheese pizza (81g) contained 10g of protein, 6g of fat and 23g of carbohydrate for a

total energy content of 180kcal. The cooked pizzas were weighed and cut into four equal

pieces before serving, and the amount left after the meal was subtracted from the initial

weight and converted to kilocalories to provide a measure of FI. These pizzas lack an outer

crust resulting in a uniform energy content thus mitigating the opportunity for the participants

to eat the energy-dense filling and leave the outside crust of the pizza. The additional water

consumption was determined by weight.


Analysis of testosterone in plasma using gas chromatography-mass spectrometry

Plasma concentrations of total testosterone were measured by gas chromatography-mass

spectrometry (GC-MS) using a modified stepwise procedure for electron capture negative

chemical ionization adapted from the methods of Fitzgerald, Griffin, and Herold appropriate

for both plasma and serum samples [181]. Briefly, 2 ng of 16,16,17-d3 testosterone (Sigma-

Aldrich, St. Louis, MO, USA #T5536-1ML) internal standard was added to 250 µL of

plasma. 2mL of ethyl acetate was added to each sample and samples were then vortexed for 2

min each followed by centrifugation at 1,850 g for 10 min. The organic layer was then

transferred into clean tubes and evaporated under nitrogen at 40°C. Pentafluorobenzyloxime

derivatization were done by adding 50 µL of pyridine and 50 µL of Florox reagent and heated

at 70°C for 1 h. Following which, samples were evaporated under nitrogen at 40°C prior to

trimethylsilylation with BSTFA. Samples were then evaporated under nitrogen at 40°C and

resuspended in 50 µL of heptane for analysis by GC-MS. GC-MS analysis of testosterone

was carried out on an Agilent 5977A single-quadruple mass spectrometer coupled to an

64

Agilent 7890B gas chromatograph (Agilent Technologies, Mississauga, ON) in negative

chemical ionization mode. Derivatized samples were injected via an Agilent 7693

autosampler (Agilent Technologies) into a DB-1 15 m x 0.32 mm (i.d.) x 0.25 µm film

thickness capillary column (J&W Scientific from Agilent Technologies #123-1012)

interfaced directly into the ion source. Injector port and interface temperatures were held at

260°C and 280°C, respectively. The oven temperature was programmed to 160°C for a hold

time of 1 min, followed by a ramp to 280°C at 20°C per minute for a hold time of 3 min,

followed by a ramp to 315°C at 25°C per minute for a hold time of 4 min. The final ramp and

temperature hold was programmed to remove sterol contaminants from the capillary column

between runs. The inlet was operated in splitless mode and helium carrier gas flow rate was

constant at 1.8 mL per min. The reagent gas for negative chemical ionization was methane

(99.999% purity) with a gas flow of 40%. The mass spectrometer was operated in select ion

monitoring, scanning for m/z 535.2 ± 0.5 and m/z 538.2 ± 0.05 corresponding to testosterone

and 16,16,17-d3 testosterone, respectively. Dwell times for each ion were set to 0.1 sec. The

mass spectrometer was calibrated daily and a gain factor of 3.33 was applied. Testosterone

was quantified against a 4-point standard (200ng/dl, 400ng/dl, 800ng/dl and 1200ng/dl) curve

prepared daily. The lower limit for detection of testosterone with this method is 5 ng/dl and

the interassay coefficient of variation (inter-CV) is <5.8%.

Biochemical assays

Concentration of glucose in the plasma was measured using a glucose oxidase kit (Teco

Diagnostics, Anaheim, CA, USA; intra-CV: <4.8%; inter-CV: <4.3%; Cat. # G519-1L).

Insulin was measured using the enzyme-linked immunosorbent assay (ELISA) (ALPCO,

Salem, NH, USA; intra-CV: <10.3%; inter-CV: <16.6%; Cat. # 80-INSHU-E10.1). LH was

also assayed with ELISA (Diagnostics Biochem Canada, London, ON, Canada; intra-CV:

<4.5%; inter-CV: <9.2%; Cat. # 80-CAN-LH-4040). Human active GLP-1 (intra-CV: <8%;

inter-CV: <12.8%; Cat. # EGLP-35K) and acylated ghrelin referred to herein as active

ghrelin (intra-CV: <7.5%; inter-CV: <12.9%; #EZGRA-88K) were also measured with

ELISA kits (Millipore, Billerica, MA, USA).

65


Sample size required to detect a treatment effect on testosterone was assessed using PS Power

and Sample Size Calculations (Version 3.0.43, 2009, Vanderbilt University). The data from

three studies in adult males showed decreases of testosterone of 24% following an OGTT

[10, 365, 366]. Based on these data, and by using mean and standard deviation values from a

study that measured testosterone in boys from T1 to T5 puberty [208], 13 boys were required

to achieve power of 0.8 at α = 0.05. A similar sample size was estimated for FI, based on a

previous study from our laboratory [281] showing effects on caloric intake at a meal 60 min

later when similar glucose and protein beverages were provided to normal and overweight

obese boys. The usual requirements of probability of Type-1 error (α) of 0.05 and Type-2

error (β) of 0.1, i.e., power =0.8, indicated a sample size of 24 participants was required.

However, because a sample size of 22 is required for measures of subjective appetite and FI

[386], a total of 23 participants were recruited.

Statistical analyses were conducted using SAS version 9.4 (SAS Institute Inc, Cary, NC,

USA). For analyses of all measurements, the baseline value was subtracted from postprandial

responses to account for between-subject differences. All data were tested for normality. Due

to the diurnal variation of sex hormones in adolescent males, statistical analysis and

presentation of the results are based on change from baseline. Analysis of covariance

(ANCOVA) of the outcome with the baseline as covariate, and analysis of variance

(ANOVA) of change from baseline are both current and unbiased methods of analysis

employed in randomized studies [387]. ANCOVA is often preferred as it has more power, but

it also assumes absence of a baseline difference. Because the protein group differed

significantly from control (p=0.0293) and from glucose (p=0.0426) at pretest (baseline) for

luteinizing hormone, in order to minimize bias, ANOVA of change from baseline was chosen

as preferred statistical test. Differences in sex hormones (testosterone, LH), gastrointestinal

hormones (GLP-1, ghrelin), glycaemic markers (blood glucose and insulin) and appetite

scores changes from baseline were examined using a two-way repeated measures analysis of

variance via PROC MIXED procedure (PROC GLIMMIX for non-normal data) followed by

Tukey-Kramer post-hoc test to analyze the main effects of treatment, weight and time and its

interactions. When a treatment by time interaction was found, one factor ANOVA was used

followed by Tukey-Kramer post-hoc test to compare the effect of treatments at each time of

measurement. Results were pooled for normal weight and overweight adolescents, unless a

66

significant weight effect was found. Food intake was analyzed by one-factor ANOVA.

Pearson correlation coefficients (for normal data) and Spearman’s rank coefficients (for non-

normal data), were calculated via PROC CORR to detect the associations between

testosterone and LH with appetite scores, GLP-1, ghrelin, blood glucose and insulin. All

results are presented as mean±standard error of the mean (S.E.M.). Statistical significance

was determined at p< 0.05.

5.4 Results

Participant characteristics are presented in Table 5.1. A total of 23 adolescent males age 12

to 18 that were classified in the mid-late pubertal stages completed the study and were

included in the subsequent analysis. Eleven were overweight/obese and were approximately

50% higher in body weight with a three-fold higher fat mass but similar FFM compared to

the 12 normal weight participants.

Average baseline levels of appetite- and sex-related hormones are shown in Table 5.2.

Overweight/obese participants had higher fasting blood glucose and insulin concentration

than the normal weight participants and lower baseline testosterone levels.

Effects of treatments over time on responses in hormones and glucose concentrations

Sex hormones

Plasma testosterone concentrations were affected by treatment (F = 3.59, p = 0.0382) and

weight status (F = 5.26, p = 0.0322), but not by time (F = 1.49, p = 0.2365). An interaction

was found between weight status×treatment (F = 7.38, p = 0.0021). No interactions were

found between treatment×time (F = 0.78, p = 0.5428), weight status×time (F = 0.55, p =

0.5812), or treatment×time×weight status (F = 1.34, p = 0.2654) (Figure 1). Testosterone

concentrations were decreased from baseline over time. The decrease in testosterone after

protein ingestion was prolonged to 65 min. However, the peak response after glucose

occurred at 20 min and was sustained at that concentration to 65 min.

Overall, normal weight subjects had a greater decrease of testosterone when compared to

overweight subjects (-153.31 ± 30.35 ng/dl vs. -51.39 ± 32.44 ng/dl, p = 0.0322) (Figure 5.2

B). The significant interaction between treatment and weight status was attributed to the

67

response (presented as the change from baseline, Figure 5.2 A) in the control as well as

the response to whey and glucose. In the overweight subjects, testosterone response to both

protein and glucose treatments were different from control (p=0.0205, p=0.0022

respectively); however, testosterone response to glucose and protein were not different

(p=0.6223). For normal weight subjects, there were no differences of testosterone response

between any of the treatments (p >0.05).

Plasma LH was affected by treatment (F = 21.37, p <0.0001) but not time (F = 1.26, p =

0.2943) or interaction (F = 1.23, p =0.3051) (Figure 5.3). Both glucose (p = 0.0195) and

protein (p < 0.0001) treatments reduced LH when compared with control, but more after

protein than after glucose (p = 0.0016). The response to protein, but not glucose was apparent

at 20 min.

Glucose, Insulin, GLP-1, and Ghrelin

Plasma glucose concentrations were affected by treatment (F = 118.70, p<0.0001), time (F =

5.08, p = 0.0107) and a time×treatment interaction (F = 4.54, p = 0.0035) (Figure 5.4). Post-

treatment plasma glucose concentrations were higher at all times after the glucose treatment

when compared to both protein and control beverages (p < 0.05) with no differences between

protein and control treatments.

Plasma insulin (Figure 5.5) was affected by time (F = 6.49, p = 0.0036), treatment (F =

115.96, p < 0.0001) and a time×treatment interaction (F = 4.67, p = 0.002). Post-treatment

plasma insulin concentration was higher after glucose, than after protein and higher after both

than control all time points (p < 0.05).

GLP-1 was affected by treatment (F = 18.26, p < 0.0001) but not by time (F = 1.48, p = 0.24)

(Figure 5.6). There were no significant interactions between treatment×time (F = 2.09, p =

0.09). GLP-1 (active) average concentrations were similarly increased by both the glucose

and protein beverages when compared to the control beverage (p = 0.0003, p <0.0001,

respectively). Glucose and protein were not different (p = 0.2495).

There was an effect of treatment (F = 29.72, p < 0.0001) on mean active ghrelin, with no

effect of time (F= 0.37, p = 0.69) and no interactions of treatment×time (F = 1.23, p < 0.3)

(Figure 5.7). Ghrelin (active) was decreased by glucose (p < 0.001) and protein (p < 0.001)

68

when compared to control with no difference between glucose and protein treatment (p

= 1308).

Appetite

There was an effect of time (F = 9.05, p = 0.0005) and treatment (F = 5.09, p = 0.0103) on

appetite (Figure 5.8) with no treatment×time interaction (F = 0.26, p = 0.904). The glucose

beverage, but not protein, lowered post-treatment subjective appetite when compared with the

control (p = 0.0198). Additionally, glucose treatment appetite score was lower than after the

protein treatment (p =0.0247).

Food Intake

There was no effect of the treatment (F = 1.79, p = 0.782) or weight status (F = 2.23, p =

0.14) on total caloric intake. Mean FI for the control, glucose and protein treatments were

1409.93 ± 97.7 kcal, 1280.18 ± 95.2 kcal and, 1325.18 ± 95.9 kcal, respectively.

Correlations

Post-treatment decrease of testosterone was correlated with decreased LH (r = 0.27, p =

0.0050) and was inversely correlated to increases in GLP-1 (r = -0.39, p = 0.0004). Lower

post-treatment testosterone concentrations over time did not correlate with ghrelin (r = 0.20, p

= 0.1531), glucose (r = 0.25, p = 0.1398), or insulin (r = -0.22, p = 0.1455) (Table 5.3).

Testosterone decrease was not correlated to average appetite (r = 0.05, p = 0.7456). Neither

was testosterone change from baseline before the meal with FI (r = 0.14, p = 0.2800). LH was

not correlated to any other parameter but testosterone (Table 5.3).

69

5.5 Discussion

The results of this study support our hypothesis that macronutrient composition of food

affects the acute post-ingestion response of testosterone in adolescent boys. However, they

provide no support for the hypothesis that these responses contribute to regulation of appetite

or FI.

The testosterone decrease in response to protein and glucose ingestion is consistent with

previous research. A decrease in testosterone has been reported in adult males after glucose

ingestion [10] and in overweight adolescent boys given a combined protein/glucose beverage

[380]. The novel contribution of this study is that it shows that whey protein suppresses

testosterone, and may have a longer duration of effect than glucose suppression, indicating

that the effects of food ingestion are not only explained by energy sensing. Further support

for a greater role of protein over carbohydrate ingestion and its greater duration of effect

arises from observations of lower fasting testosterone concentrations in adult males following

high protein (low carbohydrate) diets [334, 335]. The slight but statistically insignificant

change of testosterone in the control treatment is concordant with normal diurnal rhythms of

testosterone in adolescent males who typically display early morning peak testosterone and

late evening nadir [203]. Furthermore, obesity has been shown to alter diurnal patterns of

testosterone in males and lower morning testosterone values [388]. Though both groups had

elevated LH levels during the control treatment, overweight subjects had significantly lower

testosterone at baseline. It is therefore possible that overweight boys are more responsive to

increased LH, however this cannot be concluded from these results.

The decrease of testosterone may be independent of LH and initiated by the intake of glucose

or amino acids, particularly leucine, which stimulates rapamycin (mTOR) signaling and

subsequent protein synthesis [389]. Leucine and glucose also inhibit adenosine

monophosphate-activated protein kinase (AMPK) [390, 391], which is upstream of mTOR

and prevents protein biosynthesis [392] in addition to inhibiting androgen receptor (AR)

mRNA expression [393]. Inhibition of AMPK through glucose or protein intake may increase

AR expression leading to greater testosterone uptake by the muscle tissue which would lower

plasma testosterone levels.

No associations were found between the testosterone and appetite responses. Lower post

treatment mean testosterone concentrations over time cannot be interpreted as support for any

70

effect of testosterone on appetite. Pre-meal testosterone concentrations were not

correlated to appetite or FI. However, the appetite responses are consistent with the known

effects of carbohydrate ingestion on short term appetite [382], but contrast to other reports

that protein increases satiety in both adults [383] and adolescents [281]. In overweight and

obese boys, appetite decreases after both protein and glucose ingestion [357], but glucose

suppresses FI more than whey protein whereas normal weight boys respond equally to both

[281].

The lack of effect of the treatments on FI is surprising because the doses were adjusted for

body weight to accommodate the range of bodyweights of the participants. In a previous

study, protein and glucose beverages provided at 1g/kg body weight to 12 normal and 12

overweight obese boys, averaging 12 y of ages, reduced FI at a meal 60 min later by an

average of 17% with a greater reduction after protein [281]. Although the different results of

this and the prior study cannot be explained, the absence of effect of the treatments on FI also

provides evidence that testosterone does not function in short-term FI regulation.

Testosterone concentrations after protein were at their lowest 60 min immediately prior to the

ad libitum pizza meal, yet no correlation was found between FI and testosterone at 60 min. It

remains possible, however, that a relationship would be found by extending the measures of

the testosterone and the time of the later meal.

Although the study provides no evidence that testosterone is a regulator of short term FI, the

physiological effects of its acute decrease in response to the composition food merits further

exploration. LH was measured because of its role in testosterone production. An interaction

between treatment and time was indicated because LH increased over time in the control but

decreased over time after protein and glucose (Figure 5.3). These responses were positively

(r = 0.59, p < 0.05) associated with testosterone, consistent with the known interactions of

these hormones. LH stimulates testosterone production via the Leydig cells of the testicles

[74] and ghrelin suppresses the secretion of LH [394] and is present in Leydig cells [320].

Although few appetite hormones were measured, the pooled data show expected associations

among them and with appetite. Conversely, none associated with testosterone, adding further

doubt to any physiological significance of the association between testosterone and appetite.

Their lack of association with FI is consistent with the literature showing little predictive

strength in appetite hormone measures and overconsumption of foods in adolescent

populations [267, 395].

71

Consistent with previous reports [9], overweight/obese boys had lower testosterone

levels in comparison to the normal weight boys. Low testosterone concentrations in obese

males result from aromatization of testosterone within adipose tissue [305, 311]. Weight

status interacted with treatment effects on testosterone; normal weight subjects had a greater

decrease in testosterone compared with overweight and obese boys. This may be due to lower

baseline levels and insulin resistance. Insulin was higher at baseline with overweight/obese

boys as is commonly observed in insulin resistant subjects [396]. This could in part be

explained by obesity related insulin resistance which impedes insulin facilitated hypothalamic

GnRH secretion, further depressing testosterone production in obese subjects [397, 398].

Due to the composition of the protein powder, there were minor caloric differences between

the glucose and protein treatments; however, this difference is an unlikely factor in

determining the results. The mean of the overweight subjects in this study was 88.5± 4.7 kg

compared to normal weight 63.8± 3.5 kg resulting in treatment differences of only

approximately 23 kcal for OW and 6 kcal for NW.

Further studies of the effect of quantity and protein source (rapidly vs. slowly digested) are

also needed to explain mechanisms leading to reduction of plasma testosterone and the

physiological significance of these acute effects of food. Additionally, the decrease in LH

after protein or glucose ingestion is a novel observation, and further exploration of the acute

effect of glucose and protein on sex hormone response in girls and women is merited.

5.6 Conclusions

Protein and glucose ingestion results in acute decreases in both plasma testosterone and LH in

adolescent males. The physiological significance of this response remains to be determined as

no support for a role of testosterone in acute regulation of food intake was found.

72

Table 5.1. Participant characteristics

Normal Weight

(n = 12)

Overweight

(n = 11)

Age 14.3 ± 0.4 15.6 ± 0.5

Height (cm) 172.9 ± 2.7 171.8 ± 2.3

Weight (kg) 63.8 ± 3.5a 88.5 ± 4.7b

Fat-Free Mass (kg) 53.6 ± 3.1 59.1 ± 1.8

Fat Mass (kg) 10.3 ± 1.1a 29.4 ± 3.4b

Body Fat % 16.0 ± 1.7a 32.3 ± 2.0b

BMI (kg/m²) 21.1 ± 0.9a 29.8 ± 1.2b

BMI %ile (WHO) 67.5 ± 7.1a 97.3 ± 1.0b

1All values are means ± SEM, n = 23. Values in the same row with different superscript letters are significantly

different, p < 0.05.

Table 5.2. Baseline levels of appetite- and sex-related hormones

Normal Weight

(n = 12)

Overweight

(n = 11)

Testosterone (ng/dl) 1048.71 ± 103.21a 765.49 ± 46.12b

LH (IU/L) 3.65 ± 0.30 3.46 ± 0.38

Ghrelin, active (pg/ml) 288.37 ± 33.54 174.33 ± 29.22

GLP-1, active (pM) 4.81 ± 0.37 7.93 ± 1.51

Insulin (uIU/ml) 6.77 ± 0.78a 14.21 ± 1.38b

Glucose (mg/dl) 90.69 ± 1.96 97.15 ± 1.85

Values were calculated by averaging the baseline levels of all three of the sessions for each marker. All values

are means ± SEM, n = 23. Values in the same row with different superscript letters are significantly different, p

< 0.05.

73

Table 5.3. Relationships between testosterone and luteinizing hormone with dependent measures (Δ from

baseline means)1.

Dependent Measure Testosterone (ng/dl) Luteinizing hormone (IU/L)

Appetite score (mm) NS NS*

Luteinizing hormone (IU/L) r = 0.27338 -

GLP-1 (pM) r = -0.38993 NS*

Ghrelin (pg/ml) NS NS

Glucose (mg/dl) NS NS

Insulin (pg/ml) NS NS

1Correlation coefficients (r) indicate statistically significant associations between dependent measures, p<0.05.

NS=nonsignificant.

*Indicates Pearson correlation was calculated, otherwise Spearman’s rank coefficient was determined.

0 5 20 35 65 85 minutes

Testosterone

Luteinizing Hormone

GLP-1

Ghrelin

Glucose

Insulin

10 – 12

hour fast

Pizza meal

Treatment order

randomized

Experimental Beverage

(a) 1g/kg glucose + 500mL water

(b) 1g/kg protein + 500mL water

(c) Sucralose + 500mL water

Blood Samples

Figure 5.1. Experimental protocol. Subjects arrived in the laboratory after a 10-12h fast and were randomly

assigned one of three beverage conditions.

74

Figure 5.2. Effect of glucose and protein on plasma testosterone over time. Statistics were performed on change

from baseline. Treatment: p =0.0382, Weight Status: p = 0.0322, Weight status×Treatment p = 0.0021, Time: p

= 0.2365, TRT*Time: p =0.5428, by two-way repeated measures ANOVA. Different superscripts are

significantly different by Tukey–Kramer post hoc test, p <0.05. All values are means±S.E.M.s; Total n=22,

Normal Weight n=12, Overweight n=11.

Note: Embedded panels represent differential effects of treatments by weight status (A) and overall effect of

weight status on plasma testosterone (B).

Figure 5.3. Effect of glucose and protein on plasma luteinizing hormone. Statistics were performed on change

from baseline. Treatment: p <0.0001, Time: p = 0.2943, TRT*Time: p =0.3051, by one-way repeated measures

75

ANOVA. Different superscripts are significantly different by Tukey–Kramer post hoc test, p <0.05. All

values are means±S.E.M.s; n=23.

Figure 5.4. Effect of glucose and protein on plasma glucose. Treatment: p <0.0001, Time: p =0.0107,

TRT*Time: p =0.0035, by one-way repeated measures ANOVA. Different superscripts are significantly

different by Tukey–Kramer post hoc test, p <0.05. All values are means±S.E.M.s; n=23.

Figure 5.5. Effect of glucose and protein on plasma insulin. Treatment: p <0.0001, Time: p =0.0036,



76

Figure 5.6. Effect of glucose and protein on plasma GLP-1. Treatment: p <0.0001, Time: p = 0.2382,



Figure 5.7. Effect of glucose and protein on plasma ghrelin. Treatment: p <0.0001, Time: p = 0.6923,



77

Figure 5.8. Effect of glucose and protein on appetite score. Statistics were performed on change from baseline.

Treatment: p =0.0103, Time: p = 0.0005, TRT*Time: p =0.9042, by one-way repeated measures ANOVA.

Different superscripts are significantly different by Tukey–Kramer post hoc test, p <0.05. All values are

means±S.E.M.s; n=23.

78

Chapter 6

ACUTE DECREASE IN APPETITE IS UNRELATED TO

INCREASE IN PLASMA TESTOSTERONE DURING HIGH

INTENSITY CYCLING IN ADOLESCENT MALES

The following chapter is a reproduction of a manuscript that will be submitted for publication

to Applied Physiology, Nutrition and Metabolism

79

ACUTE DECREASE IN APPETITE IS UNRELATED TO INCREASE IN PLASMA TESTOSTERONE DURING HIGH INTENSITY CYCLING IN ADOLESCENT MALES

Alexander Schwartz1, 2, Sascha Hunschede1, R.J. Scott Lacombe1, Ruslan Kubant1, Diptendu

Chatterjee1, Diana Sánchez-Hernández1, Richard P. Bazinet1, Jill K. Hamilton1, 2, G. Harvey

Anderson1,


M5S 3E2, Canada




G. Harvey Anderson, PhD.

Department of Nutritional Sciences


1 King’s College Circle

Toronto (On), Canada, M5S 3K1

Phone: 1-416-9781832


80

6.1 Abstract

Background and Objectives: High intensity exercise (HIEX) suppresses appetite, but the

mechanism is unknown. Appetite regulating hormones have been explored. However, the

effect of aerobic HIEX on the increase in testosterone and its relationship with appetite and

appetite hormones has yet to be described. Therefore, the effect of HIEX on testosterone

levels and the relationship between testosterone responses, selected appetite hormone and

appetite was explored in teenage males.

Participants and Methods: In a randomized, crossover study, fifteen adolescent boys age

16.3 ± 0.3 completed two sessions of either: 1) three 10-minute bouts of HIEX cycling at

75% VO2peak or 2) remained at rest. Plasma testosterone, PYY, ghrelin, GLP-1 and

subjective appetite were measured before (0 min) and after (60 min) HIEX.

Results: Plasma testosterone concentrations increased by 21.5 ± 13.6% after 60 min of HIEX

when compared to rest (p = 0.0215). HIEX reduced ghrelin (p < 0.01), desire to eat (p =

0.026) and hunger (p = 0.022). However, testosterone was not correlated to appetite (r = -

0.05, p = 0.87), ghrelin (r = -0.14, p = 0.67), GLP-1 (r = -0.02, p = 0.95 or PYY (r = 0.29, p =

0.41) responses during HIEX.

Conclusion: The increases in plasma testosterone levels after aerobic HIEX in adolescent

males does not associate with the acute decrease in appetite or responses in appetite

hormones after HIEX.

81

6.2 Introduction

In adult males, both anaerobic and aerobic modes of exercise, including weight lifting [337]

or cycling [338] and running [339] increase plasma testosterone. High-intensity exercise

(HIEX) reduces appetite and alters levels of appetite hormones [272, 399, 400] and

inflammatory responses [401, 402]. However, the mechanism by which HIEX reduces

appetite is unknown, but a role for testosterone can be suggested from several lines of

evidence.

Elevated testosterone may be related to food intake (FI). It has been linked with modifying

taste acuity [378] and a reduced risk of peri and post-pubertal eating disorders [377]. A role

for testosterone in determining FI is indicated by observation that FI of pre-pubertal and

adolescent boys is higher when compared to weight, height, and aged-matched females [264].

Animal studies support a role for testosterone and FI; Orchiectomy decreases FI in rodents

[331, 332], but can be restored by testosterone injections to match normal physiological

levels [331, 332]. Injections of testosterone also increase FI in adult rats with intact testicles

[333].

Consumption of glucose or protein beverages are known to suppress appetite [281]. Recent

studies also show that testosterone decreases immediately after adults consume a glucose

beverage during an oral glucose tolerance test [10] and after children consume a mixed

protein-carbohydrate beverage [380], or adolescents consume either protein or glucose

beverages. In the latter, the decrease in testosterone was not associated with a decrease in

appetite [403]. However, associations do not show cause and effect, and it has yet to be

determined whether an increase of testosterone is associated with appetite in adolescent boys.

HIEX, based on studies in adults, provides an alternative approach to providing direct

injections of testosterone into healthy adolescents. In adolescents, HIEX decreases subjective

appetite [401] however this does not typically reflect subsequent food intake [404, 405] and

studies in populations with low or no testosterone such as obese [406] and women [407]

show varied effects on appetite after HIEX.

As with adult males [337-339] adolescent males have demonstrated increased plasma

testosterone after high intensity anaerobic exercise [344] but studies measuring the effect of

aerobic exercise in this population [340, 346] failed to yield results due to methodological

82

shortcomings such as inadequate exercise intensity and poor sensitivity for detecting

lower levels of testosterone in adolescents [408].

Therefore, the hypothesis of this preliminary study was that acute HIEX increases

testosterone and decreases appetite in adolescent boys but the responses are unrelated. The

objective of this study was to determine the effect of high-intensity repeated bouts of HIEX

cycling on testosterone levels and appetite, and the relationship between these responses, in

adolescent males aged 13 to 18.


6.3.1 Subjects

Subject recruitment was as previously described in detail [402]. Normal weight healthy

adolescent males age 13 to 18 years were recruited via print ad in the Toronto Star

newspaper.

A telephone questionnaire was employed to determine eligibility for this study. Weight status

was defined using the Center for Disease Control BMI charts (BMI for age percentile: 15th-

85th) [409]. Questions pertaining to physical activity readiness and eating habits were

administered and scored. Exclusion criteria included fear of venipuncture, dieting history, and

diabetes or other metabolic diseases. All experimental procedures have been approved by the

University of Toronto Health Sciences Research Ethics Board, and informed consent was

obtained from all participants, parents of the subjects as well as assent from the subjects

themselves. Originally 21 participants were recruited through local advertisement; however, 6

participants did not complete the study, due to a mild form of vasovagal syncope, likely due

to the IV-catheter insertion or due to difficulties scheduling the sessions.

6.3.2 Protocol

Design:

Blood samples for this secondary analysis were obtained from a study determining the effects

of high-intensity exercise (HIEX) and anti-inflammatory medication on the relationship

between inflammation and appetite [402]. In that study, 15 NW boys (aged 13-18y) were

83

randomly assigned in a crossover design to four treatments. Treatments were

1)Water+Rest; 2)Rest+Ibuprofen (IBU); 3)Water+HIEX; 4)HIEX+IBU. IBU (300mg) was

provided in 250ml water. Data reported here are for the groups when exposed to the water at

rest and water and HIEX treatments only. IBU groups were excluded due to the previously

demonstrated inhibition of testicular testosterone production after acute IBU intake resulting

in compensated hypogonadism [410].

HIEX consisted of three separate bouts of recumbent cycling between 60-70RPM at 75%

VO2Peak for 10 minutes per bout, similar to Thivel et al. [411]. Each exercise bout was

followed by 1:30min of rest. Appetite ratings and plasma biomarkers of appetite,

inflammation, stress and glucose control were measured. Upon arrival a catheter was inserted

and at time 0 visual analogue scales were administered. At five minutes the participants drank

water alone or with IBU. HIEX started at 30 min and terminated at 60 min (Figure 6.1).

Blood was collected and visual analog scale questionnaires were completed at baseline (0

min) and 65 min after the start of each session.

Participant Assessment

The assessment and protocol were as previously described in detail [402]. Initial screening of

participants was performed at the University of Toronto - Goldring Centre for High

Performance Sports. Anthropometric measures such as age, height, body-weight, BMI, BMI

for age percentile and percent body-fat were recorded and physical fitness was assessed via

indirect calorimetry. Ventilatory gases were collected using a Moxus metabolic cart (AEI

Technologies Inc.`, Pittsburgh`, PA`, USA)`, a facemask`, and a 2-way non-rebreathing valve

(Hans Rudolph`, Inc.`, Shawnee`, KS`, USA). Inspiratory ventilation was measured with a

pneumotachometer`, the O2 and CO2 contents of mixed expired gas with an S-3A Oxygen

Analyzer`, and CO2 content with a CD-3A 251 Carbon Dioxide Analyzer (AEI Technologies

Inc.`, Pittsburgh`, PA`, USA). Prior to each test`, the metabolic cart was calibrated with

known gas concentrations of 16.04% O2 and 4.06% CO2 and 20% O2 and 0.03% CO2. The

Moxus metabolic cart has been validated over wide measurement ranges using two sensors

for ventilation against the Douglas bag method [412]

The HIEX was conducted on a Kettler RE7 recumbent bicycle (Kettler, Ense-Parsit, NRW,

Germany). The VET was assessed using the ventilatory equivalent method [413]. and

VO2peak determined using the highest six consecutive breaths [414]. Body composition was

84

estimated by bioelectrical impedance analysis (RJL Systems BIA, 101Q) using the

Horlick equation the Horlick equation [381]. All sessions were conducted at the University of

Toronto Athletic Center on weekends between 9 and 10 am after a 12-h overnight fast.

Visual Analog Scales

Visual Analog Scales were provided to participants as previously described to evaluate

subjective appetite [414-416]. Four questions measuring desire to eat, hunger, fullness and

anticipated food intake were fixed at each end with contrasting terms on the 100 mm scale.

Participants marked an “x” along the 100 mm line to reflect their feelings. The scales have

been previously validated [281, 417].


Blood samples for measurement of testosterone, GLP-1, ghrelin, and Peptide YY3-36 (PYY)

were as described previously [402, 418]. Blood was collected into pre-chilled 10 mL BD

VacutainerTM (BD Diagnostics, Sparks, MD, USA) at baseline (0 min) and 65 min. Blood

collection tubes contained spray dried K2EDTA anticoagulant, and a proprietary cocktail of

protease inhibitors [e.g. DPP-IV (R-3-Amino-1-{3-(trifluormethyl)-5,6,7,8-

tetrahydro[1,2,4]triazol[4,3-a]pyrazin-7-yl}-4-(2,4,5-trifluorphenyl)butan-1-on), AEBSF (4-

(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride) and aprotinin (Trasylol)] to prevent

the proteolytic breakdown of hormones. Immediately after collection, plasma was separated

by centrifugation for 15 min at 2000 RCF at 4 °C, then aliquoted into 2 mL Eppendorf

(Eppendorf, Hamburg, Germany). Furthermore, 200 µL 1 N HCl was added to every 1 ml of

plasma collected for ghrelin analysis.

Human active GLP-1 (intra-CV: <8%; inter-CV: <5%; #EGLP-35K) and total and acylated

ghrelin (intra-CV: <2%; inter-CV: <8%; #EZGRT-89K) were also measured with ELISA kits

(Millipore, Billerica, MA, USA). Total PYY [i.e. PYY (1-36 amide) and PYY (3-36) was

also measured using commercial ELISA kits purchased from Millipore (Millipore, Billerica,

MA, USA) (intra-CV: <5.8%; inter-CV: <16.5%; #EZHPYYT66K,). Samples were stored at

-80ºC until analysis, and all samples were run in duplicate.

85

Analysis of testosterone in plasma using gas chromatography-mass spectrometry

Plasma concentrations of total testosterone were measured as previously described in

5.3.3[418] by gas chromatography-mass spectrometry (GC-MS) adapted from Fitzgerald,

Griffin, and Herold [181]. Testosterone was quantified against a 4-point standard (200ng/dl,

400ng/dl, 800ng/dl and 1200ng/dl) curve prepared daily. The lower limit for detection of

testosterone with this method is 5 ng/dl and the interassay coefficient of variation (inter-CV)

is <5.8%.


Change in testosterone was used as the primary measure to calculate sample size. Sample size

required to detect a treatment effect on testosterone was assessed using PS Power and Sample

Size Calculations (Version 3.0.43, 2009, Vanderbilt University). Published data from the

previous studies in our lab showed decreases of testosterone of 9-24% following glucose and

protein treatments [380, 418]. Based on this data, 14 boys were required to achieve power of

0.8 at α = 0.05. A similar sample size was estimated for measures of subjective appetite and

has previously been validated to be reproducible for subjective appetite measures after

exercise [386].

Paired sample t-tests were used to compare the absolute change of testosterone and other

blood measures in addition to VAS between HIEX and rest. Non-parametric paired t-tests

were used for non-normal data. Pearson correlation coefficients (for normal data) and

Spearman’s rank coefficients (for non-normal data), were calculated via PROC CORR to

evaluate testosterone compared to measures of VAS, ghrelin, PYY and GLP-1 as well as

associations among all measures. All analyses were performed with Statistical Analysis

Software, version 9.2 (SAS Institute Inc., Carey, NC, USA). The data are expressed as means

± SEM. Statistical significance was declared at P < 0.05.

86

6.4 Results

A total of 15 adolescent boys age 16.3 ± 0.3 years old completed the study. Subjects were

174.8 ± 1.9 cm tall and weighed 65.4 ± 2.3kg with 23.4 ± 2.0% body fat and were considered

normal weight under CDC BMI percentile (58.3 ± 5.1). Average VO2max values were 44.7 ±

1.2 ml/kg/min, which is considered slightly below the age average [419]. All participants had

fasting testosterone values that were expected during mid-late puberty (>150ng/dl) [215] and

no differences we detected at baseline between rest (868.16 ± 56.2 ng/dl) and HIEX (793.7 ±

50.8 ng/dl, p = 0.31).

Effects of exercise over time on responses in hormone and glucose concentrations

A. Testosterone.

Testosterone increased 21.5 ± 13.6% (126.99 ± 87.2 ng/dl) from baseline after 65 minutes of

HIEX which was greater than when compared to testosterone change after 65 minutes of rest

(-97.76 ± 90.4 ng/dl, p = 0.022) (Table 6.1). The mean testosterone increase from HIEX was

significantly greater than the mean decrease observed during rest with a difference of 256.1 ±

90.1 ng/dl (p = 0.019) (Table 6.2).

B. Ghrelin, GLP-1, PYY, Glucose, Insulin,

Changes in plasma hormones are shown in Table 6.1. The decrease in ghrelin was greater

after HIEX when compared with rest (p = 0.0017). There was no difference of change in

GLP-1 (p = 0.81) or PYY (p = 0.46) between rest and HIEX.

Glucose was not affected by HIEX and there was no difference from rest values (p = 0.37).

Though there was a trend of HIEX to decrease insulin, there was no difference of change in

insulin between rest and HIEX (p = 0.73).

C. Appetite

Mean subjective appetite change was not affected by HIEX (-3.57 ± 8.2mm) when compared

to rest (9.97 ± 5.1mm, p = 0.08). HIEX decreased the desire to eat (-2.13 ± 8.9mm) and

hunger (-0.4 ± 7.3mm), which were different from the increases seen at rest (18 ± 6.5mm, p =

0.026 and 16.8 ± 7.3mm, p = 0.0217 respectively). There were no differences in the change

of fullness (p = 0.69) or prospective FI (p = 0.17) between HIEX or rest.

87

Correlations:

Absolute testosterone values were not significantly correlated to the subjective mean appetite

score at either rest (r = 0.06, p = 0.78) or HIEX (r = -0.07, p = 0.71). Testosterone during

HIEX was correlated to PYY (r = 0.54, p = 0.007) but not at rest (r = 0.05, p – 0.84). In

addition, absolute testosterone levels during rest and HIEX were not correlated with ghrelin

(REST; r = 0.2, p = 0.32) and (HIEX; r = 0.2, p = 0.32), or GLP-1 (REST; r = 0.1, p = 0.7)

and (HIEX; r = 0.01, p = 0.95). None of the appetite hormones correlated with subjective

appetite. Mean subjective appetite during rest and HIEX was not associated with ghrelin

(REST; r = 0.005, p = 0.98) and (HIEX; r = 0.03, p = 0.88), PYY (REST; r = -0.28, p = 0.19)

and (HIEX; r = 0.09, p = 0.64), or GLP-1 (REST; r = 0.17, p = 0.43) and (HIEX; r = -0.25, p

= 0.21) respectively.

Change from baseline in testosterone during rest and HIEX was not correlated with change in

GLP-1 (REST; r = -0.16, p = 0.5) and (HIEX; r = -0.02, p = 0.95), ghrelin (REST; r = - 0.15,

p = 0.49) and (HIEX; r = -0.14, p = 0.67) or PYY (REST; r = 0.07, p = 0.78) and (HIEX; r =

0.29, p = 0.41) respectively. Furthermore, change in testosterone during both rest (r = -0.04, p

= 0.9) and HIEX (r = -0.05, p = 0.87) was not correlated to mean appetite score change.

6.5 Discussion

The results of this preliminary study show that HIEX acutely increased testosterone and

reduced appetite and ghrelin in adolescent males. However, the increases in testosterone did

not correlate with changes in appetite or appetite hormones.

The results of this study demonstrate for the first time that acute bouts of aerobic activity

increase plasma testosterone levels in teenage males. Research examining the effect of

exercise in boys has focused primarily on longitudinal interventions/exercise programs, and it

has been observed that training in young individuals may modify sex hormone responses

[341, 342]. The majority of previous studies reported chronic increases in testosterone.

Though prior research on adolescent males has shown an acute testosterone increase with

anaerobic training methods such as plyometrics and resistance training [343] or sprinting

[345], no increases have been shown with high-intensity aerobic modalities. Prior research by

Hackney et al. indicated that there were no significant differences between pre-exercise and

post exercise testosterone levels in late-pubertal males who performed 20 minutes of

88

incremental exercise to exhaustion using a cycle ergometer [346]. This study suffered

some major limitations, in particular the lack of a control session. Furthermore, the males

were only fasted for three hours before testing which could impact the baseline testosterone

levels as we and others have previously shown that food intake influences acute changes in

testosterone [10, 380]. Additionally, testosterone was measured using immuno-

chemoluminescence which may not be a reliable method to detect small changes in

testosterone as compared to the LC-MS or GC-MS [212].

The physiological mechanisms accounting for the increase in testosterone after exercise have

not been elucidated. There have been several studies using different forms of exercise which

have indicated that the level of LH (gonadotropins) in plasma does not change after exercise

[11-13]. However, lactate production may be a factor. With both anaerobic and aerobic

exercise, there is an increased production of lactate by glycolysis glycogenolysis within

skeletal muscle tissue [347]. In male rats, infusion of lactate resulted in a dose-dependent

increase of testosterone synthesis from production by 25-OH-cholesterol in the Leydig cells.

In pubertal males, short-term exercise elicits a gradual increase in maximal levels of muscle

and plasma lactate but the increases are dependent on pubertal maturation [420-422]. Thus, it

has been proposed that lactate directly affects the Leydig cell cAMP production of

testosterone and that P450scc is a target of lactate [14]. This hypothesis has been supported

by a report showing that exercise-induced lactate production resulted in dose-dependent

increases in testosterone and testicular cAMP in rats [348].

The reduction in ghrelin after HIEX in adolescent males when compared to rest is consistent

with previous work from our laboratory [401] and that of others in adults [399]. The

reductions in ghrelin from HIEX were accompanied with reductions of subjective appetite,

though there was no effect on GLP-1 or PYY. Appetite hormones have been hypothesized to

be responsible for the post-exercise reduction in appetite in males [267, 423] though direct

associations have not been reported. However, this hypothesis was not supported in this study

due to a lack of correlations between appetite hormones and subjective appetite.

The decrease in ghrelin suggests it may play a more important role in appetite of male

adolescents during HIEX. Others have reported that as a consequence of exercise, ghrelin

decreases [424-429] with a concomitant increase in the anorexigenic hormones PYY [428,

430] and GLP-1 [431]. However, as in this study, others have not found the concurrent

responses with PYY and GLP-1 [400, 432, 433] suggesting that there is a lack of compelling

89

evidence any single appetite hormone contributes to appetite suppression during exercise

[267].

Furthermore, this is the first study to examine the relationship of appetite with testosterone

and exercise in adolescent males. As hypothesized, HIEX increased plasma testosterone,

allowing us a further test of previous studies suggesting the decrease in testosterone and

appetite after food ingestion indicates it has a role in food intake regulation [380, 403].

However, the lack of relationship between HIEX testosterone and appetite, and the noted

increase in testosterone while appetite decreased rather than increased, contrasts with our

previous study. Furthermore, though absolute values of testosterone were correlated with

PYY during HIEX only, there was no correlation between the changes of these hormones.

Taken together, there is little support for testosterone in short-term appetite regulation,

leaving unexplained why it responds to food intake. Alternatively, HIEX may not be a

suitable model for examining appetite mechanisms.

The lack of an association between testosterone, appetite and appetite hormones may also be

explained by disrupted splanchnic blood flow during HIEX. Splanchnic blood flow is

increased postprandially to aid digestion, releasing appetite regulating hormones ghrelin,

PYY and GLP-1 to signal nutrient availability and satiety [434-438]. In contrast, during

HIEX, splanchnic blood flow is decreased from the gut due to the increased oxygen demand

of contracting skeletal muscle [439].

Furthermore, more frequent blood measures during the exercise sessions may have provided

a greater picture of testosterone change and how it may relate to appetite during exercise.

However, due to blood sampling restrictions from the University of Toronto Research Ethics

Board in adolescents, this was not possible.

6.6 Conclusions

In conclusion, aerobic HIEX acutely increases plasma testosterone concentrations in males

but the increase in testosterone from HIEX was not related to appetite or appetite hormones.

90

Table 6.1. Plasma testosterone, GLP-1, PYY, ghrelin, glucose and insulin in response to rest and HIEX1.

0 - 65

Rest HIEX

Testosterone(ng/dl) -97.76 ± 90.4 a 126.99 ± 87.2 b

GLP-1 (pM) -0.18 ± 0.4 -0.15 ± 0.5

PYY (pg/ml) -4.61 ± 4.7 4.1 ± 3.0

Ghrelin (pg/ml) -12.7 ± 23.8 a

-210.41 ± 46.9 b

Glucose (mmol/l) -0.15 ± 0.1 -0.46 ± 0.1

Insulin (mg/dl) 1.25 ± 5.2 -6.82 ± 6.5

1Values are means ± SEM, n = 10 for testosterone, n = 15 for all other hormones. Statistics were performed on

change from baseline. Values from the same categorized column in the same row with different superscript

letters are significantly different, P < 0.05.

91

Table 6.2- Effect of HIEX and rest on plasma testosterone levels between 0 and 65 minutes on each individual

subject. Paired sample t-tests were performed to determine differences in change between rest and HIEX.

from 0 - 65

DIFFERENCE

(HIEX - Rest)

SUBJECT Rest

(ng/dl)

HIEX

(ng/dl)

AS17 -392.1 134.1 526.3

AU07 -183 197.1 380.1

AW02 31.0 124.5 93.5

BC15 . . -

BT03 . . -

CN05 248.6 935.5 686.6

JH01 -26.8 220.8 247.6

JJ10 . 99.5 -

KP14 47.6 . -

KR16 71.3 157.8 86.5

NC12 -795.6 -369.6 426.0

NH09 -150.3 -82.4 67.9

SJ18 417.8 102.9 -314.8

TG04 . -76.4 -

VL06 -281.0 80.1 361.1

MEAN DIFFERENCE (HIEX - Rest)

256.1 ± 90.1 ng/dl1

1The mean increase of testosterone change was significantly higher (p = 0.019) following HIEX when compared

to the mean decrease from rest with a difference of 256 ± 90.1ng/dl between HIEX and rest. All values are

means ± SEM, n = 10.

92

Figure 6.1- Experimental protocol as described in Hunschede et al. [402]. Subjects arrived in the laboratory

after a 12h fast and were randomly assigned to either rest or high intensity exercise (HIEX).

93

Chapter 7

GENERAL DISCUSSION

94

GENERAL DISCUSSION

The hypothesis that testosterone has an acute response to food intake and HIEX, and has

interactive relationship with the short-term regulation of appetite and food intake in

adolescent males was partially supported by these studies (Figure 7.1). They are the first to

show an acute response of testosterone to food ingestion and HIEX, but did not demonstrate

that the testosterone response has an interactive relationship with short-term food intake and

appetite regulation. Testosterone was suppressed by both protein and glucose consumption,

though the suppression was more sustained after protein (Chapters 4 and 5) and was elevated

after high-intensity aerobic cycling when compared with rest (Chapter 6). The responses of

plasma testosterone were not associated with a decrease in appetite and FI or appetite-related

hormones.

Figure 7.1- Summary of experimental results. Consumption of both protein and glucose resulted in decreased

testosterone levels and the decrease persisted after protein intake when compared to glucose (Study 1 and 2).

Testosterone was elevated after high-intensity aerobic cycling when compared with rest (Study 3). Plasma

testosterone response was not associated with appetite or food intake (FI).

95

To date, the only study published on single-macronutrient effect of testosterone

depression in humans was performed on adult males looking exclusively at glucose with no

calorie-free control [10]. The findings of chapter 5 confirm that protein as well as glucose

intake diminishes acute testosterone levels but adds the novel finding that this occurs in

adolescent males. Additionally, the decrease of testosterone was greater in normal weight

than overweight subjects showing that their significantly lower testosterone levels at baseline

diminished the effect of food intake. It has been previously established that obese males have

lower testosterone at morning than normal weight males [388]. The greater decrease of

testosterone caused by protein may be initiated by the intake of amino acids, particularly

leucine, which stimulates rapamycin (mTOR) signaling and subsequent protein synthesis

independent of insulin [389]. In animal studies, leucine also inhibits adenosine

monophosphate-activated protein kinase (AMPK) [391], which is upstream of mTOR and

prevents protein biosynthesis [392] in addition to inhibiting androgen receptor (AR) mRNA

expression in cancer cells [393]. Leucine induced inhibition of AMPK through protein intake

may increase AR expression leading to increased testosterone intake into the muscle tissue

from the plasma thereby lowering plasma testosterone levels. The influx of testosterone into

the muscle serves to stimulate muscle protein synthesis by regulating nuclear gene expression

[103, 104]. Though an attractive explanation, this is only speculative, as this current study

cannot determine the direct pathways responsible for the observed changes of testosterone.

This research is also the first to show an acute increase of testosterone after intervals of HIEX

aerobic training in this population and is consistent with the current body of literature

showing increases of testosterone in teenage males after acute bouts of anaerobic and weight

bearing exercise [343, 345]. It is unique in that the exercise applied was aerobic and non-

weight bearing which may be a more accessible form of exercise training for overweight

adolescent males. Thus, the mode of exercise may be less important than the intensity for the

increase in testosterone production in pubertal males; greater lactate production increases

Leydig cell cAMP production of testosterone in a dose-dependent manner [14, 348].

Furthermore, the reduction in appetite after HIEX in adolescent males is in agreement with

previous findings [401], though these did not correlate to observed changes in testosterone. A

direct effect of appetite increasing from chronic elevated testosterone levels has been

previously established [331-333, 379] but Chapter 6 is the first study of its kind to show that

acute elevated testosterone levels from exercise are not related to post-exercise appetite in

adolescent males.

96

Overall, the results of this research suggests that testosterone does not have an

interactive relationship in determining acute food intake, and it remains unclear whether a

relationship exists between testosterone, appetite regulation and energy balance in adolescent

males. However, there are a number of limitations to consider when interpreting the result of

these studies. First, these studies focused on short-term testosterone responses which may

have limited application given that testosterone primarily exerts its effects through chronic

exposure, thus chronic levels of testosterone are more relevant to its physiological role in

modifying. However, there is growing evidence that androgens may exert acute changes on

other structures and systems such as altering intracellular calcium regulatory mechanisms

[115] and rapid targeting of cardiovascular structures [118-123]. Second, fat intake was not

assessed or compared with glucose and protein intake. There is paucity of data on the direct

effect of acute fat intake on testosterone levels and this is likely because of palatability issues.

One study found no difference in the effect of fat using heavy whipping cream when

compared with a liquid dextrose beverage [440]. Furthermore, the type of fat may impact the

change seen in testosterone. Meals containing plant-based fat decreased post-prandial

testosterone levels more than those with animal fat [375]. A third limitation of this research

was the preparation of the experimental beverages; 1g/kg bodyweight of glucose

monohydrate or 1g/kg bodyweight of whey protein isolate powder. This meant that there was

a caloric discrepancy given that protein powder is not composed of pure protein. Independent

third-party analysis of the protein demonstrated 90.4% of the powder was protein, with 5.7%

moisture, 2.2% ash, 1.18% fat, and 0.6% carbohydrates. Given these percentages, the protein

drink is 3.74 kcal/kg bodyweight and the glucose drink is 4 kcal/kg bodyweight with control

being 0 kcal/kg bodyweight. Since the mean of our overweight subjects was 88.5kg, this

presents a difference of approximately 23 kcal between the glucose and protein treatments

(354 kcal glucose; 331 kcal protein). Previous work comparing a 300kcal preload to a

600kcal preload showed little difference between the two with subsequent subjective appetite

and ad libitum energy intake in a buffet meal [441]. Finally, given that most research has

examined chronic exposure of exogenous testosterone in animals, the change in testosterone

in Chapter 5 and 6 may not have been great enough or sustained long enough to effect

appetite and food intake.

A significant strength of this research was provided by the development and use of GC-MS

for measurement of testosterone in Chapter 5 and 6. It was a modification (Appendix 2) of a

method [181] considered to be the gold standard compared with past research using RIA and

97

ELISA to detect acute testosterone change. This method allows for detecting modest

acute changes of testosterone in adolescent males who have normal pubertal fluctuations of

testosterone in addition to expected diurnal rhythms. This may have been a limiting factor in

the past for measuring acute change in this population.

This research brings forth novel physiological concepts that could support the currently

accepted recommendations of exercise and post-exercise nutrition in adults and may extend

them to adolescent males. The International Society of Sports Nutrition (ISSN) recommends

ingestion of a post-exercise carbohydrate and protein beverage to maintain a favourable

anabolic hormone profile, heighten muscle glycogen recover and mitigate change in muscle

damage markers [442]. Combined with findings from animal research, this current research

lends strength to the idea that in adolescent males, high intensity exercise increases plasma

testosterone (Chapter 6), it is proposed that consumption of a glucose and protein beverage

(Chapter 4 & 5) may drive increased plasma testosterone into damaged muscle tissue,

possibly through mTOR signaling and increased AR expression after protein intake [389,

393]. Though the exact mechanism could not be determined through the current research

presented herein and the fate of postprandial testosterone in adolescent males remains to be

determined, this novel concept provides new important questions to pursue.

7.1 Significance and Implications

The rise in adolescent obesity and the attenuation of testosterone production from excess

adiposity necessitates a greater understanding of how testosterone is related to energy

balance, appetite and food intake. More insight into the association of nutrition and

testosterone physiology was provided by these studies.

In addition to garnering a greater understanding of testosterone physiology and its relation to

adolescent male growth and nutrition, this research, combined with findings from animal

physiology, proposes new concepts for validation of current sports nutrition

recommendations for post-exercise protein and glucose ingestion. This research may add to

ongoing evidence of the importance of high intensity exercise and post-workout protein

consumption for promoting muscle growth. Though the research presented here does not

directly address sports nutrition, the observed diminished testosterone levels after a high-

98

protein beverage and increased testosterone from high-intensity exercise suggest this

response requires further research to support current recommendations.

7.2 Conclusion

Acute testosterone concentrations are affected by food intake and exercise in adolescent

males, but a relationship of this response to appetite and food intake was not established.

99

Chapter 8

FUTURE DIRECTIONS

100

FUTURE DIRECTIONS

Future research should consider examining the effect of testosterone dynamics and change

over a longer period to determine if testosterone plays a role in appetite regulation of

adolescent males. Though previous work has established a role of testosterone in food intake

regulation [331-333, 379], the acute nature of this current research may indicate that

testosterone mediates appetite and food intake through long-term exposure. Testosterone

typically targets cell nuclei to effect transcription leading to protein synthesis, a process that

takes several hours with greater changes occurring through chronically elevated levels that

extend well beyond the time period of this research [107-112]. Studies in adult males have

consistently demonstrated that the scale of increased lean muscle tissue and decreased fat

mass from supraphysiologic doses of testosterone is correlated with the prevalent testosterone

concentrations [134]and that significant increases of lean muscle tissue occur after 20 weeks

of regular dosing [137] supporting the chronic effects of testosterone.

Further research should also observe adolescent males with hypogonadotropic hypogonadism

who are receiving testosterone injections to stimulate pubertal development as this may be

informative. Studying the testosterone treatment response of this population would help

determine whether testosterone plays a role in both acute and long-term appetite and food

intake in adolescent males and, unlike Chapter 4,5 and 6, demonstrate a direct effect of

testosterone through exogenous administration.

Furthermore, it would be of interest to determine if intake of protein and/or glucose changes

the increase of testosterone from high-intensity exercise and the role of lactate. A study

combining Chapter 5 and 6 measuring HIEX followed by consumption of protein and glucose

may be proposed. Intake of protein immediately after intense exercise is recommended for

stimulus of muscle protein synthesis as recommended by the ISSN [442]. Whether this post-

exercise postprandial protein synthesis is related to changing levels of testosterone remains

unknown. Additionally, as lactate has been shown to play a role in testosterone production

[14, 348], demonstrating a direct relationship of HIEX lactate production and testosterone

could help guide exercise designs aimed at improving testosterone levels.

Finally, future research should examine sex differences in adolescent populations that could

help provide dietary and physical activity guidelines which promote healthy development

into adulthood. Given the role estradiol may play in appetite [443] and the changes that occur

101

from exercise that are not accounted for through weight loss [444], a study of the

interaction between food intake and exercise and estradiol in adolescent females may be

informative.

102

Chapter 9

REFERENCES

103

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the initial phase of digestion. Am J Physiol Regul Integr Comp Physiol, 2008. 294(6):

p. R1790-6.

436. Qamar, M.I., et al., Transcutaneous Doppler ultrasound measurement of coeliac axis

blood flow in man. Br J Surg, 1985. 72(5): p. 391-3.

437. Qamar, M.I. and A.E. Read, Effects of ingestion of carbohydrate, fat, protein, and

water on the mesenteric blood flow in man. Scand J Gastroenterol, 1988. 23(1): p. 26-

30.

438. Sidery, M.B., et al., Cardiovascular responses to high-fat and high-carbohydrate

meals in young subjects. Am J Physiol, 1991. 261(5 Pt 2): p. H1430-6.

439. Eriksen, M. and B.A. Waaler, Priority of blood flow to splanchnic organs in humans

during pre- and post-meal exercise. Acta Physiol Scand, 1994. 150(4): p. 363-72.

440. Alleman, R.J., Jr. and R.J. Bloomer, Hormonal response to lipid and carbohydrate

meals during the acute postprandial period. J Int Soc Sports Nutr, 2011. 8: p. 19.

441. de Graaf, C. and T. Hulshof, Effects of weight and energy content of preloads on

subsequent appetite and food intake. Appetite, 1996. 26(2): p. 139-51.

442. Jager, R., et al., International Society of Sports Nutrition Position Stand: protein and

exercise. J Int Soc Sports Nutr, 2017. 14: p. 20.

443. Butera, P.C., Estradiol and the control of food intake. Physiol Behav, 2010. 99(2): p.

175-80.

444. Ennour-Idrissi, K., E. Maunsell, and C. Diorio, Effect of physical activity on sex

hormones in women: a systematic review and meta-analysis of randomized controlled

trials. Breast Cancer Res, 2015. 17(1): p. 139.

445. Blasco, M., et al., A quantitative HPLC-MS method for the simultaneous

determination of testosterone, 11-ketotestosterone and 11-beta

hydroxyandrostenedione in fish serum. J Chromatogr B Analyt Technol Biomed Life

Sci, 2009. 877(14-15): p. 1509-15.

446. Hsing, A.W., et al., Reproducibility of serum sex steroid assays in men by RIA and

mass spectrometry. Cancer Epidemiol Biomarkers Prev, 2007. 16(5): p. 1004-8.

133

Chapter 10

APPENDICES

134

APPENDIX 1. Pubertal Assessment Flow Chart

Figure A2.1- Proposed pubertal cut off values for hormonal assay values and testicular

volume. Luteinizing Hormone values are derived from the work of Chada et al. and Resende

et al. [204, 210]. Luteinizing hormone is the most sensitive indicator of onset of puberty,

however, if values fall between 0.12 - 0.44 IU/L and 1.6 – 2.39 IU/L, secondary measures of

testosterone become necessary to determine the stage of puberty of the subject. Testosterone

values are derived from Mouritsen et al. and Konforte et al. [214, 215] using the LC/GC MS

methods and IA method respectively. Tertiary measures of testicular volume become

necessary if the values derived from the secondary measure of testosterone fall between 35-

150 ng/dl. Pre-pubertal values are defined as those < 3.0 ml, whereas values falling between

3.0-12.0ml indicate onset of early puberty and mid-puberty. A testicular volume of >12ml

can be defined as late/post puberty.

135

APPENDIX 2. GCMS Method Development and Validation

For measurements of total serum testosterone most clinical laboratories use ELISA methods.

There were different ELISA kits available in market for serum testosterone assay. Like all

ELISA procedures, there are some limitations of the testosterone ELISA kits. Recently,

several research laboratories developed Liquid Chromatography-Tandem Mass Spectrometry

(LC-MS) assay of serum testosterone. This new mass spectroscopy assay techniques and

some improved radio immunoassay (RIA) methods for testosterone assay exposed the

limitations of the market available ELISA methods for serum testosterone assay. ELISA

techniques measuring testosterone have some disadvantages such as low precision, cross-

reactivity, limited linear range and poor correlation. Relatively low circulating testosterone

concentrations especially in children and women lead to limited precision, unacceptable

cross-reactivity and result in positive bias [445].

The most important limitations of all the ELISA assays of serum testosterone become

obvious when used in low and high serum testosterone samples. Christina Wang et al [187]

findings were supported by several laboratories afterwards and showed can be seen from

Table 4 of that article that 19.8, 25.7, 39.6, 39.6, 48.5, and 50.4% of the samples fell outside

the 20% range of the LC-MSMS generated serum T value by DPC-RIA, RocheElecsys,

Ortho Vitros-Eci, HUMC-RIA, Immulite and Bayer, respectively- all commonly used for

clinical measurement of testosterone. This difference was especially noted in the samples

with testosterone values less than 100 ng/dl obtained by the six different immunoassays, the

majority (55.5–90.0% of the samples) fell outside the 20% range of those obtained by LC-

MS. On the other hand, linearity and mean values were calculated from serial dilution of the

highest calibrator (16;8;4;2;1;0.5;0.25;0.125;0.06;0.03 ng/mL). Even when the concentration

is very low (1 ng/dL) or high (1600 ng/dl) the linearity is not affected.

Wang et al demonstrated that the ELISA methods commonly used in different clinical

laboratories gave serum testosterone levels ranging from a low of 200 ng/dl to the highest

maximum 420 ng/dl. But the introduction of RIA and LC-MS changed the upper limit of

serum testosterone level to even 1500 ng/dl.

The LC-MS assay underwent vigorous validation with a linear calibration curve spanning 20–

2000 ng/dl, and had accuracy between 96.6 and 110.4% and precision of less than 10% at all

points. The range of serum Testosterone values obtained in 17 normal men ages 18–50 yr in

this study was 302–905 ng/dl by the LC-MSMS method [445]

The DPC-RIA (DPC-Coat-a-Count) is the most common RIA used in hospital or reference

laboratories and appears to show the best agreement with serum Testosterone values

measured in male serum by LC-MSMS. The normal range given by the manufacturer for this

assay (286– 1510 ng/dl) had a similar low male reference range as other methods but with an

extremely high upper limit. This suggests that the adult male range might not have been

generated by each laboratory and both the lower and the upper limit of the reference

range might have to be adjusted. The Bayer Centaur assay on the other hand showed the

reference range for adult men with this instrument is reported as 241–827 ng/dl – the

upper limit of this assay was almost half of that determined by RIA or LC-MS methods.

This is mostly because of the much better linearity of RIA or LC-MS methods compared to

the ELISA methods [446].

136

A GC-MS modified stepwise procedure was developed by Fitzgerald et al. and utilized

for study 2 and 3[181]. The procedure was carried out on an Agilent 5977A single-quadruple

mass spectrometer coupled to an Agilent 7890B gas chromatograph (Agilent Technologies,

Mississauga, ON) in negative chemical ionization mode at the Department of Nutritional

Sciences at the University of Toronto and was validated by Dr. Diptendu Chatterjee, Dr.

Richard Bazinet and Scott Lacombe. All extractions and sample preparations were performed

by Alexander Schwartz and Dr. Chatterjee and GC-MS analyses were run by Scott Lacombe.

After completing analysis of the samples, we wanted to ensure that we were getting real

values and not overestimating testosterone levels in our subjects. We sent out stock

testosterone standards of 200, 600 and 1200ng/dl to the rapid response laboratory at The

Hospital for Sick Children (HSC) (Table A3.1 and Figure A3.1). This analysis indicates a

that although there is excellent reliability, there is discordance at higher levels between HSC

standards and that of our own.

Table A3.1- Comparison of stock standards given to The Hospital for Sick Children Rapid

Response Laboratory with the Department of Nutritional Sciences GCMS values from the

same standards.

DNS GCMS values HSC LCMS Values

Sample ID (T) Testosterone (ng/dl) (nmol/l) (ng/dl)

1 200 7 201.73

2 600 16 461.10

3 1200 30 864.55

4 200 7 201.73

5 600 16 461.10

6 1200 30 864.55

137

Figure A3.1- Comparison of stock standards given to The Hospital for Sick Children Rapid

Response Laboratory with the Department of Nutritional Sciences GCMS values from the

same standards. Note that the values from HSC do not cross zero due to linearity breaks at

higher values.

138

APPENDIX 3. Study 2 Sub-Analysis of Pre-Early Pubertal

Subjects

Results for Pre-Early Subjects from Chapter 5: Acute decrease in serum testosterone and

appetite after glucose and protein beverages in adolescent males (Experiment 2).

Participant characteristics are presented in Table A4.1. A total of 11 adolescent males

age 9 to 13 that were classified in the pre-early pubertal stages completed the study and were

included in this secondary analysis. Four were overweight/obese and were approximately

44% higher in body weight with a five-fold higher fat mass and 25% greater FFM compared

to the 7 normal weight participants.

Average baseline levels of appetite- and sex-related hormones are shown in Table

A4.2. Overweight/obese participants had higher fasting FSH and lower fasting active ghrelin

than the normal weight participants.

Effects of treatments over time on responses in hormone and glucose concentrations A. Sex hormones a) Mean responses

Mean plasma testosterone concentrations (Table A4.3) were detectable in 5 subjects.

Plasma testosterone concentrations were not affected by time (F = 2.33, p = 0.16) or weight

status (F = 0.01, p = 0.91) but were affected by treatment (F = 8.22, p = 0.012) with no

interactions. The glucose beverage decreased plasma testosterone by an average 34.1% (29.9

± 9.7 ng/dl, p < 0.0043) compared to control (45.4 ± 9.8 ng/dl). The protein beverage

decreased testosterone by 21% in comparison to control, though this was not statistically

significant (36.1 ± 9.8 ng/dl, p < 0.11). There was no difference between the effect of protein

vs. glucose (p = 0.15) (APPENDIX Figure 1).

b) AUC responses

Due to the low testosterone values in the pre-early puberty subjects, the AUCs of testosterone

could only be determined in one subject for all three of the experimental sessions and were

therefore not included in the results.

B. Glucose, Insulin, GLP-1, and Ghrelin

a) Mean responses

Plasma glucose concentrations were affected by treatment (F = 19.85, p < 0.0001) and

time (F = 7.53, p = 0.0011). Plasma glucose concentrations were higher after the glucose

treatment when compared to both protein and control beverages (p< 0.0001) with no

differences between protein and control treatments.

139

Plasma insulin was affected by time (F = 8.42, p = 0.0005) and treatment (F =

54.14, p < 0.0001). Significant interactions were found in time×treatment (F = 5.29, p =

0.0009), treatment×weightstatus (F = 15.07, p < 0.0001) and treatment×time×weightstatus

(F = 3.6, p = 0.01). Plasma insulin concentrations over 65 minutes were highest after glucose

consumption when compared to both protein (p <0.0001) and the control (p<0.0001). Protein

also caused a greater increase of insulin when compared to control (p = 0.0008). In response

to the glucose treatment only, overweight subjects had a greater insulin increase than normal

weight (p = 0.0002).

GLP-1 was not affected by treatment (F = 1.92, p = 0.15), time (F = 0.61, p = 0.55) or

weight status (F = 0.21, p = 0.65). There were no significant interactions between

treatment×time (F = 0.88, p = 0.48), time×weight status (F = 0.42, p = 0.66) or

treatment×time×weight status (F = 0.39, p = 0.82). GLP-1 (active) average concentrations

were similarly increased by glucose (6.2 ± 0.5pM), protein (7.9 ± 0.6pM) and control

beverage (6.8 ± 0.6pM) (p< 0.0001).

There was an effect of treatment (F = 21.71 p < 0.0001) on mean active ghrelin, with

no effect of time (F = 0.56, p = 0.57) or weight status (F = 2.03, p = 0.16). Ghrelin (active)

was decreased by glucose (p < 0.001) and protein (p < 0.001) when compared to control, but

there was no difference in ghrelin decrease between the glucose and protein (p = 0.09).

b) AUC response

Glucose AUC was greater after the glucose treatment (p < 0.05) when compared to

protein and control. Additionally, AUC glucose was lower after protein treatment when

compared with control (p = 0.019).

Insulin AUCs were larger after protein and glucose treatments (p< 0.01) when compared

with the control. However, the AUC of insulin after glucose was higher than after protein

(p = 0.001). AUC for insulin was greater in overweight than in normal weight participants

after the glucose treatment only (p = 0.012).

There was no effect of treatment (F = 0.8, p = 0.47) or weight status (F = 0.01, p =

0.91) on GLP-1 AUC. Additionally, there were no treatment×weight status interaction (F =

0.34, p = 0.72).

Ghrelin AUC was lower (p< 0.05) but similar after both the protein and glucose

treatments when compared with the control (Table A4.6). There was no treatment×weight

status (F = 0.11, p = 0.05).

C. Appetite

a). Mean response

Overall, there was a time effect (F = 4.26, p = 0.018) but no treatment effect (F =

0.92, p = 0.4) on appetite (Table A4.5). Regardless of treatment, appetite was lower (p =

0.011) at baseline (63.9 ± 3.0mm) when compared with 65 min (71.3 ± 3.5mm). In addition,

appetite was also lower at 20 min (65.6 ± 2.8mm) when compared to 65 min (p = 0.02).

c) AUC response.

140

There was no difference in the average appetite score AUCs due to treatment (p

=0.57) or weight status (p = 0.2).

D. Food Intake

There was no effect of the treatment (F = 1.78, p = 0.2) or weight status (F = 0.42, p =

0.5) on total caloric intake. Mean FI for the control, glucose and protein treatments were

839.3 ± 134.4kcal, 744.2 ± 128.1kcal and, 706.0 ± 121.5kcal, respectively. There was no

effect of treatment (F = 1.8, p = 0.19) or weight status (F = 0, p = 1.0) on FI corrected for

FFM (kcal/kg). Mean intakes for the control, glucose and protein treatments were 29.6 ± 4.8,

26.3 ± 4.4 and, 24.5 ± 4.0/ kg, respectively.

Correlations:

Post treatment mean testosterone concentrations over time (Table A4.3) did not

correlate with average appetite (r = -0.06, p = 0.7), desire to eat (r = -0.1, p = 0.6),

prospective FI (r = 0.06, p = 0.7) or fullness scores (r = 0.12, p = 0.5) (Table 5). No

correlation was found with FI at the meal.

AUCs for average appetite were positively correlated with food intake expressed

relative to FFM (r = 0.68, p < 0.0001) and total caloric intake (r = 0.67, p < 0.0001). Appetite

AUCs did not correlate to GLP-1 AUCs (r = -0.1, p = 0.6) or to AUCs of glucose (r = -0.07,

p = 0.7), ghrelin (r = 0.17, p = 0.35), and insulin (r = -0.07, p = 0.7). No within treatment

correlations were found.

The AUCs for LH were positively associated with caloric intake expressed relative to

FFM (r = -0.5, p = 0.04) but were not associated with subjective appetite (r = -0.42, p = 0.09),

insulin (r = 0.12, p = 0.66), active ghrelin (r = 0.08, p = 0.8), glucose (r = -0.07, p = 0.8), or

GLP-1 (r = -0.06, p = 0.84).

141

Table A4.1. Participant characteristics

Normal Weight

(n = 7)

Overweight/Obese

(n = 4)

Age 10.0 ± 0.3 10.8 ± 0.8

Height (cm) 138.2 ± 2.7 149.4 ± 8.0

Weight (kg) 31.0 ± 1.7a 55.2 ± 6.8b

Fat-Free Mass (KG) 26.5 ± 1.0 a 35.2 ± 4.5b

Fat Mass (KG) 4.4 ± 0.7a 20.0 ± 3.4b

Body Fat % 13.9 ± 1.7a 36.0 ± 3.2b

BMI (kg/m²) 16.2 ± 0.5a 24.5 ± 1.1b

BMI %ile (WHO) 33.9 ± 10.1a 97.9 ± 0.7b

1All values are means ± SEM, n = 11. Values in the same row with different superscript letters are significantly

different, P < 0.05.

Table A4.2. Baseline levels of appetite- and sex-related hormones

Normal Weight

(n = 7)

Overweight

(n = 4)

Testosterone (ng/dl) 26.5 ± 1.0 35.2 ± 4.5

LH (IU/L) 1.1 ± 0.3 1.1 ± 0.4

FSH (mIU/ml) 2.6 ± 0.2 a 5.1 ± 0.8 b

Ghrelin, active (pg/ml)2 355.5 ± 55.9 a 309.5 ± 116.6 b

GLP-1, active (pM) 6.2 ± 0.6 4.7 ± 1.2

Insulin (uIU/ml) 6.2 ± 0.7 9.2 ± 2.6

Glucose (mg/dl) 108.8 ± 2.5 102.3 ± 6.0

1Values were calculated by averaging the baseline levels of all three of the sessions for each hormone. All

values are means ± SEM, n = 11. Values in the same row with different superscript letters are significantly

different, P < 0.05. 2 Ghrelin values are higher than that of Mid-Late Puberty. This is in agreement with previous

literature [268, 356].

142

Table A4.3. Testosterone and luteinizing hormone response to treatments 1

Control Glucose Protein

Time (min) 0 20 35 65 0 20 35 65 0 20 35 65

Testosterone2

(ng/dl)

49.1

±14.5

76.3

±28.4

34.7

±10.5

68.7

±43.7

38.1

±7.6

29.3

±3.7

22.7

±3.7

34.7

±11.8

47.6

±5.6

34.0

±4.0

25.0

±3.0

31.9

±6.1

LH

(IU/L)

2.2

±0.2

2.7

±0.3

2.3

±0.4

2.9

±0.3

1.8

±0.2

1.5

±0.2

2.1

±0.2

2.5

±0.6

2.1

±0.6

1.1

±0.6 N/A N/A

1All values are means ± SEM, n = 4. Values in the same row with different superscript letters are different, P <

0.05 (beverage effect using ANCOVA with proc mixed procedure, Tukey’s post-hoc).

2 Treatment affected testosterone (F = 8.22, p = 0.012).

Table A4.4. Ghrelin (active), GLP-1 (active), insulin and glucose in response to

treatments1


Time

(min) 0 20 35 65 0 20 35 65 0 20 35 65

Ghrelin

(active)2

(pg/ml)

303.7

±61.4

236.2

±45.5

268.5

±57.2

333.5

±53.2

374.6

±68.9

258.5

±40.1

212.0

±44.4

245.5

±55.5

359.6

±63.7

193.2

±24.7

183.7

±32.6

175.5

±283

GLP-1

(active)3

(pM)

4.0

±0.7

5.8

±1.3

6.0

±1.2

6.7

±1.4

6.6

±1.4

7.9

±1.3

7.1

±1.5

6.3

±1.7

5.4

±1.2

8.1

±1.4

7.8

±1.4

7.0

±1.6

Insulin4

(pg/ml)

7.0

±1.1a

6.5

±0.9a

7.2

±1.3a

6.8

±0.1a

6.9

±0.9a

60.8

±13.2b

61.2

±16.9b

26.9

±5.7c

7.9

±2.1a

30.7

±5.9c

27.3

±4.9c

23.9

±4.4c

Glucose5

(mg/dl)

109.4

±5.8

116.3

±3.8

112.6

±4.4

112.0

±3.3

107.3

±2.6

153.2

±9.4

154.4

±13.1

121.1

±8.4

103.1

±3.2

114.9

±5.1

92.5

±6.8

99.0

±8.9

1Values are means ± SEM, n = 11. Values in the same row with different superscript letters are significantly

different, P < 0.05 (beverage effect using ANCOVA with proc mixed procedure, Tukey’s post-hoc).

2 Treatment (F = 21.71, p = <0.0001) affected ghrelin with no effect of time (F = 0.56, p = 0.57) or weight status

(F = 2.03, p = 0.16).

3 No effect of treatment (F = 1.92, p = 0.15), time (F = 0.61, p = 0.55) or weight status (F = 0.21, p = 0.65) on

GLP-1.

4Treatment (F = 54.14, p< 0.0001) and time (F = 8.42, p = 0.0005) affected insulin with no effect of weight

status (F = 3.25, p = 0.076) in addition to significant interactions of time×treatment (F = 5.29, p = 0.0009),

treatment×weight status (F = 15.07, p< 0.0001), time×weight status (F = 3.13, p = 0.0497) and

treatment×time×weight status (F = 3.6, p = 0.01).

5Treatment (F = 19.85, p<0.0001) and time (F = 7.53, p = 0.0011) affected glucose.

Table A4.5. Mean VAS for subjective appetite in response to the treatments1.

143


Time2 (min) 0 20 35 65 0 20 35 65 0 20 35 65

Appetite

(mm)

61.8

±6.2

66.8

±7.0

71.1

±6.0

74.4

±6.2

66.2

±3.5

65.5

±4.8

63.5

±6.0

70.7

±5.7

63.6

±6.2

65.6

±6.2

63.8

±7.6

68.6

±6.6

1All values are means ± SEM, n = 11. Values based on subjective measure using a100 mm scale composed of

four questions anchored at each end with contrasting terms related to desire to eat, hunger, fullness and

prospective food consumption. Mean subjective appetite (appetite) is an aggregate score of these four measures.

2There was a significant effect of time (F = 4.26, p = 0.018) on appetite.

Table A4.6. Area under the curve (AUC) for testosterone, LH, ghrelin (active), GLP-1

(active), insulin, glucose and AUC mean appetite score in response to treatments 1,2.


LH

(IU/L)

61.9 ±10.4 56.9 ±12.2 68.0 ±8.5

Ghrelin, active

(pg/ml)

17078.5 ±3229.55a 15140.1 ±2884.6b 11845.3 ±1796.2b

GLP-1, active

(pM)

370.6 ±73.3 421.9 ±84.1 472.8 ±89.6

Insulin

(uIU/ml)

413.1 ±55.8a 2745.8 ±604.4b 1489.5 ±231.0c

Glucose

(mg/dl)

6774.8 ±133.8a 8426.54 ±548.9b 6078.14 ±213.4c

Mean Appetite Score

(mm)

4180.0 ±341.6 3975.8 ±275.1 3981.7 ±361.2

1All values are means ± SEM, n = 11. AUC are calculated as change from baseline over 65 min. Values in the

same row with different superscript letters are significantly different, P < 0.05 (beverage effect using ANCOVA

with proc mixed procedure, Tukey’s post-hoc).

144

Figure A4.1. Effects of treatments on plasma levels of testosterone in pre-early pubertal

males (means ± SEM, n = 11). Repeated measures ANCOVA with baseline as a covariate

was performed followed by post-hoc analysis. Plasma testosterone was decreased after

glucose (p = 0.0043) when compared to the non-caloric control drink. Different letters

indicate significant difference (p < 0.05).

145

APPENDIX 4. Consent Forms

Chapter 5: Acute decrease in serum testosterone after a mixed glucose and protein beverage

in obese peripubertal boys (Study 2)

FitzGerald Building, 150 College Street, 3rd Floor

Toronto, ON M5S 3E2

CANADA

The acute effect of protein or carbohydrate intake on testosterone levels and food intake

in children and adolescent boys

Study Information Sheet and Parent’s Consent Form

Investigators: Dr. G. Harvey Anderson, Principle Investigator

Department of Nutritional Sciences, University of Toronto

Phone: (416) 978-1832

Email: [email protected]

Dr. Jill Hamilton, Associate Professor

Department of Paediatrics, University of Toronto

Phone: (416) 813-5115


Mr. Alexander Schwartz, Ph.D. Student

Department of Nutritional Science, University of Toronto

Phone: (647) 390-2893


Ms. Mukta Wad, Lab Manager


Phone: (416) 978-6894






146

Purpose of Research:

The purpose of this study is to determine the effects of carbohydrate and protein drinks on testosterone levels and food intake in 9-18 year-old boys. This experiment is being conducted through the Department of Nutritional Sciences at the University of Toronto by Alexander Schwartz (PhD student), Dr. G. Harvey Anderson and Dr. Jill Hamilton (Supervisors). Your son will be required to attend one screening session to explain the study in detail

and then attend three experimental sessions (to measure testosterone in addition to blood sugar and insulin) conducted over a 3-week period for a total of 4 visits to The University of Toronto. Experimental sessions will last a maximum of 2 hours.

The purpose of our research is to develop an understanding of factors affecting the control of food intake and testosterone level changes in overweight and obese boys. Knowing the determinants of the regulation of food intake on testosterone levels in boys will allow us to understand how various types of food impact appetite and hormone levels in the body.

Study Visits:

1. Screening visit:

For those parents who express interest in having their sons participate, some information about your son is requested by telephone.

You will be asked to attend a screening session at the university research lab so that the researcher will explain the full details of the study. If you agree to consent for your son to participate, and your son also assents to being in the study, we will do some measurements and questionnaires at the screening visit.

At the screening session we will measure your son’s height, weight, and blood pressure.

Your son will have measurements of the waist and arms using a tape measure. We will

estimate the amount of muscle and fat tissue in your son’s body, using a technique called the

Phone 416-978-2747 FAX 416-978-5882

Website: www.utoronto.ca/nutrisci

147

bioelectrical impedance analysis (BIA). BIA is based on the measurement of electrical

resistance in the body to a tiny current (that he cannot feel). Your son will be asked to briefly

lie down after removing his shoes, socks and any jewelry. Source electrodes are placed on

the backside of his right hand and the top of his right foot and at least 5 cm away from to the

receiving electrodes, which are placed on the wrist and on the ankle.

Your son will then be asked to rank his preference for pizza that will be served as the lunch meal at each session. In addition, your son will be familiarized with the scales that we will use throughout the study sessions in which he places a pencil mark to describe various questions about hunger and appetite in addition to his preference for pizza.

Your son’s physical activity and eating habits will be assessed with Physical Activity Questionnaire and your son’s eating behaviours will be assessed with the Dutch Eating Behaviour Questionnaire. He will be asked to fill these out at the screening session visit.

The total time for the screening session visit will be approximately 60 minutes.

2. Study visits 1, 2 and 3

Your son will be asked to come to The University of Toronto, for three individual morning sessions, each lasting about 3-4 hours. These sessions will be held on weekends, over three weeks so that each visit is one week apart. Boys should be brought to the laboratory and returned home by parents only.

On each of the three test days, your son will arrive to the laboratory in the FitzGerald Building at 8:45 am. He should not have anything to eat or drink from 9pm the previous night, except for water, which he can drink until 8 am the day of the visit. After 8 am we would ask that he not have anything to eat or drink until the study begins.

On only the first visit, your son will have a short physical exam including an evaluation of

puberty. This involves the nurse doing a quick check of the size of your son’s testicles. If

your son does not feel comfortable, he will have a choice to report it himself by answering a

questionnaire relating to puberty. The boys will be presented with cartoon pictograms of

different stages of pubertal development (e.g., pubic hair, genitalia) and the boys will be

asked to pick the picture that best represents their stage of puberty. These pictograms have

148

been used extensively in youth and adolescents. They will also fill out a brief

questionnaire about puberty and changes in their bodies. We are checking this as appetite

may change during puberty and may be regulated by testosterone levels. If for any reason the

boys are not comfortable with this, they have the option of asking their parents to answer the

questionnaire and select the pictograms for them. If you have additional questions about this

part of the study, before agreeing to participate, you have the option of discussing this with

Dr. Jill Hamilton (416-813-5115 or email:[email protected]). She is a pediatric

endocrinologist at SickKids who specializes in growth and puberty.

Upon arrival, during each of the sessions your son will be given a drink (protein and water, a

sugar-sweetened beverage or an equally sweet beverage with no calories). After 60 minutes,

he will be offered McCain pizza. He will be told that he may eat as little or as much pizza as

he likes. The amount of food eaten by your son will be measured.

Throughout the morning, he will also be requested to complete scales on which he places a

pencil mark to describe his desire to eat (“Very weak” to “Very strong”), hunger (“Not

hungry at all” to “As hungry as I’ve ever felt”), fullness (“Not full at all ” to “Very full”),

how much food he could eat (“A large amount” to “Nothing at all”). He will also be asked to

complete similar scales on how much he likes the drinks and the pizza. He will complete

these scales during the information session, in order to become familiar with the questions.

Also at each study session, blood will sampled and used to measure blood sugar and appetite-

controlling (hunger) hormones. Four blood samples (10 ml or approximately 2 teaspoons)

will be taken during each experimental session. The total volume of blood collected at one

session will be 40 ml (8 teaspoons) and the total volume of blood collected within three

weeks will be 120 ml (24 teaspoons). To obtain blood samples, a nurse will insert a catheter

(a needle attached to a plastic tube or IV) into a vein in your son’s arm so that only one poke

is needed per session to collect all three blood samples. The catheter will remain in his arm

and be used to sample blood in small amounts during the session. After the nurse collects the

first sample at baseline (0 minutes), your son will consume one of the drinks within five

minutes. After he finishes the drink, we will collect a blood sample at 15, 30 and 60 minutes

after baseline. After the third blood measure, your son will be allowed to eat as much pizza as

he wishes for 20 minutes.

All youth and adolescents will be fully supervised during the study sessions. They will be

engaged in age appropriate entertainment (as distraction) e.g.: reading, puzzles, cards, before

lunch. The study session will end shortly after the pizza meal.

Confidentiality:


149

Records relating to participants will be kept confidential in a locked cabinet in the

Department of Nutritional Sciences and no disclosure of personal information of the boys or

parents will take place except where required by law. Participants will have a code and a

number that will identify them in all documents, records and files to keep their name

confidential. All data will be entered into Microsoft Excel files, available only to

investigators. Each participant will have a file, also only available for investigators. All

blood samples will be stored in a safe and secure refrigerator and may be used for future

analyses of other hormones related to appetite regulation. All forms and printouts will be

stored in the individual files and clearly labeled. Data stored outside of a secure server will be

electronically encrypted to ensure protection of subject identification. All documents will be

kept for a minimum of five years following completion of the study and then securely

destroyed with the exception of withdrawn data, which will be destroyed immediately upon

withdrawal of the participant. Withdrawal of data will be impossible exactly one year after

the final completed session.

Risks:

There is very little risk related to this study. The provided test beverages are commercially

available and safe for human consumption. In addition, the pizza that boys will be also asked

to consume are prepared hygienically in the kitchen at the time of the session. Boys may feel

dizzy following the overnight fast, but this is rare. If this happens, they will likely feel fine

once they drink the test beverage provided. There is the possibility of a small amount of

bruising, pain and the possibility of infection associated with blood collection, but this risk is

minimal as all proper sterilization precautions will be met. The pubertal staging questionnaire

may also present some psychological and social risks as the cartoon pictures may potentially

be embarrassing. In order to reduce this possible risk, participants will given the option to be

in a private room with no other individuals present with the exception of the study nurse.

Benefits:

As some of the factors causing obesity remain unclear, the potential benefits from this study

will be a better understanding of the regulation of food intake in youth and adolescents and

may help us to provide new recommendations for the prevention of obesity in children and

teenagers.

Questions and further information:

150

Participation is completely voluntary and failure to participate will not have any

consequences. Also, you and your son have the option to stop participating or skip any

step/question at any time.

If you have any questions or would like further information concerning this research project,

please do not hesitate to call: Mr. Alexander Schwartz (647) 390-2893, Dr. Jill Hamilton

(416) 813-5115 or Dr. Harvey Anderson at (416)-978-1832.

If you have questions or concerns about your rights as a research participant, please contact

Dr. Rachel Zand, 416 946 3389, [email protected].

Dissemination of findings:

A summary of results will be made available to you to pick up after the study is completed.

The summary of the data will include data from all of the participants in this study and will

be anonymous.

Consent:

I acknowledge that the research procedures described above and of which I have a copy, have

been explained to me and that any questions that I have asked have been answered to my

satisfaction. I know that I may ask additional questions now or in the future. I am aware that

participation in the study will not involve any health risk to my son.

I understand that for purposes of the research project, if my son or I choose to withdraw from

the study at any time, we may do so without prejudice.

Upon completion of each study session, my son will receive a $25 gift certificate to the

theatre, bookstore or mall after each of the first three sessions and a $125 gift certificate after

completing the fourth session and, if in high school, receive volunteer hours. The final

summary and results of the study will be available for me to pick up from the Department of

Nutritional Sciences, University of Toronto. I am aware that the researchers may publish the

study results in scientific journals, keeping confidential my son’s identity.

I hereby consent for my son, _____________________________________, to participate in

this study.


151

___________________________________ _____________________________

(Name of parent or guardian) (Signature of parent or guardian)

__________________________________ ___________________________

(Name of witness) (Signature of witness)

Date: ______________ (dd/mm/yy)

152


Toronto, ON M5S 3E2

CANADA



Participant’s Assent Form

This study will help to find out how good various drinks are for boy’s health. My weight,

height, and body fat will be measured without pain during the screening visit. I will be asked

to fill out a questionnaire that is related to my stage of puberty (changes in my body as I grow

up). I will also be asked to be examined by a nurse or to look at some cartoon pictures and

pick the one that looks most like me. I can ask my parents to answer these questionnaires and

pick the picture for me. I will also be asked to drink a drink, complete special scales to show

if I am hungry or full and have four blood samples taken by the trained nurse (one before the

drink and three after). The nurse will put in a small IV or tube into the vein with the first poke

so that there will be no extra needles needed to collect the blood samples. I will also be

provided with a pizza lunch 60 minutes after finishing the drink during each study session

(that I will eat in The University of Toronto). I will be provided with the pizzas of my choice

and be allowed to eat as much pizza as I would like for 20 minutes. All the experimental

sessions will be on weekends, school holidays or summer break, so I don’t need to be absent

from school.

I know that my participation in the study will not involve any health risk to me.

I will be asked to come for the study three times, but if at any time I decide to stop

participating, that will be O.K and I have the choice to not answer any question at any time. I

understand that the information related to me will be securely stored and not be given to

anyone from outside who is not engaged with this study. I know that as a “thank you” for my

participation, I will receive a $25 gift certificate to the theatre, bookstore or mall after each of

the first three sessions and a $125 gift certificate after completing the fourth session. I will

also receive volunteer hours for high school after completion of each study session.

“I was present when ______________________________read this form and gave his/her

verbal assent.”

153

_____________________________ Signature

Name of the person who obtained assent: _______________________________

154


Toronto, ON M5S 3E2

CANADA



Study Information Sheet and Participant Consent Form

Investigators: Dr. G. Harvey Anderson, Principle Investigator


Phone: (416) 978-1832



Department of Paediatrics, University of Toronto

Phone: (416) 813-5115


Mr. Alexander Schwartz, Ph.D. Student


Phone: (647) 390-2893


Ms. Mukta Wad, Lab Manager


Phone: (416) 978-6894



Phone 416-978-2747 FAX 416-978-5882

Website: www.utoronto.ca/nutrisci





155

The purpose of this study is to determine the effects of carbohydrate and protein drinks on testosterone levels and food intake in 9-18 year-old boys. This experiment is being conducted through the Department of Nutritional Sciences at the University of Toronto by Alexander Schwartz (PhD student), Dr. G. Harvey Anderson and Dr. Jill Hamilton (Supervisors). You will be required to attend one screening session to explain the study in detail and then attend three experimental sessions (to measure testosterone in addition to blood sugar and insulin) conducted over a 3-week period for a total of 4 visits to The University of Toronto. Experimental sessions will last a maximum of 2 hours.

The purpose of our research is to develop an understanding of factors affecting the control of food intake and testosterone level changes in overweight and obese boys. Knowing the determinants of the regulation of food intake on testosterone levels in boys will allow us to understand how various types of food impact appetite and hormone levels in the body.

Study Visits:

1. Screening visit:

For those who express interest in participating, some information about you is requested by telephone.

You will be asked to attend a screening session at the university research lab so that the researcher will explain the full details of the study. If you agree to consent to participate we will do some measurements and questionnaires at the screening visit.

At the screening session we will measure you height, weight, and blood pressure. You will

have measurements of the waist and arms using a tape measure. We will estimate the amount

of muscle and fat tissue in your body, using a technique called the bioelectrical impedance

analysis (BIA). BIA is based on the measurement of electrical resistance in the body to a tiny

current (that he cannot feel). You will be asked to briefly lie down after removing yout shoes,

socks and any jewelry. Source electrodes are placed on the backside of yout right hand and

the top of your right foot and at least 5 cm away from to the receiving electrodes, which are

placed on the wrist and on the ankle.

You will then be asked to rank your preference for pizza that will be served as the lunch meal at each session. In addition, you will be familiarized with the scales that we will use

156

throughout the study sessions in which he places a pencil mark to describe various questions about hunger and appetite in addition to his preference for pizza.

Your physical activity and eating habits will be assessed with Physical Activity Questionnaire and your eating behaviours will be assessed with the Dutch Eating Behaviour Questionnaire. You will be asked to fill these out at the screening session visit.

The total time for the screening session visit will be approximately 60-90 minutes.

2. Study visits 1, 2 and 3

You will be asked to come to The University of Toronto, for three individual morning sessions, each lasting about 3-4 hours. These sessions will be held on weekends, over three weeks so that each visit is one week apart. Boys should be brought to the laboratory and returned home by parents only.

On each of the three test days, you will arrive to the laboratory in the FitzGerald Building at 8:45 am. You should not have anything to eat or drink from 9pm the previous night, except for water, which you can drink until 8 am the day of the visit. After 8 am we would ask that you not have anything to eat or drink until the study begins.

On only the first visit, you will have a short physical exam including an evaluation of

puberty. This involves the nurse doing a quick check of the size of your testicles. If your do

not feel comfortable, you will have a choice to report it yourself by answering a questionnaire

relating to puberty. You will be presented with cartoon pictograms of different stages of

pubertal development (e.g., pubic hair, genitalia) and you will be asked to pick the picture

that best represents your stage of puberty. These pictograms have been used extensively in

youth and adolescents. You will also fill out a brief questionnaire about puberty and changes

in your body. We are checking this as appetite may change during puberty and may be

regulated by testosterone levels. If for any reason you are not comfortable with this, you have

the option of asking your parents to answer the questionnaire and select the pictograms for

you. If you have additional questions about this part of the study, before agreeing to

participate, you have the option of discussing this with Dr. Jill Hamilton (416-813-5115 or

email:[email protected]). She is a pediatric endocrinologist at SickKids who

specializes in growth and puberty.


157

Upon arrival, during each of the sessions you will be given a drink (protein and water, a

sugar-sweetened beverage or an equally sweet beverage with no calories). After 60 minutes,

you will be offered McCain pizza. You will be told that he may eat as little or as much pizza

as he likes. The amount of food eaten by you will be measured.

Throughout the morning, you will also be requested to complete scales on which you place a

pencil mark to describe your desire to eat (“Very weak” to “Very strong”), hunger (“Not

hungry at all” to “As hungry as I’ve ever felt”), fullness (“Not full at all ” to “Very full”),

how much food you could eat (“A large amount” to “Nothing at all”). You will also be asked

to complete similar scales on how much you like the drinks and the pizza. You will complete

these scales during the information session, in order to become familiar with the questions.

Also at each study session, blood will sampled and used to measure blood sugar and appetite-

controlling (hunger) hormones. Four blood samples (10 ml or approximately 2 teaspoons)

will be taken during each experimental session. The total volume of blood collected at one

session will be 40 ml (8 teaspoons) and the total volume of blood collected within three

weeks will be 120 ml (24 teaspoons). To obtain blood samples, a nurse will insert a catheter

(a needle attached to a plastic tube or IV) into a vein in your arm so that only one poke is

needed per session to collect all three blood samples. The catheter will remain in your arm

and be used to sample blood in small amounts during the session. After the nurse collects the

first sample at baseline (0 minutes), you will consume one of the drinks within five minutes.

After you finish the drink, we will collect a blood sample at 15, 30 and 60 minutes after

baseline. After the third blood measure, you will be allowed to eat as much pizza as you

wishes for 20 minutes.

All youth and adolescents will be fully supervised during the study sessions. You will be

engaged in age appropriate entertainment (as distraction) e.g.: reading, puzzles, cards, before

lunch. The study session will end shortly after the pizza meal.

Confidentiality:

Records relating to participants will be kept confidential in a locked cabinet in the

Department of Nutritional Sciences and no disclosure of personal information of the boys or

parents will take place except where required by law. Participants will have a code and a

number that will identify them in all documents, records and files to keep their name

confidential. All data will be entered into Microsoft Excel files, available only to

investigators. Each participant will have a file, also only available for investigators. All

blood samples will be stored in a safe and secure refrigerator and may be used for future

analyses of other hormones related to appetite regulation. All forms and printouts will be

stored in the individual files and clearly labeled. Data stored outside of a secure server will be

electronically encrypted to ensure protection of subject identification. All documents will be

158

kept for a minimum of five years following completion of the study and then securely

destroyed with the exception of withdrawn data, which will be destroyed immediately upon

withdrawal of the participant. Withdrawal of data will be impossible exactly one year after

the final completed session.

Risks:

There is very little risk related to this study. The provided test beverages are commercially

available and safe for human consumption. In addition, the pizza that you will be also asked

to consume are prepared hygienically in the kitchen at the time of the session. You may feel

dizzy following the overnight fast, but this is rare. If this happens, you will likely feel fine

once you drink the test beverage provided. There is the possibility of a small amount of

bruising, pain and the possibility of infection associated with blood collection, but this risk is

minimal as all proper sterilization precautions will be met. The pubertal staging questionnaire

may also present some psychological and social risks as the cartoon pictures may potentially

be embarrassing. In order to reduce this possible risk, participants will given the option to be

in a private room with no other individuals present with the exception of the study nurse.

Benefits:

As some of the factors causing obesity remain unclear, the potential benefits from this study

will be a better understanding of the regulation of food intake in youth and adolescents and

may help us to provide new recommendations for the prevention of obesity in children and

teenagers.


Participation is completely voluntary and failure to participate will not have any

consequences. Also, you and your son have the option to stop participating or skip any

step/question at any time.

If you have any questions or would like further information concerning this research project,

please do not hesitate to call: Mr. Alexander Schwartz (647) 390-2893, Dr. Jill Hamilton

(416) 813-5115 or Dr. Harvey Anderson at (416)-978-1832.

159

If you have questions or concerns about your rights as a research participant, please

contact Dr. Rachel Zand, 416 946 3389, [email protected].


A summary of results will be made available to you to pick up after the study is completed.

The summary of the data will include data from all of the participants in this study and will

be anonymous.

Consent:

I acknowledge that the research procedures described above and of which I have a copy, have

been explained to me and that any questions that I have asked have been answered to my

satisfaction. I know that I may ask additional questions now or in the future. I am aware that

participation in the study will not involve any health risk to me.

I understand that for purposes of the research project, if I choose to withdraw from the study

at any time, I may do so without prejudice.

Upon completion of each study session, I will receive a $25 gift certificate to the theatre,

bookstore or mall after each of the first three sessions and a $125 gift certificate after

completing the fourth session and, if in high school, receive volunteer hours. The final

summary and results of the study will be available for me to pick up from the Department of

Nutritional Sciences, University of Toronto. I am aware that the researchers may publish the

study results in scientific journals, keeping confidential my identity.

I _____________________________________ hereby consent to participate in this study.

___________________________________ _____________________________

(Name of parent or participant) (Signature of participant)

__________________________________ ___________________________


Date: ______________ (dd/mm/yy)


160

Chapter 6: Acute increase in plasma testosterone after high intensity cycling and

appetite in adolescent males (Study 3)

Recruitment Letter for Parents

The acute effects of exercise induced inflammation on appetite and energy intake in lean boys.

Dear Parent,

The University of Toronto is investigating the physiological and environmental determinants of

energy intake regulation on the health of children and young adolescents. We are trying to understand

the food intake in children. Our ultimate goal is to find ways to address the problems of overeating

and obesity that are becoming a concern among Canadians.

We are asking 12-18 year old male participants to take part in a research study. Your child’s

participation is straightforward, height, weight, body-fat, physical fitness and questionnaires regarding

puberty, eating behaviour and physical activity levels will be assessed during a screening visit. During

the other four experimental sessions, your child will be asked to consume water and complete either

30 minutes of resting or 30 (3 x 10 min + 1:30 min rest) minutes of exercise at a moderate intensity,

on four separate mornings. The exercise will be conducted on a recumbent bicycle, followed by 60

min of resting (130 min in total). Within each experimental session, your child will be asked to

provide 3 teaspoons (30 ml) of blood samples over the course of 2 hours. A registered nurse will take

the blood samples. The study will take place on two weekends at the Warren Stevens Building,

Faculty of Physical Education and Health Athletic Centre at 55 Harbord Street (on the southeast

corner of Harbord and Spadina, Room WS 2030).

There are criteria for participation that you need to be aware of, the child must:

• be 12-18 years of age and

• be healthy and not be taking medications

• have previously taken ibuprofen without any adverse reactions

http://www.physical.utoronto.ca/FacilitiesAndMemberships/Athletic_Centre/Location.aspx

161

As a token of consideration your son will receive a gift certificate after each completed session

(movie pass, gift cards to the bookstore or gift vouchers to the mall). He will receive a $25 for the

screening session, $60 for each of the other four sessions ($265 in total). Should he choose to

withdraw from the study before its completion, he will only be compensated for the sessions he

already attended, but not for the remaining sessions. In addition, parents will be reimbursed for

travel/parking expenses ($12/session).

The University of Toronto Health Sciences ethics review committee has reviewed this study.

If you would like your son to participate, or to get further information beyond that provided in this letter,

please contact Mr. Sascha Hunschede principal investigator at (647) 686-2045 or Dr. G. Harvey Anderson,

Professor (416) 978-1832 at the University of Toronto (Department of Nutritional Sciences).

Thank you for your support in this important research.

Sincerely,

Dr. Harvey Anderson, Department of Nutritional Sciences, University of Toronto.

Mr. Sascha Hunschede, Department of Nutritional Sciences, University of Toronto.

Dr. Scott Thomas, Faculty of Physical Education and Health, University of Toronto.

162

Study Information Sheet and Parent’s Consent Form.

The acute effects of exercise induced inflammation on appetite and energy intake in lean boys.

Investigators: Dr. G Harvey Anderson


Phone: (416) 978-1832


Mr. Sascha Hunschede


Phone: (647) 686-2045



Department of Pediatrics, University of Toronto

Phone: (416) 813-5115


Dr. Scott Thomas

Faculty of Physical Education and Health, University of Toronto

Phone: (416) 978-6957


Mr. Alexander Schwartz


Phone: (416) 978-6957





mailto:%[email protected]


163


The purpose of this study is to determine the effects of exercise induced inflammation on appetite,

energy intake and metabolic markers of appetite in normal-weight. This experiment is conducted through

the Department of Nutritional Sciences, at the University of Toronto by Dr. G. Harvey Anderson

(supervisor), Sascha Hunschede (principal investigator) and Dr. Scott Thomas (co-investigator). You will

be required to attend four sessions and one screening testing session conducted over a 4-week period, for a

total of 4 visits (4 experimental sessions + 1 visit to measure physiological parameters) to the University of

Toronto campus. Each visit will last approximately two hours.

Procedure:

Screening

Fitness Testing/Screening:

If your child expresses interest in participating, information about your child will be requested by telephone, by the principal investigator, Sascha Hunschede (647) 686-2045, [email protected]). If your child is healthy and does not receive any medications, an information session will be arranged.

You and your child will be asked to attend a screening session at the University of Toronto Athletic Center at 55 Harbord Street so that the researcher will explain the full details of the study. If you agree to consent for your son to participate, and your son also assents to being in the study, we will do some measurements and questionnaires at the screening visit.

During the information session, the researcher will explain the full details and risks of the study. Parents and participants who give consent will sign a consent form. You will receive copies of consent forms and of the study information sheet. If your child wishes to participate, your child’s weight, height, and body fat using painless techniques, will be measured.

We will assess your child’s physical fitness at the Cardiorespiratory Fitness Testing Center located in the Goldring Center of the University of Toronto campus on a seated bicycle using a simple, non-

invasive technique. During the test, a facemask will be worn to facilitate the collection of ventilator gases. In addition, your child’s heart rate (Polar Monitor) and mechanical workload will be monitored

throughout the test. The test will be conducted on a bicycle and every two minutes the resistance will be increased. The test will be stopped when your child request it (signal is thumbs down) or when you

reach your maximum aerobic capacity, or if the supervisor halts the test. The exercise time on the bicycle will be approximately 10 to 15 minutes. Your child will be asked to abstain from eating,

drinking caffeine-containing beverages for 10 hours prior to the tests.

164

There will be four individuals present during the fitness testing process including: a technician who will operate the exercise equipment and computer software, Mr. Sascha Hunschede, principal investigator, Dr. Scott Thomas who is an expert in the area of cardiorespiratory fitness testing in children and a research assistant.

Activity Assessment:

If your child consents to participate in this experiment, he will be asked to fill out a Physical Activity Questionnaire (PAQ) to assess your participation in physical activity. The PAQ will be filled out during each visit to the University of Toronto. The answers will be strictly confidential and will only serve to assist in the analysis of the data collected. Subsequent to the start of the experiment, any relevant changes in health status should be reported as soon as possible to Mr. Sascha Hunschede (principal investigator), Dr. Thomas, or Dr. Anderson.

165

Session Visits:

You and your son will be required to come to the University of Toronto Athletic Centre at 55 Harbord Street after a 10-hour overnight fast for four study visits. Each visit will be separated by one week apart Participants will receive 2 exercise treatments and 2 resting treatments with either water or

300mg of Ibuprofen, one week apart. Drink treatments will be blinded with 2g cool aid to mask the taste of them Children’s PERRIGO® Suspension. Your child is encouraged to bring homework or reading materials during the 4 experiemental sessions. The exercise protocol is described in Exercise Protocol. Please make sure to bring exercise attire for each session.

The treatment order will be randomized:

• 250ml Water with 2g of cool aid and rest (control)

• 250ml Water with 2g of cool aid and 30 min of exercise on a seated bicycle at 70% VO2max.

• 235ml Water with 15ml of Children’s PERRIGO® Suspension (Medical Ingredient: 300mg Ibuprofen)

• 235ml Water with 15ml of Children’s PERRIGO® Suspension (Medical Ingredient: 300mg Ibuprofen) + 30 min of exercise on a seated bicycle at 70% VO2max

Exercise Protocol:

Your child will cycle on a seated bicycle, at a high (70% VO2max) intensity, for 30 minutes in 3 x 10 minute periods with 1:30 minute breaks in between.

Preload treatments:

Two of the preload treatments will include 300mg Ibuprofen. Ibuprofen is a member of the

class of agents commonly known as nonsteroidal anti-inflammatory drugs (NSAIDs). Ibuprofen is

commonly used for the temporary relief of minor aches and pains in muscles, bones and joints, headache, fever, the aches and fever due to the common cold or flu, immunizations, toothache (dental

pain), sore throat, earache. Ibuprofen was chosen because it has the most favourable gastrointestinal

safety profile of all NSAIDs with a very low risk of causing serious events, according to the Food and

Drug Administration (FDA) and the Non-prescription Drug Advisory Committee. It also often used by athletes for decrease muscle soreness and better recovery. Exercise itself causes a natural occurring

inflammatory response that may affect appetite. Ibuprofen will be given to your child to suppress this inflammatory response. Your child will only be eligible to participate in this study, if he has taken

Ibuprofen previously and displayed no adverse reactions to the drug. The dosing recommendation for the age group, in this study, is 400 mg every 4 to 6 hours as needed. In this study we will give one

166

dose of 300 mg of a child-approved version of ibuprofen Children’s PERRIGO® Suspension to minimize side effects.

Risks of the preload ibuprofen treatment:

Common (3-9%) side effects of ibuprofen can include nausea, dyspepsia, gastrointestinal ulceration/bleeding, raised liver enzymes, diarrhea, constipation, nosebleed, headache, dizziness, rash, salt and fluid retention, and hypertension. Ibuprofen may (<1%) cause an allergic reaction as indicated by (wheezing; facial swelling or hives; shortness of breath; shock; fast, irregular heartbeat) if any of these reactions occur, stop the use and get medical help immediately. If any of these reactions occur,

the study will be stopped and get medical help will be seeked immediately. A full list of side effects is attached with this document.

167

Risks of exercise:

Dr. Scott Thomas has many years of experience assessing physical fitness. The fitness testing process will utilize state of the art equipment. There are minimal risks associated with any workout or maximum exercise test, however these risks can be lowered substantially with proper precautions. Your child’s vital signs will be closely monitored throughout the test and it is important that your child communicates about any symptoms to staff, including chest pain, difficulty breathing, dizziness, nausea, headaches, double vision and neck rigidity.

Appetite Assessment:

You will also be requested to complete scales on which they will place a pencil mark to describe their desire to eat (“Very weak” to “Very strong”), hunger (“Not hungry at all” to “As hungry as I’ve ever felt”), fullness (“Not full at all ” to “Very full”), how much food you could eat (“A large amount” to “Nothing at all”), sweetness of the drinks (“Not sweet at all” to “Extremely sweet”). They will complete these questionnaires during the information session, in order to become familiar with the test instruments.

Blood measurements:

There is minimal risk to your son. Four 10 ml intravenous blood samples will be taken

throughout each experimental session, one sample is the equivalent of approximately two teaspoons of blood. A trained nurse will be on sight to conduct the blood sampling and to ensure the safety of

our participants. The volume of blood taken presents a minimal risk to subjects. There is a moderate risk of infection from insertion of the catheter or venous puncture. However, a sterile indwelling

catheter will be used and the area will be swabbed with alcohol to decrease the risk. There is the possibility of a small bruising, pain and the possibility of infection associated with blood collection.

The nurse will offer topical anaesthetic spray to your son, to numb the skin prior to inserting the

needle into your son’s vein. There is a small risk of discomfort during the study period. However, your child will be able to stop the study at any point of time.

Body Composition Assessment:

Bioelectrical Impedance Analysis:

Bioelectrical impedance analysis (BIA), a recently developed technique for measuring body fat content in both adults and children, is simple and painless and is an effective method for measuring body fat. BIA is based on measurement of electrical resistance in the body to a tiny current (that the subject cannot feel). The principle of BIA lies in that muscle mass in the body is a better conductor of electricity than fat which has lesser amounts of water and electrolytes.

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Confidentiality:

Confidentiality will be respected and no information that shows your identity will be released or published without your permission unless required by law. Your name, personal information and

signed consent form will be kept in a locked filing cabinet in the investigator’s office. Your results will not be kept in the same place as your name. Your results will be recorded on data sheets and in

computer records that have an ID number for identification, but will not include your name. Your results, identified only by an ID number, will be made available to the study sponsor if requested.

Only study investigators will have access to your individual results. The information you share with me will be kept private except for information that leads me to believe that a Person under the age of

16 has suffered or is at risk physical, sexual harm or neglect. I have a legal duty to report abuse to the Children’s aid society under Ontario’s child and family services act.

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Benefits:

Community benefits from this project by potential new strategies that can improve energy homeostasis in children and adults by investigating the effects of fat oxidation during exercise on post exercise food intake. As the causes of obesity remains undefined, the potential benefits from this study will be a better understanding of the regulation food intake in children and might contribute to the prevention of obesity in children.


If you have any questions or would like further information concerning this research project, please do not hesitate to call: Sascha Hunschede principal investigator at (647) 686-2045 or Dr. Harvey Anderson, supervisor at (416)-978-1832.

If you have questions or concerns about your rights as a research participant, please contact the Office of Research Ethics, [email protected] or at 416-946-5806.


A summary of results will be made available to you after the study is concluded.

Consent:

I acknowledge that the research procedures described above and of which I have a copy, have been

explained to me and that any questions that I have asked have been answered to my satisfaction. I have read

and understood the recruitment letter, and information sheet including the drug information sheet related to the

risk of taking ibuprofen. I hereby acknowledge that my child has taken ibuprofen previously and did not have

any adverse reactions to Ibuprofen or any other painkillers. I know that I may ask additional questions now or

in the future. All the risks associated with this study have been explained to me and I am fully aware of the

health risks involved in this study.

I understand that for purposes of the research project, if I choose to withdraw consent and/or my son from the study at any time, we may do so without prejudice.

As a token of consideration for participating in research, at each session your son will receive a

gift certificate (movie pass, gift cards to the bookstore or gift vouchers to the mall). A $25 gift certificate

will be given to your son for the screening session and a $60 gift certificate will be given for each of


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experimental sessions. Should your son choose to withdraw from the study before its completion, he will

only be compensated for the sessions he attended, but not for the remaining sessions.

. The final summary and results of the study will be available for me to pick up from the Department of Nutritional Sciences, University of Toronto. I am aware that the researchers may publish the study results in scientific journals, keeping my identity confidential.

I hereby consent, _____________________________________, to participate in this study.

___________________________________ _____________________________

(Name of guardian or parent of participant) (Signature of guardian or parent of participant)

__________________________________ ___________________________


Date: ______________ (dd/mm/yy)

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APPENDIX 5. Screening Questionnaires

Telephone Screening Questionnaire

Recruitment Screening & Food Acceptability Questionnaire

Eating Habit Questionnaire

Physical Activity Questionnaire

Puberty Questionnaire

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5.1. Telephone Screening Questionnaire

Name:

Age: years DOB (d/m/y) Term baby? Yes / No

Height: cm Weight: kg Normal birth weight? Yes / No

Has your child gained or lost weight recently? Yes / No (circle correct answer)

Does your child usually have breakfast? Yes / No

Does your child like (foods that will be used in study

Milk/protein smoothies Yes / No

Sugary Drinks Yes / No Pizza Yes / No

Is your child following a special diet? Yes / No

Does your child have food allergies or sensitivities? Yes / No

Health problems? Yes / No

If yes, which problem?

Medication/s? Yes / No

If yes, which medication/s?

Education: Grade:

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Skipped or repeated grade? Yes / No Learning difficulties/problems? Yes / No

Behavioral or emotional problems Yes / No

If yes, which problem?

Include in study? Yes / No

If not, why?

Appointment date: (d/m/y)

Investigator: Date: (d/m/y)

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5.2. Recruitment Screening Information Questionnaire

Subject ID :

TEST0______

Subject Name:

Child’s Date of Birth

Child’s Date of Birth

Parent 1 Weight:

Kg / lb Height:

Parent 2 Weight:

Kg / lb Height:

(Circle correct unit)

Contact Information

Address:

Home Phone #:

Parent 1 Name:

Cell Phone #:

Work Phone #:

Email

Preferred Method of Contact Home Phone Cell Phone Email

Parent 2 Name:

Cell Phone #:

Work Phone #:

Email

Preferred Method of Contact Home Phone Cell Phone Email

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Source of referral:


Food Acceptability List


Pre-meal Beverage, Testosterone and Food Intake in Overweight and Obese boys

Name: Birth Date:

BEVERAGE

Please indicate whether you will be able to drink the beverages below:

Flavoured Protein Smoothie (500 mL) Flavoured Juice (500 mL) Flavoured Sweetened Juice (500 mL)

Yes / No Yes / No Yes / No

(Circle one Yes OR No)

LUNCH

You will be given a pizza lunch on the day of the study. For us to provide you with a lunch you will enjoy, please circle what you would like to eat (circle a, b OR c):

(a) All PEPPERONI pizza (cheese & pepperoni (b) All CHEESE pizza (3-cheese: mozzarella, cheddar and parmesean) (c) A COMBINATION of pepperoni & 3-cheese pizza

If you answered (c), please circle what you would like more: pepperoni OR 3-cheese (Circle One)

BLOOD

We will require two blood samples for each session in this investigation. Please indicate whether you will be able to provide us with blood samples.

Yes No

TANNER STAGING

The study nurse will discuss pubertal Tanner staging with you. Please indicate whether you agree to have this examination performed.

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Yes No

Subject ID:



Pre-meal Snacks, Satiety and Food Intake in Children

Body Measurements

Subject Code:

Date of Birth: (dd/mm/yy)

Age:

Weight: Kg Height cm

BIA

Resistance

Reactance

Body Fat %

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5.3. Eating Habit Questionnaire

Dutch Eating Habits Questionnaire Please complete sections 1 and 2 and then turn over.

1. Subject and test details

Name:

Date of birth:

Age:

Gender: male female

Today’s date:

2. Your weight, height, etc.

A. Current weight (kg):

B. Current height (cm):

C. Has your body weight been constant over the past six months?

yes, my weight did not change much

no, I lost kg

no, I gained kg

no, sometimes I gained weight and sometimes I lost weight

D. Have you ever had an episode of eating an amount of food that others would regard as unusually large?

yes

no

Please do not mark below this line

BMI (please take the age of the child into account):

DEBQ scale Raw score Number of items Scale score Classification

Emotional eating 7

External eating 6

Restrained eating 7

Please turn over >>>>>

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Instructions

Below you’ll find 20 questions about eating.

Please read each question carefully and tick the answer that suits you best.

Only one answer is allowed. Don’t skip any answer.

There are no incorrect answers; it’s your opinion that counts.

1. Do you feel like eating whenever you see or smell good food?

No Sometimes Yes

2. If you feel depressed do you get a desire for food?

No Sometimes Yes

3. If you feel lonely do you get a desire for food?

No Sometimes Yes

4. Do you keep an eye on exactly what you eat?

No Sometimes Yes

5. Does walking past a candy store make you feel like eating?

No Sometimes Yes

6. Do you intentionally eat food that helps you lose weight?

No Sometimes Yes

7. Does watching others eat make you feel like eating too?

No Sometimes Yes

8. If you have eaten too much do you eat less than usual the next day?

No Sometimes Yes

9. Does worrying make you feel like eating?

No Sometimes Yes

10. Do you find it difficult to stay away from delicious food?

No Sometimes Yes

11. Do you intentionally eat less to avoid gaining weight?

No Sometimes Yes

12. If things go wrong do you get a desire for food?

No Sometimes Yes

13. Do you feel like eating when you walk past a restaurant or fast food

restaurant?

No Sometimes Yes

14. Have you ever tried not to eat in between meals to lose weight?

No Sometimes Yes

15. Do you have a desire to eat when you feel restless?

No Sometimes Yes

16. Have you ever tried to avoid eating after your evening meal to lose

weight?

No Sometimes Yes

17. Do you have a desire for food when you are afraid?

No Sometimes Yes

18. Do you ever think that food will be fattening or slimming when you

eat?

No Sometimes Yes

19. If you feel sorry do you feel like eating?

No Sometimes Yes

20. If somebody prepares food do you get an appetite?

No Sometimes Yes

PLEASE CHECK, TO BE SURE THAT YOU TICKED EVERY QUESTION.

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5.4. Physical Activity Questionnaire

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5.5. Puberty Questionnaire

Would you say that your growth spurt (height)?

1. there has been no development

2. development has barely begun

3. development is definitely underway

4. development is already completed

And regarding hair growth (under your arms, your pubic hair), would you say that:

1. there has been no development

2. development has barely begun

3. development is definitely underway

4. development is already completed

Have you noticed changes in your skin (e.g. acne)?

1. there have been no changes

2. changes have barely begun

3. changes are definitely underway

4. changes are already complete

Have you noticed that your voice has changed (lowered)?





Have you started to see hair on your face?





Tanner stage exam to be performed by MD or health care practitioner trained in Tanner staging. If

subject refuses exam, self-stage with cartoons and tanner beads.

181

182

183

184

APPENDIX 6. Study Day Questionnaires

Baseline/Recent Food Intake Questionnaire

Motivation to Eat VAS

Physical Comfort VAS

Treatment and Test Palatability

Test Meal Record

185

6.1. Baseline/Recent Food Intake Questionnaire

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6.2. Motivation to Eat VAS

Visual Analogue Scale Motivation to Eat

These questions relate to your “motivation to eat” at this time. Please rate yourself by placing a

small “x” across the horizontal line at the point which best reflects your present feelings.

1. How strong is your desire to eat?

Very Very

WEAK _______________________________________________ STRONG

2. How hungry do you feel?

NOT As hungry

Hungry ________________________________________________ as I have

at all ever felt

3. How full do you feel?

NOT VERY

Full ________________________________________________ Full

at all

4. How much food do you think you could eat?

NOTHING A LARGE

at all ________________________________________________ amount

5. How thirsty do you feel?

NOT As thirsty

Thirsty _____________________________________________ as I have

at all ever felt

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6.3. Physical Comfort VAS

Visual Analogue Scale Physical Comfort

This question relates to your “physical comfort” at this time. Please rate yourself by placing a

small “x” across the horizontal line at the point which best reflects your present feelings.

1. How well do you feel?

NOT VERY

well _________________________________________________ Well

at all

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6.4. Treatment and Test Palatability

Visual Analogue Scale Pleasantness

This question relates to the palatability of the drink you just consumed. Please rate the

pleasantness of the drink by placing a small “x” across the horizontal line at the point which

best reflects your present feelings.

1. How pleasant have you found the drink?

NOT ________________________________________________ VERY

at all pleasant

pleasant

This question relates to the palatability of the drink you just consumed. Please rate the

sweetness of the drink by placing a small “x” across the horizontal line at the point which best

reflects your present feelings.

1. How sweet have you found the drink?

NOT ________________________________________________ VERY

sweet sweet

at all

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6.5 Test Meal Record

Subject ID: ______________ Pizza Preference: ____________________________ Amount of whey for treatment: ___________________(g) Amount of glucose for treatment: __________________(g) Amount of sucralose needed for treatment:

Session: ________ Treatment: _____________

Investigator: ______________Date: __________

Before After Number

Tray 1

Pepperoni (g)

3-cheese (g)

Tray 2

Pepperoni (g)

3-cheese (g)

Tray 3

Pepperoni (g)

3-cheese (g)

Session: ________ Treatment: _________________

Investigator: ______________ Date: ______________

Before After Number

Tray 1

Pepperoni (g)

3-cheese (g)

Tray 2

Pepperoni (g)

3-cheese (g)

Tray 3

Pepperoni (g)

3-cheese (g)

Session: ________ Treatment: _________________

Investigator: ______________ Date: ______________

Before After Number

Tray 1

Pepperoni (g)

3-cheese (g)

Tray 2

Pepperoni (g)

3-cheese (g)

Tray 3

Pepperoni (g)

3-cheese (g)

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