The Effects of Substrate Oxidation on Post-Exercise Food Intake in. Pre-pubertal, Normal-weight Boys and Men.

Transcription

1 The Effects of Substrate Oxidation on Post-Exercise Food Intake in Pre-pubertal, Normal-weight Boys and Men. By Sascha Hunschede A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of Nutritional Sciences University of Toronto Copyright by Sascha Hunschede 2013

2 The Effects of Substrate Oxidation on Post-Exercise Food Intake in Pre-pubertal, Normal-weight Boys and Men. Master of Science, 2013 Sascha Hunschede Graduate Department of Nutritional Sciences University of Toronto ABSTRACT The relationship between substrate oxidation (RER) and food intake (FI) is undefined. This study examined the effects of RER modified by a glucose pre-load (GL), exercise (EX) and GL with EX on, FI and energy balance (NEB) in normal-weight boys (9-12 y) and men (20-30 y). Subjects (15 boys, 15 men) were randomized with treatments of either water or GL followed by either EX or rest. Measures included RER, energy expenditure (EE)(kcal/kg), subjective appetite, FI(kcal/kg) measured at a pizza lunch and NEB (kcal/kg). FI(kcal/kg) was reduced by GL(p < ), and further decreased with GL ingested prior to EX(p = ). RER was increased with GL(p < ) and EX(p = ), and was higher in men compared to boys (p = 0.007). There was no association between RER and FI(kcal/kg). In conclusion, there was no relationship between RER and FI, suggesting that FI is not affected by substrate oxidation. ii

3 ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor, Dr. Harvey Anderson, whose expertise, understanding, and patience, added extensively to my graduate experience. His vast knowledge in many areas (e.g., nutrition, exercise and the interaction of food intake and energy expenditure), without whose assistance and motivation and encouragement I would not have considered pursuing further steps in nutritionals sciences. It was under his guidance that I developed a greater focus and became more interested in obesity and the prevention of it. A very special thanks goes out to Dr. Scott Thomas, who encouraged and assisted me with the design and practical implementation of the project and he was always available to exchange concepts, knowledge, skills, and helped me venting of frustration during my graduate program, which helped to enrich the experience. He provided me with direction, technical support and became more of a mentor and friend, than a professor. It was through his, persistence, understanding and kindness that I completed this project and applied for the Ph.D. program. I doubt that I will ever be able to convey my appreciation fully, but I owe him my highest gratitude. I would like to thank Dr. Thomas Wolever who is the third member of my committee, for taking time out from his busy schedule and providing assistance at all levels of the research project. I must also acknowledge Dr. Sophie Antoine-Jonville who joined the Department of Nutritional Sciences, University of Toronto on her sabbatical, Dr. Antoine-Jonville supported me with her understanding and the clinical applications of the substrate measurements and the metabolic cart. Further thanks goes to Dr. Dalia El Khoury who was always available to discuss ideas and give me feedback and suggestions for my graduate work. iii

4 I would also like to thank my family for the support they provided me through my entire life and in particular the last two years, I must acknowledge my significant other and best friend, Katherine, without whose love, encouragement and editing assistance, I would not have finished this thesis. In conclusion, I recognize that this research would not have been possible without the financial assistance of CIHR and the University of Toronto, Department of Nutritional Sciences, which I would like to express my gratitude. iv

5 TABLE OF CONTENTS ABSTRACT... ii ACKNOWLEDGEMENTS... iii TABLE OF CONTENTS... v LIST OF TABLES... x LIST OF FIGURES... xii LIST OF EQUATIONS... xiii LIST OF ABBREVATIONS... xiv 1. INTRODUCTION LITERATURE REVIEW Overweight and Obesity Physical Activity and Obesity Physical Activity and Food Intake Regulation Physical Activity and Food Intake Regulation in Adults Physical Activity and Food Intake Regulation in Children Energy Balance and Obesity Carbohydrate Balance Fat Balance Protein Balance v

6 2.5. Energy Balance and Substrate Oxidation Metabolic Flexibility and Obesity Metabolic Flexibility and Physical (In)Activity Physical Activity, Substrate Utilization and Food Intake Regulation SUMMARY AND STUDY RATIONALE HYPOTHESIS Primary Hypothesis OBJECTIVES Overall Objective Specific Objective MATERIALS AND METHODS Experimental Design Participants Screening Session Experimental Sessions Preload Treatment Exercise Treatment Exercise Protocol for Children Exercise Protocol for Adults Resting Protocol vi

7 7. MEASURES AND DATA ANALYSIS Food Intake Blood Glucose Measurements Collection of Ventilatory Gases Measurement of Physical Fitness Maximum Oxygen Consumption Ventilation Threshold Substrate Oxidation and Energy Expenditure Assessment of Body Fat Percentage Estimation of Percentage of Maximum Heart Rate Visual Analog Scales Subjective Appetite Subjective Physical Comfort Subjective Energy/Fatigue and Stress Subjective Palatability Subjective Sweetness Statistical Analysis RESULTS Descriptive Measures Food Intake vii

8 8.3. Energy Expenditure Net Energy Balance Substrate Oxidation Carbohydrate Oxidation Fat Oxidation Heart Rate Water Consumption Net Area Under the Curve Blood Glucose Measurements Blood Glucose Measurements Visual Analog Scale Analysis Subjective Appetite Thirst Physical Comfort in Boys Physical Comfort in Men Preload Palatability in Boys Preload Palatability in Men Pizza Meal Palatability in Boys Pizza Meal Palatability in Men Correlation Analysis Correlations with Food Intake viii

9 Associations with Net Energy Balance DISCUSSION FUTURE DIRECTIONS Metabolic Flexibility and Food Intake Regulation in Obesity Explore the Effects of Exogenous and Endogenous Carbohydrate Oxidation on Food Intake Regulation Control for Appetite Hormones in Lean and Obese Subjects Standardization of the Time to Meal Control for Daily Physical Activity Levels and Diet Long-term Intervention Study SUMMARY & CONCLUSIONS SUMMARY CONCLUSION REFERENCES APPENDENCIES ix

10 LIST OF TABLES Table 7-1 Thermal equivalents of oxygen for the non-protein respiratory exchange ratio. 36 Table Mean subject characteristics of boys and men Table 8-2 Baseline characteristics for 15 boys Table 8-3 Baseline characteristics for 15 men Table Food intake (kcal/kg) in boys and men Table Energy expenditure (kcal/kg) in boys and men Table Net energy balance (kcal/kg) in boys and men Table Respiratory exchange ratio in boys and men Table Carbohydrate oxidation (kcal/kg) in boys and men Table Fat oxidation (kcal/kg) in boys and men Table Heart rate (bpm) in boys and men Table Water consumption (kg/ml) in boys and men Table nauc blood glucose levels (min*mmol/l) in boys and men Table Average Blood Glucose Concentrations (mmol/l) for boys and men Table Average appetite ratings (mm) in boys and men Table Average thirst ratings (mm) in boys and men Table Average physical comfort (mm) in boys Table Average physical comfort (mm) in men x

11 Table Average preload palatability (mm) in boys Table Average preload palatability (mm) in men Table Average pizza palatability (mm) in boys Table Average pizza palatability (mm) in men Table Associations with food intake (kcal/kg) Table 8-23 Associations with net energy balance (kcal/kg) xi

12 LIST OF FIGURES Figure 6-1 Study design Figure 7-1 VET (arrow) of an adult subject identified by the VE/VO2 (solid line) over VE/VCO2 (dashed line) method Figure 7-2 VET (arrow) of an adult subject identified by the V-Slope method xii

13 LIST OF EQUATIONS Equation 6-1 ASCM running equation for the determination of VO2 in boys and men Equation 6-2 Converted ASCM running equation for the determination of grade (%) in boys and men Equation 7-1 RQ equation for glucose and fat Equation 7-2 Energy expenditure for 40 minutes of gas exchange measurements Equation 7-3 Equations to calculate CHOox and FATox Equation 7-4 Durnin and Womersley equations for body density Equation 7-5 Siri equation for prediction of fat mass xiii

14 LIST OF ABBREVATIONS ANOVA - Analysis of Variance AUC - Area under the Curve ASCM - American College of Sports Medicine BF - Body Fat BG - Blood Glucose BPM - Beats per Minute BIA - Body Impedance Analysis BMI - Body Mass Index BW - Body-weight CDC - Centre for Disease Control CHOox - Carbohydrate Oxidation CO2 - Carbon Dioxide CON - Control DEV - Physical Development EB - Energy Balance EE - Energy Expenditure EI - Energy Intake EX - Exercise EXCN - Exercise with Control EXGL - Exercise with Glucose FATox - Fat Oxidation FI - Food Intake xiv

15 FQ - Food Quotient FP - Food Palatability GL - Glucose GLT-4 - Glut 4 Transporter GLP-1 - Glucagon-like-peptide 1 HR - Heart Rate HRmax - Maximum Heart Rate LD - Long Duration MTE - Motivation To Eat NEB - Net Energy Balance NW - Normal-weight OB - Obese OW - Overweight OXM - Oxyntomodulin PA - Physical Activity PAL - Physical Activity Level PC - Physical Comfort PS - Preload Sweetness PP - Preload Palatability PP - Pancreatic Polypeptide PYY - Peptide YY RER - Respiratory Exchange Ratio RQ - Respiratory Quotient xv

16 RO - Resting Oxygen Consumption ROb - Resting Oxygen Consumption Boys ROm - Resting Oxygen Consumption Men SECN - Sedentary Condition with Water SEGL - Sedentary Condition with Glucose SED - Sedentary Behaviour/Rest SD - Short Duration SEM - Standard Error of the Mean SKF - Skinfold SI - Suprailiac Speedb - Running Speed Boys Speedm - Running Speed Men SS - Subscapular TRT - Treatment TVV - Television Viewing VAS - Visual Analog Scale VE - Ventilatory Equivalent VET - Ventilatory Threshold VO2 - Oxygen Consumption VCO2 - Carbon Dioxide Consumption VO2peak - Peak Oxygen Consumption WHO - World Health Organisation xvi

17 1 1. INTRODUCTION The World Health Organisation (WHO) defines obesity as, abnormal or excessive fat accumulation that presents a risk to health. It is caused by a disturbance in the equilibrium of energy intake (EI) and energy expenditure (EE) and obesity is one of the top ten preventable global diseases [1]. From 1978 to 2004, the number of obese Canadian adults has more than doubled and childhood obesity has tripled [2]. In particular, class III obesity, which is defined with a body mass index (BMI) of greater than 40, has witnessed an increase from 0.4% to 1.3% from 1990 to 2003 in Canada [3]. Overweight and obesity now characterize 31 % of children and adolescents in the US and Canada [4]. Additionally, obese children and adolescents are at a greater risk of staying overweight throughout maturation and developing severe forms of obesity into adulthood [5]. This rapid increase is predicting enormous public health costs due to the potential long-term health impacts of obesity. The increased consumption of high-caloric foods and decrease of EE has been associated with the increased phenomenon of obesity, which now characterizes more than half of the Canadian population [6-9]. Obesity plays an essential role in the pathophysiology of cardiovascular diseases such as hypertension, atherosclerosis [10] and metabolic impairments such as insulin resistance, dyslipidemia, and type 2 diabetes mellitus [11]. As part of the metabolic syndrome, obesity is highly correlated with greater mortality [12], and an increased risk for liver, intestinal, pulmonary, endocrine and reproductive dysfunctions. The annual cost of obesity related conditions has been estimated to be at 4.6 billion dollars in Canada alone. [13, 14].

18 2 Countless physical activity (PA) and dietary programs have been designed to counteract this epidemic by creating a behavioural change without much success. Less attention has been given to understanding of physiological relationships of EE, energy balance (EB), food intake (FI), appetite regulation and their impact on obesity. Therefore the focus of this research is on the physiology of appetite regulation. The following sections provide a brief description of the obesity epidemic, followed by a discussion of the physiological interrelationship of PA, EB, and substrate oxidation on FI regulation.

19 3 2. LITERATURE REVIEW 2.1 Overweight and Obesity Obesity is a chronic medical condition characterized by an accumulation of excess body fat is presented with pathophysiologic consequences. A person with a BMI 25 kg/m 2 is considered overweight; a person with a BMI of 30 kg/m 2 is considered obese. Those classified as class I obesity have a BMI of , class II a BMI of and class III a BMI of 40 [15]. In children, the diagnosis of obesity is based on percentile charts established by the CDC BMI-for-age growth charts [16]. It is the standard method to classify obesity and overweight for children and adolescents from 2 to 20 years of age. A child is considered overweight if he/she is above the 85 th percentile and obese if above the 95 th percentile for the age specific BMI [17]. In 1997, the WHO officially named obesity as a worldwide epidemic [18]. As of 2005, the WHO estimated that at least 400 million adults are obese, and obesity rates are increasing [18]. Particularly, Canada, United States and Australia show a greater increase in obesity when compared with the global rate. Research conducted in the Canadian Community Health Survey reported that 61.1 % of Canadians older than 18 years were overweight, of which 23 % were obese. In children and adolescents aged 5 to 17 years of age, 31.5 % were overweight, of which 11.7% were classified as obese [19]. It is of concern that these proportions have tripled over the past 25 years in children and adolescents [20]. Obesity is related to a number of complex and multifactorial diseases. It can lead to ischemic heart disease [21], congestive heart failure and high blood pressure [22]. Endocrinologic and reproductive conditions associated with obesity include diabetes mellitus type II, infertility

20 4 and birth defects [22] and various types of cancer such as breast, ovarian, liver, pancreatic, prostate and colorectal cancers [23]. Gastrointestinal and respiratory disorders involve sleep apnea, asthma [24], gastrointestinal reflux disease [25], fatty liver disease and cholelithiasis [22]. Additionally, obese and overweight individuals often suffer from depression and/or social stigmata [22]. An imbalance between EI and EE is thought to play a key role in the development of obesity. The over-consumption of calories combined with decreases in PA are considered essential underlying causes for the development of obesity. Therefore, the interaction of PA and appetite regulation is explored in the following sections. 2.2 Physical Activity and Obesity In recent decades, there has been a large shift towards a decrease in PA and an increase in EI and fat intake [26-28]. In adults, PA has declined due to less physical demanding work and a greater use of modern conveyance [26, 29, 30]. PA is defined as any movement that results in an increase of EE when compared to resting conditions. PA occurs in daily life and includes occupational, sports, conditioning, household and other activities. Exercise (EX), as a subgroup of PA is defined as a planned form of PA, which incorporates structured body movements designed to enhance physical fitness [31]. EX includes occupational sports, conditioning and recreational activities. Evidence that highlights the significance of PA and EX in maintaining cardiovascular health and preventing diseases has been increasing over the past decades [32]. It shows that physically active individuals are less likely to develop stroke [33], some forms of cancer [34], type 2 diabetes [35] and obesity [36]. Conversely, a decrease in PA is strongly correlated with an increase in body mass index (BMI) and waist-hip

21 5 ratio and waist circumference [37, 38], which is accompanied by a loss of aerobic performance [39]. The decrease in PA is also directly linked with increased occurrence of various risk factors related to metabolic syndrome and cardiovascular diseases [40]. Some research suggests that sedentary (SED) behaviour is an independent predictor of metabolic risk, even if the individual s PA meets current guidelines [41]. The WHO recommends that adults aged 18 or older, participate in at least 150 minutes of moderate to vigorous activity a week or the equivalent of 30 minutes of daily activity [42]. Currently, just over 15% of Canadian adults are meeting the PA guidelines. In obese populations, these numbers are even lower [43]. Obese men in Canada only achieve 19 minutes of daily activity. Daily physical activity levels (PAL) are evaluated by a person s daily energy expenditure divided by his or her basic metabolic rate [44]. Some studies of PALs in sedentary and obese individuals found that they have PALs, of which is comparable to PALs in bed resting individuals [45, 46]. Bed resting and low PALs increase ectopic fat storage, impair lipid trafficking, increase insulin resistance and decrease fat oxidation (FATOX) [45]. Following a similar trend, the decrease in PA is associated with weight gain in children [47]. According to the current Canadian PA guidelines, 50% of boys and 68% of girls are categorised as inactive. Children participating in organized sports are unlikely to meet current PA recommendations of 16,500 steps a day [48]. To achieve this, PA equivalent of at least 60 minutes of moderate to vigorous PA per day is suggested for healthy development during childhood [48]. The decline in PA among children has been attributed primarily to decreased time walking and increased time spent playing video games and television viewing (TVV) [49, 50]. In Canada in 2004, 36% of all children aged 6-11 spent more than two hours per day in front of a screen. Obesity

22 6 rates in children with two or more hours of screen time per day are doubled compared to those who were exposed to screen time for one hour or less [20]. The contribution of reduced PA to obesity pathophysiology has been suggested to occur via two pathways: firstly through decreasing EE, and secondly by a failure to compensate in EI. 2.3 Physical Activity and Food Intake Regulation The interaction between PA, EX and FI regulation is multifactorial and very complex. Factors such as age, caloric intake, obesity, metabolic function and physical fitness are all involved in regulation of FI. Additional difficulties in interpreting and finding consistency in the literature occur because of the different study treatments prior to measuring FI, including variations in composition and quantity of the preload, time to next meal, control of FI prior to study sessions, the EX mode, intensity, duration, frequency and the individual s training status. It is generally accepted that habitual PA counteracts obesity and helps to maintain a healthy body weight in children and adults. In a 12 month study of the effects of military training, substantial improvements were found in body weight, waist circumference, BMI, body composition measured by bioelectrical impedance analysis (BIA) and aerobic performance [51]. Similarly, in a longitudinal study in the eating routines of 5-year-old children (e.g. eating together as a family, having the TV on during meals, duration of meals, etc.), PA and TVV behaviour with weight status development until age 8 were studied. Both TVV and SED patterns significantly increased the risk for becoming overweight at an early age and more physically active children

23 7 were less prone to develop obesity [52]. A review of key factors reducing abdominal fat also found that regular EX reduces body adipose tissue deposits both in obese and overweight subjects [53]. PA and EX exert a beneficial impact on body weight beyond what can be attributed to their energy cost alone. Studies as early as the 1930s found an increase in energy metabolism that persists for many hours following EX [54, 55]. Decades later, studies demonstrated that the resting metabolic rate was greater in endurance-trained individuals than that predicted by their body weight [56]. Therefore, it would be expected that the energy cost of PA and the related increase in post-ex metabolic rate would induce body weight loss if no compensation in EI occurred over time. Consequently, there has been considerable investigation of the impact of both short- and long-term effects of PA and EX on the regulation of EI and EB. PA has been associated with a more accurate regulation of FI and equilibrium in EB [57]. One of the earliest reported studies on this effect was conducted in an Indian male population which measured FI in workers carrying out SED and medium to hard work [58]. The study found that EI and EE were mismatched in individuals carrying out SED work. EI and body-weight were higher in workers performing SED work when compared to workers with medium to high demanding occupations. The matching homeostasis between EI and EE in SED individuals compared to individuals who are active, is in accordance with findings from other studies [59, 60]. One other study showed improvements in FI regulation after an EX intervention. SED adults who exercised for six weeks, were given either a high- or a low-caloric drink followed by an ad libitum buffet meal 60 minutes following EX [61]. After the EX intervention, the participants showed a greater average compensation of FI (79.5%) for the caloric content of the drinks when compared with their SED response (8.9 %) [61].

24 Physical Activity and Food Intake Regulation in Adults Most studies in adults show that in the short-term, large energy deficits, due to high levels of PA, lead to increased FI and are tolerated in part by decreasing other daily activities. No change in EI at a subsequent meal was reported when lean men exercised once at 70% of their maximum oxygen uptake (VO2peak) for 50 minutes, expending an average of 1191 kcal [62]. Even exercising for seven days did not increase EI in men who increased EE by 765 kcal/day by completing three 40 minute period of cycling per day [63]. The lack of adjustment in FI was explained by a decrease in non-structured daily activities, such as taking the elevator instead of stairs. This compensational decrease in EE accounted for 25-30% of the EE induced by EX [63]. Similarly, females failed to compensate for the EE of EX. When women expended an additional 453 kcal/day or 812 kcal/day by cycling for seven days, total daily EE decreased over time during their EX interventions. This is likely due to the same behaviour changes that were observed in men [64]. Partial compensation of only 30% of the EE was accounted for by the reduction of non-structured PA [64]. However, another study with a similar approach found that women tolerated an EE of 907 kcal per day for 14 days but compensated only partially (30%) by increasing FI [65]. This responsiveness in FI regulation to PA may depend on habitual PAL and weight status. Woo et al. conducted two similar studies which investigated the effect of 19 days of a SED, mild EX and moderate EX condition on FI regulation and found that lean active women match EE by increasing EI and maintained a stable EB for all three treatments [66]. However, obese women did not [67].

25 Physical Activity and Food Intake Regulation in Children As noted previously, low levels of PA may desensitize FI regulatory mechanisms and precede weight gain. However, it is unclear if EB is primarily determined by EE or FI in children [68]. Based on PA information collected twice a year by accelerometer studies in children, the children with the highest PA had significantly lower BMIs and sum of skinfolds (SKF) than the low or moderate activity groups [68]. Shorter studies modified PA patterns in children for three weeks by increasing sedentary behaviours, such as the time spent watching TV and playing video games, found increased EIs [69-72]. Conversely, a reduction of SED activities (TV viewing, video games) resulted in lower EI and higher PA in healthy body weight boys when compared to boys with lower BMI Z scores [69-72]. In obese boys, PA decreased when SED activities decreased [71]. An elevation of daily EB was observed with increase in SED behaviours (350 kcal), leading to an increase of BW of 0.32 Kg per week [72]. One study did show differences in EI but induced a significant negative EB after EX [73]. The study of 19 girls assessed differences in EI following one SED and two equicaloric EX protocols performed one week apart. The EX protocols consisted of cycling at a low (50% VO2peak) and high (75% VO2peak) intensity until an EE of 360 kcal was reached [73]. The sessions lasted between 38 and 56 minutes according to the intensity. FI, measured at an ad libitum lunch and dinner, either 75 or 105 minutes after the completion of the EX or SED session, did not differ between the different intensities. Showing that FI may not be responsive to EX in children [73]. Other acute short-duration studies have also failed to show an effect of EX on FI. When 9 to 14 year old boys were asked to EX at their ventilatory threshold (VET) for 12 minutes, expending

26 10 50 kcal, an increase in appetite was found. However, FI was not measured [74]. A follow up study of moderate short (15 minutes) and long-duration EX (45 minutes) on post-ex FI in 9 to 14 year old boys and girls found no effect on FI [75]. Similarly, a third study failed to detect increases in post-ex FI in either lean or obese boys who exercised for either 15 minutes at their individual ventilatory threshold (VET) or 25% above. When the boys received either a non-caloric sweetened control or GL drink in random order 5 minutes after EX or an SED activity, it was found that FI decreased after the GL preload, but was not affected by EX [76]. As a result, EX reduced EB over the duration of the experiments in overweight/obese but not in normal-weight boys. This suggests the regulation of FI in overweight/obese boys in response to a GL drink is similar to normal-weight boys, but it may be less responsive to EX, resulting in an improved EB [76]. In summary, it is clear that increased PA increases EE but its mechanisms on FI regulation are unresolved. One reason may be due to the adaptations that occur in substrate oxidation with PA and the variability induced by body fat. It is well known that each of the macronutrients contributes to intake regulation through different mechanisms. However, the primary sources of energy during EX are glucose and fatty acids; how their oxidation impacts the effects on FI regulation has received little attention. The following section gives a description of the effects of macronutrients (protein, carbohydrate and fat) on FI and EB, and is followed by an evaluation of the role of substrate oxidation in regulation of FI Energy Balance and Obesity Individuals with low EE, low levels of PA and excess levels of EI are particularly vulnerable to weight gain. This can be explained in part by the utilization and storage of energy

27 11 from macronutrients and their effect on FI. Carbohydrates, fat and protein contribute to intake regulation through the glucostatic, lipostatic and aminostatic mechanisms, respectively Carbohydrate Balance Carbohydrates are stored as glycogen [77]. Daily EI in form of carbohydrates accounts for up to % of the total energy stored as glycogen. Therefore carbohydrate oxidation (CHOOX) is decreased or increased, in order to keep the energy stored as glycogen in balance [78]. However, an individual s energy storage of glycogen is limited to a range of kcal [79], and shows much greater fluctuations within the day and from day-to-day than energy storage of fat and protein. In humans, a conversion of carbohydrate to fat in the liver occurs exclusively when daily carbohydrate intake exceeds total daily energy expenditure [80]. Carbohydrates provide signals to FI regulatory systems by several mechanisms including their stimulation of gut peptides and endocrine signals [81]. Their effects are highly correlated with blood glucose which led to the glucostatic theory of appetite control more than 50 years ago. This theory states that decreased glucose utilization or metabolic hypoglycemia occurs at the point where the peripheral arteriovenous difference in blood glucose becomes negligible. As a result, glucose entering metabolizing cells and conversion to energy is decreasing, signalling hunger. This signalling process is thought to account for the short term control of hunger, satiety and satiation [81].

28 Fat Balance Compared with other macronutrients, fat is the largest energy store in the body. In healthy individuals, energy stored as fat is approximately 140,000 kcal and six times larger than the energy stored as protein. In obese individuals, it can be several times larger than that [82]. Adaptations in substrate utilization in response to dietary fat intake are slow to take place. In conditions of overfeeding fat intake is readily stored as body fat since FATOX does not adjust rapidly to the dietary intake [82] and increased FATOX may take up to 7 days [83]. As a result, fat balance is virtually equal to total EB [82]. The lipostatic feedback theory of FI regulation, proposed by Kennedy in 1953, is based on the idea that fatty acids signal the amount of body fat to the brain. In return, the brain compares the current level with a desired target level (the set-point), regulating FI and EE according to body fat stores [84]. FATOX is believed to play a primary role in long-term but not short-term regulation of FI [81] Protein Balance Protein balance is tightly controlled, and is achieved on a day-to-day basis [77]. The total energy derived from energy stored as protein represents about kcal [82]. Unlike energy stored as fat, higher protein intake than required does not increase the amount of energy stored as protein. Only growth stimuli, such as growth hormone, androgens, physical training and weight gain will increase storage of energy as protein [85].

29 13 The aminostatic theory of FI regulation, as first proposed by Mellinkoff in 1956 [86], is based on appetite suppression that is triggered due to a rise of serum amino acid levels. In the past few decades, the regulation of FI based on amino acid sensing systems in the brain has been investigated [87]. Many studies have focused on the role of amino acids such as tryptophan, tyrosine and BCAAs as hypothalamic signals controlling satiety and satiation [88]. In more recent years, it has become apparent that these theories do not fully explain the complexity of FI regulation. However, the principle that they are different in the way they stimulate FI regulatory mechanisms and that metabolism affects EI and EB remains fundamentally sound. Of interest to the present research is that while the role of substrate oxidation has been explored as a factor in physical performance, its role in FI regulation has received little attention. The interaction of EB with substrate oxidation is discussed in the following sections, followed by a description of disturbances in substrate oxidation and metabolic flexibility in obesity and the role of PA in their modulation Energy Balance and Substrate Oxidation Although obesity is a complex and multifactorial problem in origin, a decreased ability of obese and overweight individuals to adequately oxidize substrates may also contribute to obesity [89-91]. The role of fuel utilization in the control of FI was of interest in the 1990s. However, the focus subsequently shifted towards the study of pre-absorptive hormones and brain mechanisms in response to macronutrient ingestion as the primary factors in control of FI, as a more promising cause of obesity than the role of fuel utilization [92]. Although the interest in the role of fuel utilization has declined, the issue was never resolved [93].

30 14 Substrate oxidation in humans as measured by the respiratory quotient (RQ) or the respiratory exchange ratio (RER), was described as early as the 1930s [94]. The RQ (RER) is usually measured by indirect calorimetry and calculated as the ratio of carbon dioxide production to oxygen consumption. Depending on the net metabolic needs of the body at a given moment, the ratio ranges from 0.7 to 1. The ratio is determined by the composition of substrates that are utilized: 1.0 for 100% CHOOX and 0.7 for 100% FATOX. With normal activity levels, the ratio ranges between 0.80 and In conditions that elicit a high production of CO2, such as overfeeding, it can be as high as and indicates lipogenesis [95]. In the 1980s, the RQ : Food Quotient (FQ) concept was proposed. The concept was based on the hypothesis that under normal conditions, the body matches substrate oxidation to the macronutrient composition of the ingested food [89, 96], thus describing the variations in EB and its correlation to variations in macronutrient balance [56]. Due to the daily variation of EE and EI, changes in substrate homeostasis occur constantly. In response to an increased carbohydrate intake, the oxidation of carbohydrates may shift rapidly towards an increased CHOOX while FATOX is decreased, resulting in an increased RER [89, 97]. In very low carbohydrate diets, CHOOX will decrease while FATOX is increased [98]. Fat utilization responds relatively slowly to dietary fat and is therefore stored directly as adipose tissue [83, 89]. These changes in substrate utilization are reflected by the RER [97]. Substrate utilization during EX can vary greatly and is determined by many factors including intensity, type, duration, fitness levels, body weight, whether the person is exercising in a fasted state and whether carbohydrates are ingested during the EX. Additionally, the ability to utilize GL during EX is not based on insulin secretion but rather on muscle contraction itself [99],

31 15 resulting in an intact CHOOX even if metabolic impairments such as insulin resistance are present [100]. In healthy individuals at low EX intensities (25% VO2peak), almost all energy is derived from plasma fatty acids [101]. When EX intensity increases to moderate intensity (50-60% VO2peak), total FATOX increases to its peak [101]. Above this crossover point, energy requirements reach levels where FATOX cannot meet them and approximately half of the energy is provided by carbohydrates [101]. In healthy individuals, the aerobic/anaerobic threshold occurs approximately at this crossover point [102]. At EX intensities > 85% VO2peak, the energy is predominately supplied by muscle glycogen and CHOOX. As soon as energy stored as carbohydrate depletes, FATOX cannot supply energy at rates sufficient for high EX intensities [101] Metabolic Flexibility and Obesity Metabolism and the utilization of substrates is generally determined by an individual s demand to generate adenosine triphosphate to maintain body temperature and movement. The adjustment of substrate utilization is called substrate shift or substrate choice. The term metabolic inflexibility as first proposed by Kelley et al., defined a metabolic deregulation that impairs the capacity to increase FATOX when fatty acid availability is increased and to switch from fat to GL as primary fuel source after a meal [103, 104]. It has also been defined as the incapability of the body or cells to match fuel oxidation to fuel availability and the endocrine environment. [85]. Impairments such as insulin resistance, hyperinsulinaemia, reduced lipid trafficking and hyperlipidemia, increased RER during EX, shift in muscle fibre type and ectopic fat storages are also often stated as characteristics of metabolic inflexibility [105].

32 16 In inactive and especially inactive obese and overweight individuals, impairments of the ability to shift substrates seem to be present [45, ]. This is described by a heavy reliance upon carbohydrates under fasted conditions [109] and an inability to increase CHOOX under insulin-stimulated conditions [110]. The capability to appropriately shift substrates is reduced in obese individuals and a consistently high fasting RER has been associated with weight gain [98, ]. Formerly obese individuals have a higher RQ than never-obese individuals and experience greater weight regain following weight loss [ ]. Obese and formerly obese individuals also display a blunted increase in FATOX following weight loss [109], potentially contributing to an individual's susceptibility to overconsumption and weight gain [ ] Metabolic Flexibility and Physical (In)Activity Some studies suggest that SED behaviour is an autonomous contributor to metabolic inflexibility, even if the current guidelines for PA have been met [118]. Physical inactivity is one of the primary augmenters in the progress of developing metabolic inflexibility [105]. Individuals who follow SED daily lifestyle patterns are more likely to develop obesity and insulin resistance [119, 120]. One study showed an increase in insulin resistance in healthy individuals after 1-3 weeks of bed rest [121]. Studies investigating three months of bed rest have also shown decreases in FATox of up to 37% and increases in CHOOX of 21% [106]. Other studies that investigated bed rest have discovered an increased RER of 4-14% [106, 107, 122]. This increase was inversely correlated with metabolic flexibility [123], which is further aggravated by excess adipose tissue [124].

33 17 In contrast, high PALs improve outcomes related to metabolic inflexibility. Several studies have shown beneficial effects, of PA and EX on metabolic flexibility and substrate utilization, in children and adults. Trained men have significantly higher aerobic capacities when compared to untrained men [125]. This indicates that trained individuals are more reliant on FATOX and are therefore more metabolically flexible. In children, Bell et al., showed that an 8 week EX program, consisting of 3 x 1 hours sessions per week improved cardiorespiratory fitness, insulin sensitivity and the lipid profile of obese children [126]. Schmitz et al., observed similar findings during a hyperinsulemic clamp study in 357 non-diabetic children. He found a strong positive correlation of PALs with insulin sensitivity and lipid profiles [110]. Another study has shown that pubescent boys display significantly higher rates of FATOX during EX when compared with their obese counterparts [127]. To summarize, PA contributes to metabolic flexibility because of an increased ability to oxidize fat. Individuals with impaired capacity to up-regulate FATOX may also signal a promotion of FI. Although it is known that EX improves insulin resistance, no study has investigated the benefits of EX intervention programs on metabolic flexibility and FI regulation Physical Activity, Substrate Utilization and Food Intake Regulation Substrate metabolism may also act as a biological determinant of eating behavior, rather than being a response to EX or FI. Although there have been few studies, substrate metabolism has been attributed to post-ex compensatory eating. Although no mean increase in EI was reported in 11 lean men following 90 minutes of cycling (60% VO2peak), when participants were retrospectively divided into 'high' or 'low' fat oxidizers based on their RQ, post-ex EI was

34 18 significantly lower in the high-fat oxidizers. EX induced a -406 kcal net energy deficit in the highfat oxidizers, but a net positive EB of a similar order in the low-fat oxidizers [48]. The group suggested that a low RQ attenuates EX-induced glycogen depletion and therefore decreases EI in the high-fat oxidizers [48]. Other studies also have reported similar results [49, 50, [128]. Obesity has been proposed to modify the metabolic control of EI due to body- and skeletal muscle-related impairments in FATOX associated with adiposity [92, 129]. As a result, metabolically inflexible individuals who display a blunted ability to up-regulate FATOX during EX may be more susceptible to compensatory eating. In addition, enhanced reliance upon CHOOX during EX could induce reductions in stored glycogen, blood glucose and consequently enhance the drive to restore availability via feeding as proposed by the glucostatic hypothesis of FI regulation. In summary, the role of substrate oxidation as influenced by metabolic flexibility and/or PA, on FI regulation has not been reprised. The objective of this thesis research is to begin to examine these relationships.

35 19 3. SUMMARY AND STUDY RATIONALE The relationship between substrate oxidation and FI has not been investigated. It is well accepted that habitual EX can help to improve metabolic flexibility [125, 130] as well as FI regulation [61, 129]. Obese and SED populations often display signs of metabolic inflexibility when compared to lean individuals such as a lower RER and the impaired ability to oxidize fat [105]. Similar to the comparison of lean and obese individuals, pre-pubertal boys have been shown to have a better metabolic flexibility and therefore lower RER values and higher rates of FATOX when compared to adults [131]. The study, as part of this thesis, will examine substrate oxidation and its relationship with FI. The study will examine RER in normal weight boys and young men, without confounders of insulin resistance and hyperinsulinaemia which are present in obese populations [105, 132]. This research will also aid in the understanding of the general effects of RER on post-ex FI.

36 20 4. HYPOTHESIS 4.1. Primary Hypothesis An elevated RER, indicating an increased carbohydrate relative to fat oxidation, is associated with an increased FI at a later meal. 5. OBJECTIVES 5.1. Overall Objective To examine the relationship between substrate oxidation and short-term FI in normalweight pre-pubertal boys and young adult men Specific Objective To describe the effects of a GL preload, EX, and GL with EX combined, on substrate oxidation and FI in normal-weight pre-pubertal boys and young adult men.

37 21 6. MATERIALS AND METHODS 6.1 Experimental Design Fifteen normal-weight boys and fifteen normal-weight male adults were recruited through posters at the University of Toronto Athletic Centre and recruitment letters which were sent to local sports clubs (Appendix 4A + 4B + 8A +8B). The experiment followed a 2 x 2 x 2 factorial repeated measures randomized design, generated with a random generator script in SAS version 9.2, with four experimental sessions separated by one week. For each session, including the screening session, subjects arrived after a 12-hour overnight fast. Subjects received four treatments, which included: 1) Water preload in a SED condition (control), 2) GL in a SED condition, 3) Water in an EX condition (control), 4) GL in an EX condition. Participants were blinded about the type of treatment. Heart rate (HR), gas exchange, subjective appetite and blood glucose (BG) were measured throughout the session. Five minutes after the completion of the SED or EX, the participants were provided with an ad libitum pizza meal. Short-term FI reflected each individual s net energy pizza consumption.

38 22 Figure 6-1 Study design EX/SED EX/SED Pizza lunch 3 GL/CON 1,2 Time (min): /95 3 MTE 4 MTE 4 MTE 4 MTE 4 MTE 4 MTE 4 PC 5 PC 5 PC 5 PC 5 PC 5 PC 5 EF 9 PS 6 EF 9 EF 9 EF 9 FP PP 7 EF 9 EF 9 Visual Analog Scale Legend g kg -1 bodyweight GL (adults) or 1.2 g kg -1 bodyweight GL (children) preload was given in an opaque covered mug with a straw and consumed within 5 min 2 250ml (children) or 350 ml (adults) water control was given in an opaque covered mug with a straw and consumed within 5 min 3 Pizza lunch and a 500 ml bottle of spring water was presented with the test meal at 85 minutes for children and 95 minutes in adults 4 A was presented with the test meal at 85 minutes for children and 95 minutes in adults 5 Motivation-to-Eat (MTE) VAS administered to determine subjective appetite and thirst 6 Physical Comfort (PC) VAS was administered to determine subjective physical well-being 7 Drink Sweetness (PS) VAS was administered to determine subjective sweetness of the preload 8 Drink Palatability (PP) VAS was administered to determine subjective palatability of the preload 9 Food Palatability (FP) VAS was administered to determine subjective palatability of the test meal 10 Energy Fatigue (EF) VAS was administered to determine subjective energy/fatigue ratings in adults

39 Participants Participants born at full-term and with normal birth weight were included in the study. Those taking any medications that could interfere with study outcomes, with significant learning, behavioural, injuries or emotional difficulties were excluded. Participants who volunteered to partake in this study were first tested for eligibility using a telephone questionnaire (Appendix 2A + 2B). Participants were also asked to select the type of pizza they would eat during the test visits. All study sessions took place on weekend mornings for children at either 9:00 am or 10:00 am and weekday mornings at either 8:00 am or 9:00 am for adults. Participants were asked to arrive at the same day and time for each of the following sessions. If subjects arrived more than 30 minutes late, the session was postponed to another day. Prior to the first study visit, the parents and child were given a tour of the facility to familiarize the child with the study rooms and minimize his apprehension during the first test visit. 6.3 Screening Session If the initial inclusion criteria were met, the adults or the children along with their parents attended a screening session at the University of Toronto Athletic Centre, where the study was explained to them. An informed written consent was obtained from the adults or the parents and their children (Appendix 5A + 5B). The BG measurements were voluntary for children. Parents as well as the child had to give their consent. Participants were asked to fill out questionnaires about food acceptability, food preference, sleep habits and previous PALs. Children additionally filled out questions about their Tanner staging (Appendix 3A).

40 24 After that, height, weight and body composition by SKF calipers were assessed (Appendix 3A + 3B). Physical fitness was measured by VO2peak and VET. To evaluate EX intensity and physical fitness, a continuous incremental walking protocol on a motorized treadmill was utilized. A continuous and progressive walking protocol based on a VO2peak of 65 ml kg -1 min-1 was employed on a motorized Trackmaster TMX 425 CP treadmill (Full Vision, Newton, KS, USA) to determine the slope and speed to maintain the EX intensity at a RER of The measurement of ventilatory gases identified the VET and VO2peak as well as the RER of 0.82, which translates into an approximate CHOOX of 40% and FATOX of 60%. The identified slope and speed was then employed during the experimental EX sessions. It required a fast walk during all stages of the protocol, but depending on participants height and running mechanics, some were more comfortable running than walking to achieve the target speed. The final stage was determined by the participants fitness and effort. To allow collection of ventilatory gases, they were fitted with a Hans-Rudolph mouthpiece/facemask with a Hans Rudolph two-way non-rebreathing valve (Hans Rudolph, Inc., Shawnee, KS, USA). A Polar Monitor was used to detect HR (Polar Electro Inc. Lake Success, NY, USA). Participants were asked to refrain from eating before the screening session. The participants were able to stop the treadmill at any time if they were uncomfortable with the protocol or the measurements. The accuracy of speed and incline of the treadmill were verified before the start of the study. The test was performed at the Human Physiology and Performance Laboratory (University of Toronto - Athletic Centre, Toronto, ON, Canada) after an overnight fast. Subjects that met the eligibility criteria were scheduled for the experimental sessions.

41 Experimental Sessions Adults started the session on a weekday morning at either 8:00 am or 9:00 am and children started their sessions on a weekend morning at either 9:00 am or 10:00 am, following an overnight fast. Subjects were allowed to consume water until one hour before each session. Each subject arrived at the same chosen time for each session. Subjects were instructed to refrain from any unusual EX and activity the day before the study sessions. Upon their arrival to the University of Toronto Athletic Centre, participants were asked to change into EX clothing so they could not anticipate the treatment. A fasting BG sample was obtained and recorded in the session sheet (Appendix 7), prior to the completion of the first visual analog scale (VAS) questionnaires assessing their Sleep Habits, Stress Factors, Food Intake and Activity Level and Feelings of Fatigue. If subjects reported significant deviations from their usual patterns, they were asked to reschedule. In this study, six participants had to reschedule due to not arriving in a fasted state and/or showing elevated fasting BG levels. Fifteen minutes before the start of the EX or SED sessions, participants consumed the preload treatment consisting of a GL solution or a water control. Subjects were then prepared for the gas exchange measurement for another 10 minutes and started exercising at 15 minutes. Participants exercised for 45 minutes in 2 x 20 minute periods with a 5-minute break at an intensity following an RER of A Polar HR monitor was used to measure HR during the 2 x 20 minute time periods. BG was measured four times via finger-pricks at 0, 15, 35 and 60 minutes. FI was measured at an ad libitum lunch meal (pizza), served 5 min after the end of the EX or SED session, and the subject was instructed to eat until he is comfortably full. Deep and Delicious pizzas (McCain Foods Ltd, Florenceville, NB, Canada) were served up to 30 minutes. Subjects had a

42 26 choice between varieties of pizza, including Three Cheese, Pepperoni and Deluxe. EI from the pizza meal was calculated based on the weight consumed and the compositional information provided by the manufacturer, whereas water intake was measured by weight. 6.5 Preload Treatment Treatments consisted of either a GL preload or water (control). In adults, the preload contained 1.0 g per kg body weight of g per kg body weight GL monohydrate (Grain Process Enterprises, Toronto, ON Canada) and 1.6 g of aspartame-sweetened orange flavored crystals (Sugar Free Kool-Aid, Kraft Canada Inc., Don Mills, ON Canada) in 350 ml water. In boys, 1.2 g per kg body weight of 1.31 g per kg body weight GL monohydrate and 1.1g of aspartamesweetened orange flavored crystals were added to 250 ml water. In adults, the amount of GL and water was altered to avoid nausea. The control consisted of 250 ml water (children) or 350 ml water (adults). Subjects were asked to consume the beverage within a period of 5 minutes Exercise Treatment The EX intensity was estimated based on a VO2peak of 65 ml. kg -1. min -1 and a resting oxygen consumption (RO) of 4.5 ml. kg -1. min -1 in boys (ROb) and 3.5 ml. kg -1. min -1 in men (ROm) [133]. The American College of Sports Medicine (ASCM) running formula was used to calculate the 9-2 minutes continuous stages (Equation 6-1) [134, 135]. To ensure the safety of the participants, the running speed for the final stage of the protocol was fixed at 150 m/min for boys (Speedb) and 250 m/min for men (Speedm). This speed was determined with a preliminary testing

43 27 at the University of Toronto Athletic centre. The formula was converted for the determination of the grade incline for the final stage of the protocol. The speed and grade incline of the final stage were partitioned to each of the nine stages to guarantee a steady incline within each stage. To prevent an overshoot of RER, both children and adults were asked to EX at 0 % incline and a speed of 3 km/h to warm up and become familiar with the treadmill prior to the start of the protocol. Equation 6-1 ASCM running equation for the determination of VO2 in boys and men VO2peak (ml/kg/min) = 0.2 Speedb (m/min) Speedb (m/min) Grade (%) + ROb VO2peak (ml/kg/min) = 0.2 Speedm (m/min) Speedm(m/min) Grade (%) + ROm Equation 6-2 Converted ASCM running equation for the determination of grade (%) in boys and men Grade boys (%) = Grade men (%) = [ 65 ml kg 1 min ml kg 1 min m min 1 ] [ 65 ml kg 1 min ml kg 1 min m min 1 ] Exercise Protocol for Children The protocol for children started with 2-minute warm-up followed by a 2-minute rest period. After the warm-up and rest period the treadmill started at 4.5% incline and a speed of 3 km/h. Speed and incline increased every 2 minutes by 1.5% in incline and 1 km/h in speed. Children were asked to EX on the treadmill until a RER >1.15, a HR of > 205 BPM or a voluntary exhaustion was achieved.

44 Exercise Protocol for Adults The protocol was designed similar to that for children, based on a different resting oxygen consumption. The protocol started at a speed of 5 km/h. Adult men were also asked to EX on the treadmill until a RER >1.15, a HR >195 BPM or a voluntary exhaustion was achieved Resting Protocol During resting periods participants remained sedentary and engaged in quiet games (Sudoku, word puzzles, reading, Jenga, Dominoes, and Checkers) or reading. However, they were allowed to get up to use the washroom. Participants were monitored by a volunteer or a research assistant at all times who avoided the topic of food.

45 29 7. MEASURES AND DATA ANALYSIS 7.1. Food Intake Two varieties of five-inch diameter pizza (McCain Foods Ltd, Florenceville, NB, Canada) were offered for the test meal. Boys were served a total of nine pizzas with three pizzas per tray in regular 10-minute intervals. Men were served a total of 12 pizzas with four pizzas per tray in 6:30 minute-intervals. The intervals and numbers of pizza were determined in previous studies in our lab on adults and children. Deep and Delicious pizzas were selected due to their lack of crust and uniform composition. The boys had the option of choosing between Three Cheese, Pepperoni or a combination of both pizzas, while adults had an additional choice of Deluxe. Each pizza contained on average 180 kcal. The energy content and macronutrient information is provided by the manufacturer (Appendix 12). Pizzas were cooked and then weighed and cut into four equal pieces. Pizzas were then served and the amount left over after the meal was subtracted from the initial weight of the pizza to determine the net weight consumed in grams. Different varieties of pizza were weighed separately. The energy consumed (kcal) was calculated by converting the consumed net weight of the pizza (grams) using information provided by the manufacturer. During feeding period, participants were escorted and seated in a feeding room to minimize distractions and maintain consistent conditions. A 500 ml bottle of spring water (Danone Crystal Springs, Quebec City, QC, Canada) was served along with the pizza meal. The bottle was weighed before and after the meal to determine water intake (grams). Subjects were provided with a second bottle if they finished the first.

46 Blood Glucose Measurements Fifteen adults and six children gave their consent and completed the BG measurements. Each session, four finger-pricks were taken for a total of 16 throughout the study for each individual. Finger-prick blood samples were obtained using a Monojector Lancet Device (Sherwood Medical, St. Louis, MO, U.S.A.). One drop of blood was placed on an Accu-Chek test strip for immediate reading of the GL concentration with the Accu-chek GL meter (Accu-Chek Compact and Compact-Plus, Roche Diagnostics Canada, Laval, Quebec). The meters and test strips were standardized against Assayed Human Multi-sera (Randox Laboratories LTD, Antrim, UK). Proper procedure for obtaining blood sample was demonstrated to participants, prior to the first session during the screening interview. The adult subjects pricked their own fingers while supervised by the investigator. Children had their fingers pricked by the supervisor. Each subject was assigned the same GL meter throughout the study. There was no risk of contamination because the lancet needles were discarded after each use and each subject was provided with a sterilized monojector device (immersed overnight in ethanol, 70%) at each session. Moreover, the monojector device was wiped with an antiseptic isopropyl alcohol pad before inserting the disposable sterile lancet needle. Subjects cleaned their fingers with antiseptic isopropyl alcohol pads before pricking and were seated at a safe distance from each other to prevent crosscontamination Collection of Ventilatory Gases Ventilatory gases were collected using a Moxus metabolic cart (AEI Technologies, Inc., 300 William Pitt Way, Pittsburgh, PA 15238, USA), a facemask and a two-way non-rebreathing

47 31 valve (Hans Rudolph, Inc., 8325 Cole Parkway Shawnee, KS 66227, USA). A pneumotachometer measured inspiratory ventilation and gas analyzers measured the mixed expired gas. The O2 content was analyzed by S-3A Oxygen Analyzer and a CD-3A Carbon Dioxide Analyzer (AEI Technologies, Inc., 300 William Pitt Way, Pittsburgh, PA 15238) measured the CO2 content of the expired air. Known gas concentrations of 16.04% O2 and 4.06% CO2 and 20% O2 and 0.03% CO2, were used to calibrate the metabolic cart, prior to each test Measurement of Physical Fitness Physical fitness was determined by measuring the VO2peak and the VET. VO2peak is the maximum capacity of an individual's body to transport and use oxygen during incremental exercise, which reflects the physical fitness of the individual. The VET roughly corresponds to lactic acid threshold, at which plasma lactic acid builds at a rate faster than that at which the body clears lactate from circulation. The VET is a submaximal marker of aerobic fitness, which is used clinically to monitor patients Maximum Oxygen Consumption The VO2peak requires the individual to reach a maximum physical effort in order to achieve a plateau of oxygen consumption with no further increase of the workload. These tests are usually performed on a treadmill or a cycle ergometer. The measurement of VO2max is regarded as the gold standard for determining cardiovascular fitness of an individual [136]. A VO2max at plateau occurs in approximately 50% of all tests, and when not achieved the peak value is then called VO2peak [136]. The measurement of VO2peak can be affected by a number of factors like

48 32 age, gender, training state, altitude changes and the action of ventilatory muscles [137]. Further criteria, such as RER or HR, are used to indicate if an individual reached their VO2max. The average of six breaths of the highest achieved values is usually used to identify the plateau. There is limited evidence regarding the most appropriate oxygen uptake data averaging interval; nonetheless, the averaging interval does not seem to affect the reproducibility of VO2peak measurements [138] Ventilation Threshold The VET is an indicator of aerobic fitness. It represents a practical and non-invasive method to approximate an individual s lactic acid threshold [139, 140]. Intensities above the VET indicate a more fatiguing less sustainable level of EX, causing an individual to switch from fat to carbohydrates as predominant fuel source. The VET of healthy untrained individuals averages at 45-65% of their individual VO2peak [141]. It can be increased with habitual EX, as a study in marathon runners reported VET values as high as 76% [142]. This study used the two most common methods to determine the VET. The first method used the ratio of pulmonary ventilation (VE) divided by VO2 and VCO2. This method, identifies the VET when there is a rise of VE/VO2 without a significant increase in VE/VCO2 [143] (Figure 7-1). The second method, the V-Slope method, is used if the increase in VE/VO2 over VE/VCO2 is not conclusive. In the second method, the VET is described as the point at which a change in slope occurs if VCO2 is plotted over VO2 [144] (Figure 7-2).

49 33 45 VE/VO 2 VE/VCO 2 Ventilatory Equivalents (ml min -1 ) V E T Percentage VO 2 peak Figure 7-1 VET (arrow) of an adult subject identified by the VE/VO2 (solid line) over VE/VCO2 (dashed line) method V E T 2500 VCO 2 (ml) VO 2 (ml) Figure 7-2 VET (arrow) of an adult subject identified by the V-Slope method.

50 Substrate Oxidation and Energy Expenditure The RER is sometimes referred to the RQ. The difference is that RER is measured at the mouth and is VCO2/VO2, whereas the RQ is the ratio of CO2 produced to O2 consumed at the cellular level. Thus, RQ is dependent on the type or types of fuel substrates being used by the cell. The measurement of RER provides a means of estimating the composition of the fuels oxidized and represents the ratio of carbon dioxide exhaled to the amount of oxygen consumed by the individual (VCO2/VO2). Generally RER = RQ but if the subject is hyperventilating, has an acidbase disturbance, or is performing intense EX with an RER < 1.0, extra CO2 can result from buffering. Thus CO2 production, measured at the level of the mouth, may not accurately reflect CO2 production at the cellular level [145]. Furthermore, RER is a non-protein measurement because proteins cannot be completely oxidized into CO2 and H2O and nitrogen is additionally present. O2 consumption needed for oxidizing a protein and the resulting CO2 production could be measured, but it would not accurately reflect protein use by the body because nitrogen cannot be measured by RER. Furthermore, under normal circumstances in humans, less than 5% of the energy production comes from protein oxidation and is therefore neglected in this measurement. Although fat contains more potential chemical energy on a per unit-weight basis, carbohydrates due to their oxygen content give more energy for a given volume of O2. A RER of 0.7 indicates that only fat is being used as a substrate whereas a RER of 1.0 indicates that only carbohydrates are being used, as showing in the following equations (Equation 7-1).

51 35 Equation 7-1 RQ equation for glucose and fat a) Glucose C6H12O6 + 6 O2 6 CO2 + 6 H2O RQ = 6 CO2 produced / 6 O2 consumed = 1.0 b) Fat C57H104O O2 57 CO H2O RQ = 57/80 = 0.71 RER is also useful in interpreting EE, which was measured indirectly with a metabolic cart by analyzing respired gases such as O2 and CO2. The volume of air passing through the lungs was assessed by a pneumotachometer placed on the inspiratory side. From this value the amount of inspired and expired VO2 and VCO2 was extracted and the RER was calculated. The measurements of RER helped to calculate the EE (kcal) and the ratio of CHOOX and FATOX. The Weir equation was used to calculate EE for 40 minutes of EX or SED as shown in the following example (Equation 7-) [146]. Equation 7-2 Energy expenditure for 40 minutes of gas exchange measurements EE (kcal) = ((1.1 RQ) + 3.9) VO2) 40 minutes The percent of CHOOX and FATOX were derived from the non-protein RER table (Table 7-1) [147] and their amount contributing to EE was calculated as in Equation 7-1. Equation 7-3 Equations to calculate CHOox and FATox CHOox (kcal) = EE % kcal derived from CHO FATox (kcal) = EE % kcal derived from FAT

52 36 Table 7-1 Thermal equivalents of oxygen for the non-protein respiratory exchange ratio RER kcal per Liter O2 Uptake % kcal Derived from CHO % kcal Derived From FAT Grams per Liter O2 CHO Grams per Liter O2 FAT

53 Assessment of Body Fat Percentage A Harpenden SKF caliper was used to measure SKFs at four sites (biceps, triceps, subscapular and suprailiac crest) and recorded to 0.2 mm. The mean SKF of three measurements at each site was used to estimate body fat percentage. The typical standard error of estimate for SKF measurements in healthy individuals was previously determined at 3-5% and was also reported to be higher in younger individuals [148]. Age specific regression equations from Durnin and Womersley were used to determine percent body fat in boys and adults, as shown in Equation7-4 [149]. Body fat measurements with the SKF method are considered an inexpensive and direct procedure to assess body fat in children and adults. The density value can then be converted to percent body fat (%BF) using the Siri Equation (Equation 5) [150]. Equation 7-4 Durnin and Womersley equations for body density a) Boys Body density boys = ( ) x log (SKF biceps + SKF triceps + SKF Subscapular + SKF Suprailiac Crest) b) Men Body density boys = ( ) x log (SKF biceps + SKF triceps + SKF Subscapular + SKF Suprailiac Crest) Equation 7-5 Siri equation for prediction of fat mass 4.95 % BF = { 4.5} 100 body density

54 Estimation of Percentage of Maximum Heart Rate The maximum HR, which was measured during the screening session, was compared with each subject s estimated maximum HR (HRest), to determine if the subjects reached their VO2peak. The HR was estimated according to the formula of Mahon et al. [151], in boys (HRmax boys = 208 age 0.7) and the formula of Robergs et al., in men (HRmax men = 205 age 0.685) [152] Visual Analog Scales Standardized VASs questionnaires were used to measure subjective appetite, physical comfort, and energy/fatigue and stress as well as treatment and pizza meal palatability and sweetness. Different questionnaire versions were used for children and adults for the assessment of physical comfort and food/preload palatability and questionnaires on subjective sweetness were only administered in children, whereas energy fatigue VASs were only used for adults Subjective Appetite Motivation-to-eat VAS (Appendix 9A), which consisted of five questions, was used to assess subjective appetite and thirst. Each question was followed by a 100 mm line with opposing statements at either end. Subjective appetite questionnaires were asked at 0, 5, 15, 35, 60 and 85 in adults and 95 minutes in children. Subjects pencilled an X mark on the line to indicate their subjective perception regarding the question. Scores were assessed by measuring the distance in mm from the left of the line to the X mark. The five questions of the VAS are: How strong is your desire to eat? ( Very weak to Very strong ) How hungry do you feel? ( Not hungry at all to As hungry as I ve ever felt )

55 39 How full do you feel? ( Not full at all to Very full ) How much food do you think you could eat? ( Nothing at all to A large amount ) How thirsty do you feel? ( Not thirsty at all to As thirsty as I have ever felt ) Subjective average appetite scores were determined by adding the scores of desire to eat, hunger and how much food do you think you can eat and 100 minus fullness and dividing them by four [average appetite (mm) = (desire to eat + hunger + (100 fullness) + how much food do you think you can eat)/4] [153]. Appetite scores have been calculated in previous studies [ ] Subjective Physical Comfort Visual Analog Scales for physical comfort were assessed by How well do you feel? with a range of Not well at all to Very well in children (Appendix 9B). Physical comfort in adults was assessed by a number of questions such as Do you feel nauseous? with a range of Not at all to Very much, Does your stomach hurt? with a range of Not at all to Very much, How well do you feel? with a range of Not well at all to Very well, Do you feel like you have gas? with a range of Not at all to Very much and Do you feel like you have diarrhea? with a range of Not at all to Very much (Appendix 10B). Subjective physical comfort was assessed in children and adults at 0, 5, 15, 35, 60 and 85 in adults and 95 minutes in children. Subjective average physical comfort scores were determined by adding the scores of do you feel nauseous, does your stomach hurt, how do you feel like having diarrhea, do you feel like having gas as well as 100 minus how well do you feel, and dividing them by five [physical comfort (mm) = (do you feel nauseous + does your stomach hurt + how do you feel like having diarrhea + do you feel like having gas + (100 how well do you feel))/5].

56 Subjective Energy/Fatigue and Stress. Subjective energy/fatigue and stress was assessed only in adults. Subjects subjective energy perception was assessed by the question How energetic do you feel right now? with a range of Not at all to Very energetic, How tired do you feel right now? with a range of Not at all to Very tired and How anxious do you feel right now? with a range of Not at all anxious to Very anxious (Appendix 10C). Subjective energy/fatigue and stress scores were assessed at 0, 5, 15, 35, 60 and 85 minutes Subjective Palatability The enjoyment of the beverage/meal was assessed by the VAS palatability score by asking questions such as How pleasant have you found the food? with a range of very pleasant to Not pleasant at all (Appendix 9D). Adults were asked questions such as How pleasant have you found the beverage/food? with a range of Not at all pleasant to Very pleasant, How tasty have you found the beverage/food? with a range of Not at all tasty to Very tasty and How did you like the texture of the beverage/food? with a range of Not at all to Very much. Subjective palatability was only assessed after the preload and the pizza lunch at 5 minutes in children and adults and 85 minutes in adults and 95 minutes in children. Palatability scores for adults were determined by adding the scores of how pleasant have you found the beverage/food, does your stomach hurt, how tasty have you found the beverage/food and how did you like the texture of the beverage/food and dividing them by four [Subjective palatability (mm) = (How pleasant have you found the beverage/food + does your stomach hurt + How tasty have you found the beverage/food + How did you like the texture of the beverage/food)/4] (Appendix 10D).

57 Subjective Sweetness The sweetness VAS was only assessed in children at 5 minutes. Subjects subjective sweetness perception was assessed by the question How sweet have you found the beverage? with a range of Extremely sweet to Not sweet at all (Appendix 9C) Statistical Analysis Statistical analyses were conducted using SAS version 9.2 (Statistical Analysis Systems, SAS Institute Inc., Carey, NC). All results are presented as mean ± standard error of the mean (SEM). Statistical significance was concluded with a 2-tail p-value less than Repeated measures ANOVA analysis were conducted with the Proc Mixed procedure. A 3-factor ANOVA was used to determine the effects of GL, EX and age (AGE) on FI, NEB, RER, EE, and HR. The effect of AGE and AGE interaction with GL, EX and GL with EX was used to identify different FI and NEB responses between groups. Separate ANOVA analysis for boys and men were conducted if there was an interaction for AGE*GL, AGE*EX or AGE*GL*EX. A 4-factor ANOVA was used to determine the effect of GL, EX, TIME and AGE on average BG and VAS scores for pre-meal subjective feelings of appetite, thirst and physical comfort. The pre-meal results for the min times are expressed as change from baseline, and represent the time between completion of either the SED or EX condition and the the test meal. Pearson correlation coefficients were calculated to identify associations between FI, NEB and RER, HR, CHOOX, FATOX and EE.

58 42 8. RESULTS 8.1. Descriptive Measures The study was completed with 30 participants, with 36 being initially recruited. Pooled values for subject characteristics of the 15 boys and 15 men are shown in Table 8-1. These characteristics are shown for each of the 30 participants in Table 8-2 for boys and Table 8-3 for men. The average age of boys and men was 10.9 ± 0.3 and 23.5 ± 0.8 years respectively. Baseline BMI and BMI percentile classified both age groups as normal weight, according to the CDC BMI and BMI for age guidelines (Appendix 1A + 1B). VO2peak was similar in both groups (43.5 ± 2.0 ml kg -1 min-1 in boys vs ± 1.2 ml kg - 1 min -1 in adults). However, VO2peaks indicated higher absolute fitness levels in adults when compared to children relative to their age specific norms [156, 157]. The VETs at 29.2 ± 1.6 ml kg - 1 min -1 in boys and 25.7 ± 0.9 ml kg -1 min-1 in men were not significantly different. The VET as a relative percentage of the VO2peak in children (67.6 ± 1.9 %) may indicate that they had a better ability to rely on fats during EX when compared to adults (59.2 ± 2.0 %), but adults had less body fat (13.5 ± 0.9 %) when compared with children (17.6 ± 1.0 %). HRmax and %HRest were not significantly different between boys and men

59 43 Table Mean subject characteristics of boys and men Subject Characteristics Children Adults P-value Age (years) ± ± 0.8 < Weight (kg) ± ± 2.4 < Height (cm) ± ± 1.5 < BMI 2 (kg/m 2 ) 17.1 ± ± 0.6 < BMI 2 percentile 50.2 ± 5.6 SKF body fat 3 (%) ± ± VET 4 (ml kg -1 min-1 ) ± ± 0.9 n.s. %VET of VO2peak 67.6 ± ± VO2peak 5 (ml kg -1 min-1 ) 43.5 ± ± 1.2 n.s. HRmax 6 (bpm) 183 ± ± 3.2 n.s. %HRest 7 93 ± ± 1.7 n.s. 1 Means ± SEM; n=30. A student s t-test was used to determine differences between boys and men. 2 BMI = Body mass index 3 VET = Ventilation threshold (ml min -1 kg -1 ) 4 VET = percentage VET of Vo2peak 5 VO2peak = maximum oxygen consumption (ml min -1 kg -1 ) 6 HRmax = maximum heart rate (bpm) 7 %HRest = percentage of estimated HRmax

60 44 Table 8-2 Baseline characteristics for 15 boys 1 ID Age (year s) Weigh t (kg) Heigh t (cm) BMI 2 (kg/m 2 ) BMI 2 percentil e VET 3 (ml mi n -1 kg -1 ) VET 4 % of VO2pea k VO2peak 5 (ml min - 1 kg -1 ) HR max 6 (bpm ) HRes t (%) 7 SKF body fat(%) 8 Tanne r Stage Mean SEM Means ± SEM; n=15 2 BMI = Body mass index 3 VET = Ventilation threshold (ml min -1 kg-1 ) 4 VET = percentage VET of Vo2peak 5 VO2peak = maximum oxygen consumption (ml min -1 kg-1 ) 6 HRmax = maximum heart rate (bpm) 7 %HRest = percentage of estimated HRmax 8 SKF BF = Percent body fat by SKF analysis (%)

61 45 Table 8-3 Baseline characteristics for 15 men 1 ID Age (year s) Weig ht (kg) Heigh t (cm) BMI 2 (kg/m 2 ) VET 3 (ml min - 1 kg -1 ) VET 4 % of VO2peak VO2peak 5 (ml min - 1 kg -1 ) HR max 6 (bpm) HRe st (%) 7 SKF BF (%) M ea n SE M Means ± SEM; n=15 2 BMI = Body mass index 3 VET = Ventilation threshold (ml min -1 kg -1 ) 4 VET = percentage VET of Vo2peak 5 VO2peak = maximum oxygen consumption (ml min -1 kg -1 ) 6 HRmax = maximum heart rate (bpm) 7 %HRest = percentage of estimated HRmax 8 SKF BF = Percent body fat by SKF analysis (%)

62 Food Intake Compared to CON, FI (kcal/kg) was lower after GL (p < ), but not affected by EX. FI (kcal/kg) was higher in boys when compared to men (AGE, p = ). A significant interaction was found for GL*EX (p = ), showing an additional suppression of FI (kcal/kg) by GL combined with EX. No other significant interactions were found (Table 8-4). Table Food intake (kcal/kg) in boys and men Activity: Sedentary Exercise Pooled Drink: Control Glucose Control Glucose Boys 24.4 ± ± ± ± ± 0.8 Men 16.7 ± ± ± ± ± 0.7 Pooled 20.6 ± ± ± ± Mean ± SEM (kcal/kg); pooled n=30. ANOVA analysis (GL, p < ; EX, p = ; GL*EX, p = ; AGE, p = ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

63 Energy Expenditure Compared to CON, EE (kcal/kg) was increased by GL (p = ) and EX (p < ). EE (kcal/kg) was higher in boys when compared to men (AGE, p = ). AGE*EX did show a trend without reaching statistical significance (p = 0.087). No other significant interactions were found (Table 8-5). Table Energy expenditure (kcal/kg) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 1.21 ± ± ± ± ± 0.20 Men 0.85 ± ± ± ± ± 0.20 Pooled 1.03 ± ± ± ± Mean ± SEM (kcal/kg); pooled n=30. ANOVA analysis (GL, p = ; EX, p < ; GL*EX, p = ; AGE, p = ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

64 Net Energy Balance Compared to CON, NEB (kcal/kg) was increased by GL (p = ) and decreased by EX (p < ). NEB (kcal/kg) was higher in boys when compared to men (AGE, p = ). GL*EX showed a trend, but no significant interaction was found (p = ). No other significant interactions were found (Table 8-6). Table Net energy balance (kcal/kg) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 23.6 ± ± ± ± ± 0.8 Men 15.8 ± ± ± ± ± 0.7 Pooled 19.7 ± ± ± ± Mean ± SEM (kcal/kg); pooled n=30. ANOVA analysis (GL, p = ; EX, p < ; GL*EX, p = ; AGE, p = ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

65 Substrate Oxidation Compared to CON, RER was increased by GL (p < ) and by EX (p = ). RER was lower in boys compared to men (AGE, p = ). AGE*EX showed a trend towards statistical significance (p = 0.087). No significant interactions were found (Table 8-7). Table Respiratory exchange ratio in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 0.85 ± ± ± ± ± 0.01 Men 0.87 ± ± ± ± ± 0.01 Pooled 0.86 ± ± ± ± Mean ± SEM; pooled n=30. ANOVA analysis (GL, p < ; EX, p = ; GL*EX, p = ; AGE, p = 0.007; AGE*EX, p = 0.087; AGE*GL, p = ; AGE*EX*GL, p = ).

66 Carbohydrate Oxidation Compared to CON, CHOOX (kcal/kg) was increased by GL (p < ) and EX (p < ). A significant interaction was found for GL*EX (p = ), showing an additional increase in CHOOX (kcal/kg) with GL and EX. AGE*EX did show a trend to be statistically significant (p = ). No other significant interactions were found (Table 8-8). Table Carbohydrate oxidation (kcal/kg) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 0.61 ± ± ± ± ± 0.14 Men 0.48 ± ± ± ± ± 0.17 Pooled 0.54 ± ± ± ± Mean ± SEM (kcal/kg) pooled n=30. ANOVA analysis (GL, p < ; EX, p < ; GL*EX, p = ; AGE, p = 0.724; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

67 Fat Oxidation Compared to CON, FATOX (kcal/kg) was decreased by GL (p < ) and increased with EX (p < ). FATOX (kcal/kg) was higher in boys when compared to men (AGE p < ). A significant interaction was found for GL*EX (p = ), showing increased FATOX (kcal/kg) with GL and EX when compared with CON. Moreover, AGE*EX interaction reached statistical significance (p = ). No other significant interactions were found (Table 8-9). AGE-specific analysis found GL (p < ) to decrease and EX (p < ) to increase FATOX (kcal/kg) in boys. A significant interaction was also found for GL*EX (p = 0.004) in boys, revealing increased FATOX (kcal/kg) with GL and EX. In men, GL (p < ) decreased and EX (p < ) increased FATOX (kcal/kg). GL*EX did not interact significantly in men. Table Fat oxidation (kcal/kg) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys ± ± ± ± ± 0.11 Men ± ± ± ± ± 0.06 Pooled 0.49 ± ± ± ± Mean± SEM (kcal/kg); pooled n=30. ANOVA analysis (GL, p < ; EX, p < ; GL*EX, p = ; AGE, p < ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ). 2 Mean± SEM (kcal/kg); n=15. ANOVA analysis for boys (GL, p = ; EX, p < ; EX*GL, p = 0.004). 3 Mean ± SEM (kcal/kg); n=15. ANOVA analysis for men (GL, p = ; EX, p < ; EX*GL, p = ).

68 Heart Rate Compared to CON, HR (bpm) was increased by GL (p < ) and EX (p < ). Boys had higher HR than men AGE (p < ). No other significant interactions were found (Table 8-10). Table Heart rate (bpm) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 81 ± 2 80 ± ± ± ± 3 Men 62 ± 2 67 ± ± ± 4 89 ± 4 Pooled 71 ± 2 74 ± ± ± 3 1 Means ± SEM (bpm); n=30. ANOVA analysis (GL, p = ; EX, p < ; GL*EX, p = ; AGE, p < ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

69 Water Consumption Water consumption (ml/kg) was similar in boys and men. Neither EX nor GL affected water consumption in boys and men. No interactions were found (Table 8-11). Table Water consumption (kg/ml) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 5.7 ± ± ± ± ± 5.8 Men 3.3 ± ± ± ± ± 5.2 Pooled 4.5 ± ± ± ± Means ± SEM (ml/kg); n=30. ANOVA analysis (GL, p = ; EX, p = ; GL*EX, p = ; AGE, p = ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

70 Net Area Under the Curve Blood Glucose Measurements Compared to CON, BG nauc was increased by GL (p < ) and decreased by EX (p < ). BG measured by nauc BG was not different between boys and men. A significant interaction was found for GL*EX (p < ), showing lower BG AUC responses when GL was combined with EX. No other significant interactions were found (table 8-12). Table nauc blood glucose levels (min*mmol/l) in boys and men Activity: Sedentary Exercise Pooled Drink: Control Glucose Control Glucose Boys -15 ± ± ± 6 87 ± ± 15.7 Men -4 ± ± 18-9 ± 6 93 ± ± 16.6 Pooled -7 ± ± 14-2 ± 5 90 ± 12 1 Means ± SEM (min*mmol/l); n = 6 boys and n = 15 men. ANOVA analysis (EX p = ; GL p < ; GL*EX p < ; AGE p = ; AGE*EX p = ; AGE*GL p = ; AGE*EX*GL, p = ).

71 Blood Glucose Measurements Compared to CON, average BG levels were higher after the GL (p < ), lower after EX compared to SED (p < ), increased over TIME (p < 0.001) and were not affected by AGE. AGE*GL interaction showed a trend for statistical significance (p = ), with higher BG levels reported in adults. Significant interactions were found for EX*TIME (p < ) and GL*TIME (p = ), reflecting higher BG with GL and lower BG with EX over time, when compared with the CON sessions. EX*GL*TIME interaction showed a reduction of BG over TIME with GL with EX when compared to GL alone (p < ). No other significant interactions were found (table 8-13). Table Average Blood Glucose Concentrations (mmol/l) for boys and men Time Activity: Sedentary Exercise Drink: Control Glucose Control Glucose Boys ± ± ± ± 0.14 Men 4.95 ± ± ± ± 0.14 Boys 5.02 ± ± ± ± Men 4.87 ± ± ± ± 0.3 Boys 4.88 ± ± ± ± Men 4.9 ± ± ± ± 0.36 Boys 4.87 ± ± ± ± Men 4.84 ± ± ± ± 0.28

72 56 1 Mean SEM (mmol/l) n = 6 boys and n = 15 men. ANOVA analysis change from baseline (0 min) average BG measurements. EX conducted between 15 and 60 minutes and GL administered at 0 min; (GL p < ; EX p < ; TIME p < ; AGE p = ; EX*GL p < ; EX*AGE p = ; GL*AGE p < ; EX*TIME p < ; GL*TIME p = ; AGE*TIME p = ; EX*GL*AGE p = ; EX*GL*TIME p < ; EX*AGE*TIME p = ; GL*AGE*TIME p = ; EX*GL*AGE*TIME p = ) 2 Preload provided 3 Start of Exercise 4 End of Exercise

73 Visual Analog Scale Analysis Subjective Appetite Pre-meal ratings of appetite were affected by the GL (p = ), and (AGE, p = ), and increased over TIME (p < ). EX had no effect on subjective appetite. The interaction of TIME*AGE (p < ), is explained by the suppression of appetite by GL in men but not in boys. No other interactions were found. Post-meal minus pre-meal ratings were less in men (AGE, p = ) indicating less suppression of appetite following the meal than in boys, but were not affected by either GL or EX. No other significant interactions were found (Table 8-14). Table Average appetite (mm) in boys and men Time Activity Sedentary Exercise Drink: Control Glucose Control Glucose Pre-meal appetite scores Boys ± 0 0 ± 0 0 ± 0 0 ± 0 Men 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Boys ± ± ± ± Men ± ± ± ± 3.0 Boys ± ± ± ± Men ± ± ± ± 2.2 Boys ± ± ± ± 5.8

74 58 Men ± ± ± ± 3.9 Boys ± ± ± ± Men ± ± ± ± 4.3 Boys ± ± ± ± ,7 /95 3,7 Men ± ± ± ± 6.0 Post meal minus pre-meal scores Boys ± ± ± ± 12.4 Men ± ± ± ± Mean SEM (mm) n = 15 boys and n = 15 men. Pre-meal (0-60 minutes) ANOVA analysis change from baseline (0 min) average appetite measurements. EX conducted between 15 and 60 minutes and GL administered at 0 min; (GL p = ; EX p = ; TIME p < ; AGE p = ; EX*GL p = ; EX*TIME p =0.7734; GL*TIME p = ; AGE*TIME p = ; EX*GL*AGE p = ; EX*GL*TIME p = ; EX*AGE*TIME p = ; GL*AGE*TIME p = ; EX*GL*AGE*TIME p = ) Post-meal minus pre-meal ANOVA analysis 85 2 / minutes (GL p = ; EX p = ; AGE p = ; EX*GL p = ; EX*AGE p = ; GL*AGE p = ; EX*GL*AGE p = ) 4 Preload provided at 0 minutes 5 Start of Exercise at 15 minutes 6 End of Exercise at 60 minutes 7 Termination of Meal

75 Thirst Pre-meal ratings of thirst were not affect by GL, EX, AGE or TIME. Post-meal minus pre-meal scores were also not affected by GL, EX, AGE or Time. No interactions were found (Table 8-15). Table Average thirst (mm) in boys and men Time Activity Sedentary Exercise Drink: Control Glucose Control Glucose Pre-meal appetite scores Boys ± 0 0 ± 0 0 ± 0 0 ± 0 Men 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Boys ± ± ± ± Men ± ± ± ± 2.1 Boys ± ± ± ± Men ± ± ± ± 1.6 Boys ± ± ± ± Men ± ± ± ± 3.3 Boys ± ± ± ± Men ± ± ± ± 3.6 Boys 85 2,7 /95 3,7-6.5 ± ± ± ± 7.2

76 60 Men -7.4 ± ± ± ± 9.6 Post meal minus pre-meal scores Boys ± ± ± ± 7.8 Men ± ± ± ± Mean SEM (mm) n = 15 boys and n = 15 men. Pre-meal (0-60 minutes) ANOVA analysis change from baseline (0 min) thirst measurements. EX conducted between 15 and 60 minutes and GL administered at 0 min; (GL p = ; EX p = ; TIME p = ; AGE p = ; EX*GL p = ; EX*TIME p = ; GL*TIME p = ; AGE*TIME p = ; EX*GL*AGE p = ; EX*GL*TIME p = ; EX*AGE*TIME p = ; GL*AGE*TIME p = ; EX*GL*AGE*TIME p = ) Post-meal minus pre-meal ANOVA analysis 85 2 / minutes (GL p = ; EX p = ; AGE p = ; EX*GL p = ; EX*AGE p = ; GL*AGE p = ; EX*GL*AGE p = ) 4 Preload provided at 0 minutes 5 Start of Exercise at 15 minutes 6 End of Exercise at 60 minutes 7 Termination of Meal

77 Physical Comfort in Boys Pre-meal ratings in boys were decreased over TIME (p = ). GL and EX did not affect physical comfort in boys. No interactions were found. Physical comfort post-meal minus pre-meal ratings were not affected by neither GL nor EX. No further interactions were found (Table 8-16). Table Average physical comfort (mm) in boys Activity: Sedentary Exercise Drink: Control Glucose Control Glucose Time (min) Pre-meal scores ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 9.8 Post-meal minus pre-meal scores ± ± ± ± Mean SEM (mm) n = 15 boys. Pre-meal ANOVA analysis change from baseline (0 min) average physical comfort measurements. EX conducted between 15 and 60 minutes and GL administered

78 62 at 0 min; (GL p = ; EX p = ; TIME p = ; EX*GL p = ; EX*TIME p =0.6147; GL*TIME p = ; EX*GL*TIME p = ). Post-meal minus pre-meal ANOVA analysis 85 2 / minutes (GL p = ; EX p = ; EX*GL p = ) 2 Preload provided 3 Preload terminated 4 Test meal provided 5 Test meal terminated

79 Physical Comfort in Men Pre-meal ratings in men were decreased due to the GL (p = ). EX did not affect physical comfort in men. No interactions were found. Physical comfort post-meal minus pre-meal ratings were not affected by neither GL nor EX. No further interactions were found (Table 8-17). Table Average physical comfort (mm) in men. Activity: Sedentary Exercise Drink: Control Glucose Control Glucose Time (min) Pre-meal scores ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.6 Post-meal minus pre-meal ± ± ± ± Mean SEM (mm) n = 15 men. Pre-meal ANOVA analysis change from baseline (0 min) average physical comfort measurements. EX conducted between 15 and 60 minutes and GL administered

80 64 at 0 min; (GL p = ; EX p = ; TIME p = ; EX*GL p = ; EX*TIME p =0.6411; GL*TIME p = ; EX*GL*TIME p = ). Post-meal minus pre-meal ANOVA analysis 85 2 / minutes (GL p = ; EX p = ; EX*GL p = ) 2 Preload provided 3 Preload terminated 4 Test meal provided 5 Test meal terminated

81 Preload Palatability in Boys (Table 8-18). Boys found the GL drink significantly more pleasing compared with water (p = ) Table Average preload palatability (mm) in boys Activity: Control Glucose p-value (DRINK) 26.9 ± ± Mean± SEM (mm); n=30. A student s t-test was used to determine the difference in drink palatability (5 min). GL (p = )

82 Preload Palatability in Men Men found the GL drink more palatable than water (p = ) (Table 8-19). Table Average preload palatability (mm) in men Activity: Control Glucose p-value (DRINK) 56.3 ± ± Mean ± SEM (mm); n=30. A student s t-test was used to determine the difference in drink palatability (5 min). GL (p = )

83 Pizza Meal Palatability in Boys (Table 8-20). Neither EX nor GL had an effect on pizza palatability in boys. No interactions were found Table Average pizza palatability (mm) in boys Activity: Drink: Water Glucose Pooled Sedentary 73 ± 9 81 ± 7 77 ± 5 Exercise 89 ± 3 82 ± 5 86 ± 3 Pooled 81 ± 5 81 ± 4 1 Means ± SEM (mm); n = 15 boys. ANOVA analysis (85 min) (EX p = ; GL p = ; GL*EX p = ).

84 Pizza Meal Palatability in Men (Table 8-21). Neither EX nor GL had an effect on pizza palatability in men. No interactions were found Table Average pizza palatability (mm) in men Activity: Drink: Water Glucose Pooled Sedentary 64.2 ± ± ± 3.6 Exercise 65.1 ± ± ± 3.6 Pooled 64.6 ± ± Mean ± SEM (mm); n = 15 men. ANOVA analysis (95 min) (EX p = ; GL p = ; GL*EX p = ).

85 Correlation Analysis Correlation analysis were conducted to investigate the relationship between FI, NEB and RER, EE, HR. FATOX, CHOOX and BG levels. All measurements, except for HR, RER and BG levels were adjusted for body-weight Correlations with Food Intake Subjective appetite, expressed was correlated with FI (kcal/kg) in boys but not men (r = 0.297; p = ). No significant correlations were found for EE, Net AUC BG, HR, CHOOX and FATOX (Table 8-22). Table Associations with food intake (kcal/kg) Boys Men r p r p EE (kcal/kg) RER HR (bpm) Net AUC BG (min*mmol/l) CHOox (kcal/kg) FATox (kcal/kg) Water consumption (ml/kg)

86 70 nauc Appetite (mm*min) nauc Physical Comfort (mm*min) nauc Thrist (mm*min) Food Palatability at 85/95 min (mm) Preload Palatability at 5 min (mm) Preload Sweetness at 5 min(mm) Not assessed 1 Pearson correlation coefficients; n = 15 boys and 15 men 2 Pearson correlation coefficients; n = 6 boys and 15 men

87 Associations with Net Energy Balance A correlation was found for NEB and EE in boys (r = ; p = ) and men (r = ; p = 0.018). Subjective appetite was also correlated with NEB in boys (r = 0.277; p = ) but not men. FATOX was correlated with NEB in both boys (r = ; p = ) and men (r = ; p = ). No correlations were found for FI and EE, HR, BG levels, FATOX and CHOOX (Table 8-23). Table 8-23 Associations with net energy balance (kcal/kg) Boys Men r p r p EE (kcal/kg) RER HR (bpm) CHOox (kcal/kg) FATox (kcal/kg) nauc BG (min*mmol/l) Water consumption (ml/kg) nauc Appetite (mm*min) nauc Physical Comfort (mm*min) nauc Thrist (mm*min) Food Palatability at 85/95 min (mm)

88 72 Preload Palatability at 5 min (mm) Preload Sweetness at 5 min(mm) Not assessed 1 Pearson correlation coefficients; n = 15 boys and 15 men 2 Pearson correlation coefficients; n = 6 boys and 15 men

89 73 9. DISCUSSION The results of this study do not support our hypothesis. In contrast, several lines of evidence show that substrate oxidation was not a factor determining FI. First, RER was higher after GL, showing increased CHOOX and decreased FI. Second, EX increased RER as well but did not stimulate FI. Third, GL combined with EX did not increase RER and resulted in the greatest decrease in FI. Last, boys had a lower RER than men, but had higher FI/kg body weight. This is the first study to assess the effects of substrate oxidation on short-term FI in boys and men using a unique design to examine the links among metabolic flexibility and substrate oxidation with FI. Substrate oxidation was modified with EX, GL and their combination in two age groups because metabolic flexibility is known to decline with age [131]. This study was stimulated by former studies, investigating the effects of obesity on FI regulation [76, 153, 158] and metabolic flexibility [127, ]. However, these studies did not report if the metabolic impairments of obese populations, characterized by an increased proportion of CHOOX relative to FATOX, are related to an increased FI that perpetuates a positive energy balance. A reduction in FI with GL but no effect of EX on FI in boys and men is consistent with previous studies of the effects of GL [74, 76, 153, 155] and EX at similar EE [73, ]. However, the measures of RER allowed an examination of the relationship between substrate oxidation and FI. GL increased RER by 12 % (Table 8-7), consistent with other studies showing that carbohydrate ingestion increases RER by triggering the release of insulin, which stimulates splanchnic and peripheral glucose uptake and CHOOX [ ]. However, the interpretation of the relationship between RER and FI may be confounded by a suppression of appetite and FI that

90 74 has been found after EX. This EX-induced anorexia describes a brief suppression of appetite after long and/or high intensity EX [73, ]. Therefore, a modest level and medium duration of EX, standardized for an individual RER value below the VET, was chosen to increase RER above resting values [172] without affecting FI [75, 76]. As noted in Table 8-7, EX alone increased RER by an average of 10 % which is consistent with the literature of EX at similar intensities [173]. RER was not further increased by combining EX with GL (Table 8-7). This can be explained by the reduction of endogenous CHOOX while utilizing more exogenous carbohydrates derived from plasma [166]. FI was additionally suppressed by GL in combination with EX, showing an additional effect of EX on FI suppression when compared with GL alone, without increasing RER. Therefore, these findings did not provide evidence for an association between RER and FI (Table 8-4, Table 8-7). In support of this, we did not find any correlations between FI and RER (Table 8-22). The comparison of metabolic flexibility in boys with men provides firmer support against the hypothesis that a higher RER leads to increased FI. Men had a 13 % higher RER, indicating higher reliance on carbohydrates across all conditions, but a lower FI/kg body weight when compared with boys (Table 8-4, Table 8-7). EX showed a trend for RER to be further increased, but did not stimulate additional differences in FI between men and boys (Table8-4). The decreased RER in boys is caused mainly by their increased ability to oxidize fat (Table 8-9). Associations between RER and FATOX showed that Boys (r = ; p < ) reached higher levels of FATOX, which were additionally increased with EX (Table 8-9), by having lower levels of RER when compared with men (r = -0.31; p = ). Other studies have similarly shown higher rates of FATOX in boys compared to men [131, 174].

91 75 Why children oxidize more fat is currently not clear. Based on limited biopsy data collected from 6-yr-old children, pre-pubertal children may have an enhanced ability to oxidize fat due to higher intramuscular triglyceride stores and higher overall fat stores [175]. In support to this hypothesis, boys had a higher body fat content compared to men (Table 8-1). Higher rates of FATOX in children might also be a consequence of underdeveloped glycogenolytic and/or glycolytic systems [ ]. Children have lower lactate levels during exercise [179, 180], perhaps due to decreased capacity to utilize glucose, resulting in increased rates of FATOX. Other studies that investigated FATOX in children and adults also found lower RQ and higher rates of FATOX, but did not analyze FI behaviour [131, 174]. Data on FATOX as a major component of substrate oxidation, also confirms our conclusion that RER is not linked to FI in boys and men. First, FATOX strongly correlated with RER (Figure 8.1), but did not associate with FI in the present study. Second, FATOX was decreased with GL (Table 8-9), and this did not translate into an increase but instead a decrease in FI. GL has previously been shown to limit lipolysis to an extent that can significantly lower overall FATOX [181]. Last, EX expectedly increased FATOX (Table 8-9) but did not affect FI. CHOOX, another determinant of RER, increased with both GL and EX (Table 8-8) but did, not translate into increases in FI, as was similar to FATOX. CHOOX was positively associated with RER in boys (r = 0.553; p < ) and men (r = 0.55; p < ) but did not associate with FI in either boys or men. EX increased CHOOX to meet the increased EE of EX [182], and CHOOX increased with GL in order to keep the energy stored as carbohydrates stable [78]. In contrast to FATOX, CHOOX was not increased in boys when compared to men, reflecting the lower RER values in boys (Table 8-7, Table 8-8, Table 8-9). Expectedly, BG circulating levels showed response patterns parallel to those of CHOOX as they reflect readily available carbohydrate sources for

92 76 CHOOX [183] (Table 8-8). GL increased nauc BG levels because it is readily absorbed [183] (table 8-12). EX, on the other hand, reduced nauc BG (table 8-12) by utilizing available plasma GL in circulation to meet the increased energy demands of EX [158]. NEB regulation by substrate oxidation was another objective of the current study. NEB was calculated in this study based on EI from the GL preload and the ad libitum pizza lunch, in addition to the energy expended during EX and SED sessions. Unlike FI, EE is directly linked to substrate oxidation, and it was found to be positively correlated to CHOOX in boys (r = 0.825; p < ) and men (r = 0.963; p < ) and FATOX in boys (r = 0.708; p < ) and men (r = 0.65; p < ) in the present study. GL and EX are known to modulate EE [75, 76, 131, 153, 158, 164, 168, ]. In the current study, EX increased EE by an average of 360 % when compared to the resting condition, while GL increased EE by only 4% when compared to the water control (Table 8-5). The increased EE with GL intake has been previously described as glucoseinduced thermogenesis, with an average increased EE of 1-3 % with 50 g of GL [187]. The thermic effect in the current study reached 4 % with an average GL load of 58 g. Boys had higher EE across all conditions (Table 8-5), attributed mainly to their higher resting metabolic rates per kg body weight when compared to men [189]. In support of the higher EE in children, we found higher levels of HR in boys than men (table 8-10). The relationship between HR and EE has previously been established [190, 191], and this correlation was confirmed in the current study, in boys (r = 0.875; p < ) and men (r = 0.853; p < ). Decreased stroke volume and increased oxygen demand generally cause HR to be higher in children [192]. This is the first study to investigate the effects of substrate oxidation on NEB. Similar to FI, we did not find an association between NEB and RER, suggesting that NEB is also not affected by substrate oxidation. NEB in this study was increased by 7 % with GL intake (Table 8-6), which

93 77 resulted in higher RER values (Table 8-7). Consistent with other studies, GL ingestion was found to increase NEB [76]. Conversely, EX decreased NEB but increased RER (Table 8-7), showing that RER is not related to NEB. EE was negatively correlated with NEB in boys (r = ; p = ) and men (r = ; p = ), which is consistent with the literature on children [193, 194] and adults [62, ] (Table 8-23). This observation may not be applicable in the long term, as other studies have shown a decrease of daily non-structured activities to compensate for the increased EE with EX [198, 199]. Data on metabolic flexibility in our two age groups also support our conclusion that substrate oxidation is not linked to NEB. Boys had a greater NEB across all conditions compared to men, although they displayed lower relative RER values (Table 8-7). The greater NEB in boys compared to men was mainly caused by their higher FI (Table 8-4, Table 8-6). It is unlikely that pre-meal anticipation of pizza induced a greater FI/kg body weight in boys and consequently higher NEB values, because palatability ratings in boys and men were similar despite being assessed with different questionnaires (Table 8-20, Table 8-21). Increased palatability of food significantly promotes FI [ ]. The mechanisms underlying age-related differences in NEB have never been studied and need to be further investigated. EX alone reduced NEB but did not affect subjective appetite and FI, therefore, findings of this study do not support the concept of EX-induced anorexia. Although the concept recently received more attention as a potential modulator for NEB [203], it is difficult to discuss EXinduced anorexia with confidence, especially if other parameters such as appetite-regulating hormones were not measured. EX-induced anorexia was suggested to be the result of alterations of the circulating levels of appetite-stimulating and suppressing hormones [ ]. Two studies reported reductions in the appetite stimulating hormone acylated ghrelin and increases in the

94 78 appetite suppressing hormone peptide YY, after 60 minutes of high intensity EX, but did not measure FI [204, 205]. Another study showed increases of the appetite-suppressing hormone glucagon-like-peptide-1, after medium and high intensity EX, and increases of peptide YY only appeared after high intensity EX associated with decreases in appetite ratings and EI only after high intensity EX [206]. Stensel et al., concluded that EX-induced anorexia is mainly promoted by high but not low intensity levels of EX [203]. Although appetite hormones were not measured, the lack of effect on FI and subjective appetite scores support the notion that EX-induced anorexia was not present in the current study. This is further supported by the low-to-moderate intensity and the medium duration of our EX sessions, which were considerably below the EX intensity and/or duration of studies reporting significant changes in appetite hormone concentrations and/or FI [185, ]. Appetite has been reported to strongly predict subsequent FI in several studies [74]. Despite the limitations of VAS questionnaires, we measured subjective appetite in boys and men using similar questionnaires [208, 209]. We found positive associations of appetite with FI (r = 0.297; p = ) and NEB (r = 0.277; p = ) in boys but not men, which may have been caused by differences in sensory-specific satiety systems between children and adults (Table 8-14) [210]. However, we found a suppression of pre-meal subjective appetite with GL in both groups, which is consistent with other studies from this lab (Table 8-14) [76, 153]. The palatability of the GL preload reported to be more pleasant by boys than men (Table 8-18, Table 8-19) and may have promoted the suppression of pre-meal appetite [211] consequently leading to a lower FI with GL ingestion (Table 8-4). Preload palatability was positively correlated with appetite in men (r = 0.359; p = ) but not boys. A role of palatability has been described in appetite and FI regulation, despite controversial findings [202, 212]. EX did not affect pre-meal satiety ratings

95 79 (Table 8-14). The low-to-moderate EX intensity in our study may not have been high enough to affect appetite significantly. Other studies investigating the effect of EX intensity have found reductions in appetite only after medium- to high- intensities [170, 213]. Conversely, post-meal appetite scores decreased during all conditions being affected mainly by the large caloric consumption from the pizza meal; a finding consistent with other studies conducted on children [75, 76, 214] and adults [87, 215] (table 8-15). Palatability of the pizza meal was also assessed because it can affect the amount of food eaten at a meal [ ]. Adults are known to reward themselves for physical activity by eating more of preferred foods, which tend to be high in fat [216]. Although we used different questionnaires in boys and men, we did not find any effects of either GL or EX (Table 8-20, Table 8-21) on food palatability. FI and appetite were not correlated with food palatability in either children or adults. Thus, palatability of pizza meal did not affect FI responses to either GL and/or EX in the current study. Thirst and water intake have been strongly correlated with FI, and might also provide explanation to our observations on FI in boys and men. In both animals [217] and humans [218], FI and water intake at a meal were consistently found to be correlated [219]. FI was shown to be reduced when water intake was restricted in healthy volunteers [218]. The analysis of thirst and water consumption in the present study showed that neither thirst nor water consumption were affected by GL or EX (Table 8-15). Moreover, we did not observe any association between water intake and FI. This is inconsistent with previous studies who have found increased thirst ratings with both EX and GL [76]. Larger volumes of preloads, 250 ml for boys and 350 ml for adults, may have acted as a positive control in our study and suppressed water intake and thirst in a

96 80 confounding manner [220], likely hindering expected increases of thirst and water intake with EX and/or GL. This study has several limitations. First, only lean individuals were assessed in this study. Obese and SED individuals were not included. As a consequence of insulin resistance, obese and SED populations generally display higher levels of insulin (hyperinsulinaemia) [221], which in turn may disrupt the metabolic flexibility of insulin responsive (hepatic, muscular and fat) tissues [222]. Accordingly, higher levels of insulin may prevent lipolysis and therefore hinder FATOX [223]. Recent studies provided evidence that insulin can also have a direct effect on feeding behavior [224]. In obese and hyperinsulinemic individuals, increased insulin levels were associated with altered appetite regulation and increased FI when compared to lean individuals [225]. In the present study, although we did not measure insulin, we hypothesized that the cause of lower metabolic flexibility in adults compared to children is not related to differences in insulin levels as they were all healthy. Healthy children and adults have been previously described with similar insulin levels [132]. Therefore, the relationship between FI regulation and metabolic flexibility, involving substrate oxidation impaired by insulin, may be different in obese compared to lean individuals. Our study solely assessed metabolic flexibility by substrate oxidation on FI without insulin as a potential confounder. Nonetheless, the concept of an impaired FI regulation and substrate oxidation in obese, involving insulin resistance and hyperinsulinaemia as an underlying mechanism, needs further investigation. Second, children exhibited lower fitness levels relative to their age-specific norms and when compared with adults [157, 226] (Table 8-1). Aerobic fitness is known to affect metabolic flexibility [130], therefore, differences in metabolic flexibility between boys and men could have been altered. However, it is difficult to compare metabolic flexibility of our participants to those

97 81 of other studies due to the variety of assessment methods and study methodologies that are being used. The assessment of aerobic fitness levels and metabolic flexibility is often based on VET, FATOX and RER, as practiced in the current study, but varies greatly in units and assessment methods among studies [127, 131, 158, 186, 188, ] which makes comparison challenging. Third, the GL preload differed between 1.0 g/kg bodyweight in adults and 1.2 g/kg bodyweight in children; however, this difference was a major part of the study design in order to reduce the risk of nausea in men with a greater preload [230]. We chose these numbers based on a pre-testing where the GL load for our average adult participant would have resulted in a total GL intake of 84.6 g if the per kg values would have been the same in boys and men. We found that GL intake decreased physical comfort in men, while it did not affect boys (Table 8-16, Table 8-17). This finding suggests that men cannot tolerate GL loads on a per kg basis to the same extent as boys. Furthermore, with the small difference of 0.2g/kg GL intake averaging 10.6 g in total for our participants, we minimized the possibility that differences in GL intake between boys and men may have contributed to differences in CHOOX and consequently RER. Significant increases of 29 % in CHOOX were reported with total GL doses of 100 g compared to 50 g [231]. Fourth, we compared appetite and thirst responses of boys and men using similar VAS questionnaires. A number of studies have shown that children aged 7 years and younger do not have the cognitive ability to use VAS [232, 233]. However, questions regarding appetite and thirst were simple and easy to understand for children in our age group. In conclusion, there was no relationship between RER and FI in either age group, suggesting that FI regulation, in the short term, is not affected by substrate oxidation as modified by GL, EX, GL with EX or age.

98 FUTURE DIRECTIONS This research provides evidence that substrate oxidation is not affecting short-term FI regulation. However, a role of substrate oxidation as a short- and long-term modulator for FI regulation has been hypothesized since the early 1950 s by the glucostatic and lipostatic theory of appetite control. Although this study did not report a relationship between RER and FI regulation, it is important to investigate these theories with respect to different EX durations, intensities, modes and study populations to understand the full picture of how the oxidation of substrates is involved in linking metabolic flexibility to appetite and FI regulation and EB. Linking newer approaches based on appetite hormones with the traditional ones of the lipo- and glucostatic theories may provide useful data to develop a better understanding of FI and NEB regulation during EX [81, 84]. Additional research is required to explore the effects of EX modalities and intensities, fitness and body fat levels of individuals, and time to the next meal under both short-term and long-term conditions to better comprehend the benefits of EX interventions on FI and body composition regulation Metabolic Flexibility and Food Intake Regulation in Obesity It is important to assess the effects of substrate oxidation on FI behaviour among obese and SED participants. Studies that have investigated habitual activity levels in active and obese/sed individuals found that active individuals exhibit better aerobic fitness levels, a better metabolic flexibility [125, 234] and a better regulation of FI and body weight when compared with obese

99 83 individuals [129, 185, 235]. As previously described, those impairments in metabolic flexibility and FI regulation are often accompanied with higher levels of insulin in obese individuals [221, 225]. Therefore, it is important to investigate these relationships in obese and SED individuals to gain a better understanding of the mechanisms involving increased FI and metabolic inflexibility including insulin resistance and hyperinsulinaemia Explore the Effects of Exogenous and Endogenous Carbohydrate Oxidation on Food Intake Regulation Previous studies have shown that total as well as endogenous and exogenous CHOOX differs with EX intensity and GL intake [166, 182]. When carbohydrates are ingested, energy derived from exogenous carbohydrates was found to meet a greater proportion of the EE during rest and EX [182]. Although we did not measure exogenous and endogenous CHOOX, our study has shown a suppression of FI with GL ingestion and an even greater suppression with GL ingested prior to EX. These findings suggest that CHOOX was based mainly on the oxidation of exogenous rather than endogenous carbohydrate sources, which may have been involved in regulating FI responses. In this context, it is also important to explore various intensities of EX and their relationship with endogenous, exogenous, and total CHOOX. High intensity EX was reported to increase the amount of endogenous CHOOX compared to low intensity EX [182]. Some studies have shown a reduction of FI after high intensity EX [73, ]. Although we did not find a relationship between total CHOOX and FI regulation, it is unclear whether higher rates of endogenous and/or exogenous CHOOX promoted by higher intensity EX could have caused alterations in FI behavior.

100 84 Therefore, it is important to measure rates of endogenous and exogenous CHOOX with stable isotope tracers, in addition to total CHOOX. Future studies should investigate the components of CHOOX in response to higher EX intensities to better understand whether any of these is more involved than others in FI regulation Control for Appetite Hormones in Lean and Obese Subjects Knowing that appetite hormones are important modulators of food-intake regulating systems, it is critical to understand how appetite-related hormones respond to EX generally, and to compare these responses between normal-weight and overweight/obese individuals. There should be special focus on children because literature investigating the effects of EX on appetite hormones in children is scarce. The link between appetite hormone responses, substrate oxidation and habitual EX and the understanding of the underlying mechanisms of action should also be investigated as it would be a major asset to the formation and utilization of successful obesity prevention and treatment EX regimens Standardization of the Time to Meal Some studies reported a time dependent effect of EX on FI. The lack of effect of EX on FI in the present study may be due to the time interval between EX bout and test meal [185]. It is possible that there would have been an increase in FI after EX if the meal had been further distanced from the end of the EX session. This suggestion is secondary to the findings of studies that found decreased suppression of post-ex appetite 60 minutes later [73], and studies that showed no effect of EX on FI within 30 minutes [75, 76]. Rather than a fixed length of time to the

101 85 next meal post exercise, participants in future studies could be fed at several time points or given the option to snack or choose when they would like to have a meal. In the literature, the time to the next meal ranged from 30 min [75, 76] to 4 hours [163] Control for Daily Physical Activity Levels and Diet The measures of VO2peak and VET reflect overall habitual activity levels, but activity levels may differ on the day before the experimental sessions. Studies have shown higher rates of glycogen depletion with vigorous activity when compared to resting [236, 237]. In the same way, high caloric diets can result in more replenished glycogen stores relative to [238]. Glycogen stores have been proposed to affect the regulation of FI [239, 240]. Thus, future studies should take into account the level of physical activity and the dietary habits of participants in the days preceding the measurements. Accelerometers and HR monitors could quantify the duration and intensity of the habitual activity performed before the measurements [241] Long-term Intervention Study The most important issue is to identify and create effective long-term EX programs which guarantee ongoing weight loss and weight maintenance. This study assessed only short-term subjective appetite, FI, NEB and substrate oxidation after acute exercise sessions, which may not reflect the relationship between substrate oxidation and FI in the long-term [240]. Future studies should investigate the effects of long-term substrate oxidation on overall FI and NEB regulation using activity patterns typical to individuals real life, such as in schools and with typical feeding times such as after recess or gym class.

102 SUMMARY & CONCLUSIONS SUMMARY 1. GL increased RER and decreased FI, while EX increased RER but had no effect on FI. GL with EX combined decreased FI but did not affect RER. Boys had higher FI than men, despite lower values of RER across all conditions. 2. GL suppressed FATOX and increased CHOOX in boys and men. Boys had a lower RER and a stronger preference for FATOX across all conditions and especially during EX when compared to men. 3. NEB was increased by GL preloads, lowered by EX and showed a trend to be decreased when GL was combined with EX. CONCLUSION In conclusion, there was no relationship between RER and FI in either age group, suggesting that FI regulation, in the short term, is not affected by substrate oxidation as modified by GL, EX, GL with EX or age.

103 87 REFERENCES 1. Lopez AD, Mathers CD: Measuring the global burden of disease and epidemiological transitions: Ann Trop Med Parasitol 2006, 100(5-6): Santos MG, Pegoraro M, Sandrini F, Macuco EC: Risk factors for the development of atherosclerosis in childhood and adolescence. Arq Bras Cardiol 2008, 90(4): Katzmarzyk PT, Mason C: Prevalence of class I, II and III obesity in Canada. CMAJ 2006, 174(2): (NHANES) NHaNES: Prevalence of Childhood Overweight and Obesity The NS, Suchindran C, North KE, Popkin BM, Gordon-Larsen P: Association of adolescent obesity with risk of severe obesity in adulthood. JAMA : the journal of the American Medical Association 2010, 304(18): Del Coso J, Hamouti N, Ortega JF, Mora-Rodriguez R: Aerobic fitness determines whole-body fat oxidation rate during exercise in the heat. Appl Physiol Nutr Metab 2010, 35(6): Dietz WH: "Adiposity rebound": reality or epiphenomenon? Lancet 2000, 356(9247): Hodgkin E, Hamlin MJ, Ross JJ, Peters F: Obesity, energy intake and physical activity in rural and urban New Zealand children. Rural Remote Health 2010, 10(2): Lau DC, Douketis JD, Morrison KM, Hramiak IM, Sharma AM, Ur E, Obesity Canada Clinical Practice Guidelines Expert P: 2006 Canadian clinical practice guidelines on the management and prevention of obesity in adults and children [summary]. CMAJ 2007, 176(8):S1-13.

104 Manson JE, Willett WC, Stampfer MJ, Colditz GA, Hunter DJ, Hankinson SE, Hennekens CH, Speizer FE: Body weight and mortality among women. The New England journal of medicine 1995, 333(11): Bray GA: Obesity and surgery for a chronic disease. Obesity research 1996, 4(3): Keller U: Sekundärfolgen der Adipositas und Therapieansätze. Schweiz Med Forum 2002, 39: Schoeller DA: The energy balance equation: looking back and looking forward are two very different views. Nutrition reviews 2009, 67(5): Finkelstein EA, Fiebelkorn IC, Wang G: National medical spending attributable to overweight and obesity: how much, and who's paying? Health Aff (Millwood) 2003, Suppl Web Exclusives:W Sweeting HN: Measurement and definitions of obesity in childhood and adolescence: a field guide for the uninitiated. Nutrition journal 2007, 6: Mei Z, Grummer-Strawn LM, Pietrobelli A, Goulding A, Goran MI, Dietz WH: Validity of body mass index compared with other body-composition screening indexes for the assessment of body fatness in children and adolescents. The American journal of clinical nutrition 2002, 75(6): CDC: About BMI for Children and Teens mi.html. 18. Caballero B: The global epidemic of obesity: an overview. Epidemiol Rev 2007, 29: Canada S: Canadian Community Health Survey - Annual Component (CCHS)

105 Shields M: Overweight and obesity among children and youth. Health reports / Statistics Canada, Canadian Centre for Health Information = Rapports sur la sante / Statistique Canada, Centre canadien d'information sur la sante 2006, 17(3): Yusuf S, Hawken S, Ounpuu S, Dans T, Avezum A, Lanas F, McQueen M, Budaj A, Pais P, Varigos J et al: Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet 2004, 364(9438): Haslam DW, James WP: Obesity. Lancet 2005, 366(9492): Bianchini F, Kaaks R, Vainio H: Overweight, obesity, and cancer risk. The lancet oncology 2002, 3(9): Poulain M, Doucet M, Major GC, Drapeau V, Series F, Boulet LP, Tremblay A, Maltais F: The effect of obesity on chronic respiratory diseases: pathophysiology and therapeutic strategies. CMAJ 2006, 174(9): Anand G, Katz PO: Gastroesophageal reflux disease and obesity. Gastroenterol Clin North Am 2010, 39(1): Organisation WH: WHO: Physical Inactivity: A Global Health Problem Anderson MJ, Fasching CL, Xu HJ, Benedict WF, Stanbridge EJ: Chromosome 13 transfer provides evidence for regulation of RB1 protein expression. Genes, chromosomes & cancer 1994, 9(4): Egger GJ, Vogels N, Westerterp KR: Estimating historical changes in physical activity levels. The Medical journal of Australia 2001, 175(11-12): Organization WH: WHO: Obesity and Overweight Ness-Abramof R, Apovian CM: Diet modification for treatment and prevention of obesity. Endocrine 2006, 29(1):5-9.

106 Caspersen CJ, Powell KE, Christenson GM: Physical activity, exercise, and physical fitness: definitions and distinctions for health-related research. Public Health Rep 1985, 100(2): Blair SN, Morris JN: Healthy hearts--and the universal benefits of being physically active: physical activity and health. Annals of epidemiology 2009, 19(4): Booth FW, Gordon SE, Carlson CJ, Hamilton MT: Waging war on modern chronic diseases: primary prevention through exercise biology. J Appl Physiol 2000, 88(2): Farrell SW, Cortese GM, LaMonte MJ, Blair SN: Cardiorespiratory fitness, different measures of adiposity, and cancer mortality in men. Obesity (Silver Spring) 2007, 15(12): Helmrich SP, Ragland DR, Leung RW, Paffenbarger RS, Jr.: Physical activity and reduced occurrence of non-insulin-dependent diabetes mellitus. The New England journal of medicine 1991, 325(3): Di Pietro L, Dziura J, Blair SN: Estimated change in physical activity level (PAL) and prediction of 5-year weight change in men: the Aerobics Center Longitudinal Study. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 2004, 28(12): Gustat J, Srinivasan SR, Elkasabany A, Berenson GS: Relation of self-rated measures of physical activity to multiple risk factors of insulin resistance syndrome in young adults: the Bogalusa Heart Study. Journal of clinical epidemiology 2002, 55(10): Lakka TA, Laaksonen DE, Lakka HM, Mannikko N, Niskanen LK, Rauramaa R, Salonen JT: Sedentary lifestyle, poor cardiorespiratory fitness, and the metabolic syndrome. Medicine and science in sports and exercise 2003, 35(8):

107 Roberts CK, Freed B, McCarthy WJ: Low aerobic fitness and obesity are associated with lower standardized test scores in children. The Journal of pediatrics 2010, 156(5): , 718 e Hamilton MT, Hamilton DG, Zderic TW: Role of low energy expenditure and sitting in obesity, metabolic syndrome, type 2 diabetes, and cardiovascular disease. Diabetes 2007, 56(11): Booth FW, Lees SJ: Fundamental questions about genes, inactivity, and chronic diseases. Physiological genomics 2007, 28(2): Organization WH: Global recommendations on physical activity for health Canada S: Physical activity levels of Canadian adults, 2007 to Consultation. RoaJFWUE: Human energy requirements: Principles and Definitions. Food and Agriculture Organization of the United Nations Bergouignan A, Momken I, Schoeller DA, Normand S, Zahariev A, Lescure B, Simon C, Blanc S: Regulation of energy balance during long-term physical inactivity induced by bed rest with and without exercise training. The Journal of clinical endocrinology and metabolism 2010, 95(3): Blanc S, Normand S, Ritz P, Pachiaudi C, Vico L, Gharib C, Gauquelin-Koch G: Energy and water metabolism, body composition, and hormonal changes induced by 42 days of enforced inactivity and simulated weightlessness. The Journal of clinical endocrinology and metabolism 1998, 83(12): CFaLR I: The Canadian Physical Activity Levels Among Youth (CANPLAY) Survey Canada PHA: National Consultation on Physical Activity Guidelines

108 Gortmaker SL, Must A, Sobol AM, Peterson K, Colditz GA, Dietz WH: Television viewing as a cause of increasing obesity among children in the United States, Arch Pediatr Adolesc Med 1996, 150(4): Vioque J, Torres A, Quiles J: Time spent watching television, sleep duration and obesity in adults living in Valencia, Spain. Int J Obes Relat Metab Disord 2000, 24(12): Mikkola I, Keinanen-Kiukaanniemi S, Jokelainen J, Peitso A, Harkonen P, Timonen M, Ikaheimo T: Aerobic performance and body composition changes during military service. Scandinavian journal of primary health care 2012, 30(2): Gubbels JS, Kremers SP, Stafleu A, Goldbohm RA, de Vries NK, Thijs C: Clustering of energy balance-related behaviors in 5-year-old children: lifestyle patterns and their longitudinal association with weight status development in early childhood. The international journal of behavioral nutrition and physical activity 2012, 9: González Calvo G HSS, Pozo Rosado P, García López D.: Positive effects of physical exercise on reducing the relationship between subcutaneous abdominal fat and morbility risk. Nutr Hosp 2011 Jul-Aug;26(4): Edwards HT, Thorndike A, Jr., Dill DB: The New England Journal of Medicine, Volume 213, 1935: The energy requirement in strenuous muscular exercise. Nutrition reviews 1989, 47(5): Margaria R EH, Dill DB: The possible mechanisms of contracting and paying the O2 debt and the role of lactic acid in muscular contraction. American Journal of Physiology 1935, 106: Tremblay A, Fontaine E, Poehlman ET, Mitchell D, Perron L, Bouchard C: The effect of exercise-training on resting metabolic rate in lean and moderately obese individuals. International journal of obesity 1986, 10(6):

109 Chaput JP, Klingenberg L, Rosenkilde M, Gilbert JA, Tremblay A, Sjodin A: Physical activity plays an important role in body weight regulation. Journal of obesity 2011, Mayer J, Roy P, Mitra KP: Relation between caloric intake, body weight, and physical work: studies in an industrial male population in West Bengal. The American journal of clinical nutrition 1956, 4(2): King NA, Tremblay A, Blundell JE: Effects of exercise on appetite control: implications for energy balance. Medicine and science in sports and exercise 1997, 29(8): Martins C, Morgan L, Truby H: A review of the effects of exercise on appetite regulation: an obesity perspective. Int J Obes (Lond) 2008, 32(9): Martins C, Truby H, Morgan LM: Short-term appetite control in response to a 6-week exercise programme in sedentary volunteers. The British journal of nutrition 2007, 98(4): King NA, Stubbs RJ, Blundell JE: High dose exercise does not increase hunger or energy intake in free living males. European Journal of Clinical Nutrition 1997, 51: Stubbs RJ, Sepp A, Hughes DA, Johnstone AM, Horgan GW, King N, Blundell J: The effect of graded levels of exercise on energy intake and balance in free-living men, consuming their normal diet. European journal of clinical nutrition 2002, 56(2): Stubbs RJ, Sepp A, Hughes DA, Johnstone AM, King N, Horgan GW, Blundell JE: The effect of graded levels of exercise on energy intake and balance in free-living women. International Journal of Obesity 2002b, 26: Whybrow S, Hughes DA, Ritz P, Johnstone AM, Horgan GW, King N, Blundell JE, Stubbs RJ: The effect of an incremental increase in exercise on appetite, eating behaviour and energy balance in lean men and women feeding ad libitum. British Journal of Nutrition 2008, 100(5):

110 Woo R, Sunyer FXP: Effect of Increased Physical Activity on Voluntary Intake in Lean Women. Metabolism 1985, 34(9): Woo R, Garrow JS, Pi-Sunyer FX: Voluntary food intake during prolonged exercise in obese women. Am J Clin Nutr 1982, 36(3): Moore LL, Gao D, Bradlee ML, Cupples LA, Sundarajan-Ramamurti A, Proctor MH, Hood MY, Singer MR, Ellison RC: Does early physical activity predict body fat change throughout childhood? Preventive medicine 2003, 37(1): Epstein LH, Roemmich JN, Paluch RA, Raynor HA: Physical activity as a substitute for sedentary behavior in youth. Annals of behavioral medicine : a publication of the Society of Behavioral Medicine 2005, 29(3): Epstein LH, Saad FG, Giacomelli AM, Roemmich JN: Effects of allocation of attention on habituation to olfactory and visual food stimuli in children. Physiology & behavior 2005, 84(2): Epstein LH, Roemmich JN, Paluch RA, Raynor HA: Influence of changes in sedentary behavior on energy and macronutrient intake in youth. The American journal of clinical nutrition 2005, 81(2): Epstein LH, Paluch RA, Consalvi A, Riordan K, Scholl T: Effects of manipulating sedentary behavior on physical activity and food intake. The Journal of pediatrics 2002, 140(3): Moore MS, Dodd CJ, Welsman JR, Armstrong N: Short-term appetite and energy intake following imposed exercise in 9- to 10-year-old girls. Appetite 2004, 43(2): Bellissimo N, Thomas SG, Pencharz PB, Goode RC, Anderson GH: Reproducibility of short-term food intake and subjective appetite scores after a glucose preload, ventilation threshold, and body composition in boys. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 2008, 33(2):

111 Bozinovski NC, Bellissimo N, Thomas SG, Pencharz PB, Goode RC, Anderson GH: The effect of duration of exercise at the ventilation threshold on subjective appetite and short-term food intake in 9 to 14 year old boys and girls. The international journal of behavioral nutrition and physical activity 2009, 6: Tamam S, Bellissimo N, Patel BP, Thomas SG, Anderson GH: Overweight and obese boys reduce food intake in response to a glucose drink but fail to increase intake in response to exercise of short duration. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 2012, 37(3): Abbott WG, Howard BV, Christin L, Freymond D, Lillioja S, Boyce VL, Anderson TE, Bogardus C, Ravussin E: Short-term energy balance: relationship with protein, carbohydrate, and fat balances. The American journal of physiology 1988, 255(3 Pt 1):E Schutz Y, Flatt JP, Jequier E: Failure of dietary fat intake to promote fat oxidation: a factor favoring the development of obesity. The American journal of clinical nutrition 1989, 50(2): Acheson KJ, Schutz Y, Bessard T, Anantharaman K, Flatt JP, Jequier E: Glycogen storage capacity and de novo lipogenesis during massive carbohydrate overfeeding in man. The American journal of clinical nutrition 1988, 48(2): Hellerstein MK: De novo lipogenesis in humans: metabolic and regulatory aspects. European journal of clinical nutrition 1999, 53 Suppl 1:S Mayer J: Regulation of energy intake and the body weight: the glucostatic theory and the lipostatic hypothesis. Annals of the New York Academy of Sciences 1955, 63(1): Bray GA: Treatment for obesity: a nutrient balance/nutrient partition approach. Nutrition reviews 1991, 49(2):33-45.

112 Schrauwen P, van Marken Lichtenbelt WD, Saris WH, Westerterp KR: Changes in fat oxidation in response to a high-fat diet. The American journal of clinical nutrition 1997, 66(2): Kennedy GC: The role of depot fat in the hypothalamic control of food intake in the rat. Proceedings of the Royal Society of London Series B, Containing papers of a Biological character Royal Society 1953, 140(901): Galgani J, Ravussin E: Energy metabolism, fuel selection and body weight regulation. Int J Obes (Lond) 2008, 32 Suppl 7:S Mellinkoff SM, Frankland M, Boyle D, Greipel M: Relationship between serum amino acid concentration and fluctuations in appetite. J Appl Physiol 1956, 8(5): Anderson GH, Luhovyy B, Akhavan T, Panahi S: Milk proteins in the regulation of body weight, satiety, food intake and glycemia. Nestle Nutrition workshop series Paediatric programme 2011, 67: Fernstrom JD: Branched-chain amino acids and brain function. The Journal of nutrition 2005, 135(6 Suppl):1539S-1546S. 89. Flatt JP: Body composition, respiratory quotient, and weight maintenance. Am J Clin Nutr 1995, 62(5 Suppl):1107S-1117S. 90. Tremblay A, Almeras N: Exercise, macronutrient preferences and food intake. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 1995, 19 Suppl 4:S Tremblay A, Buemann B: Exercise-training, macronutrient balance and body weight control. Int J Obes Relat Metab Disord 1995, 19(2): Hopkins M, Jeukendrup A, King NA, Blundell JE: The relationship between substrate metabolism, exercise and appetite control: does glycogen availability influence the motivation to eat, energy intake or food choice? Sports Med 2011, 41(6):

113 Melzer K, Kayser B, Saris WH, Pichard C: Effects of physical activity on food intake. Clin Nutr 2005, 24(6): Edwards HT, Margaria Ra, Dill DB: Metabolic rate, blood sugar and the utilization of carbohydrate. Am J Physiol 1934, 108: Halsted CH: Malnutrition and Nutritional Assessment. Harrisons Internal Medicine 2005, 16th ed: Flatt JP, Blackburn GL: The matabolic fuel regulatory system: implications for protein-sparing therapies during caloric deprivation and disease. Am J Clin Nutr 1974, 27(2): Garlick PJ, Clugston GA, Swick RW, Waterlow JC: Diurnal pattern of protein and energy metabolism in man. Am J Clin Nutr 1980, 33(9): Seidell JC, Muller DC, Sorkin JD, Andres R: Fasting respiratory exchange ratio and resting metabolic rate as predictors of weight gain: the Baltimore Longitudinal Study on Aging. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 1992, 16(9): Rose AJ, Richter EA: Skeletal muscle glucose uptake during exercise: how is it regulated? Physiology (Bethesda) 2005, 20: Martin IK, Katz A, Wahren J: Splanchnic and muscle metabolism during exercise in NIDDM patients. The American journal of physiology 1995, 269(3 Pt 1):E Coyle EF: Substrate utilization during exercise in active people. The American journal of clinical nutrition 1995, 61(4 Suppl):968S-979S PO A, K R: Textbook of Work Physiology-4th: Physiological Bases of Exercise. Human Kinetics 10%, Corpeleijn E, Saris WH, Blaak EE: Metabolic flexibility in the development of insulin resistance and type 2 diabetes: effects of lifestyle. Obes Rev 2009, 10(2):

114 Kelley DE, He J, Menshikova EV, Ritov VB: Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes 2002, 51(10): Bergouignan A, Rudwill F, Simon C, Blanc S: Physical inactivity as the culprit of metabolic inflexibility: evidence from bed-rest studies. J Appl Physiol 2011, 111(4): Bergouignan A, Schoeller DA, Normand S, Gauquelin-Koch G, Laville M, Shriver T, Desage M, Le Maho Y, Ohshima H, Gharib C et al: Effect of physical inactivity on the oxidation of saturated and monounsaturated dietary Fatty acids: results of a randomized trial. PLoS Clin Trials 2006, 1(5):e Bergouignan A, Trudel G, Simon C, Chopard A, Schoeller DA, Momken I, Votruba SB, Desage M, Burdge GC, Gauquelin-Koch G et al: Physical inactivity differentially alters dietary oleate and palmitate trafficking. Diabetes 2009, 58(2): Zunquin G, Theunynck D, Sesboue B, Arhan P, Bougle D: Evolution of fat oxidation during exercise in obese pubertal boys: clinical implications. J Sports Sci 2009, 27(4): Kissileff HR, Pi-Sunyer FX, Segal K, Meltzer S, Foelsch PA: Acute effects of exercise on food intake in obese and nonobese women. The American journal of clinical nutrition 1990, 52(2): Schmitz KH, Jacobs DR, Jr., Hong CP, Steinberger J, Moran A, Sinaiko AR: Association of physical activity with insulin sensitivity in children. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 2002, 26(10): Zurlo F, Lillioja S, Esposito-Del Puente A, Nyomba BL, Raz I, Saad MF, Swinburn BA, Knowler WC, Bogardus C, Ravussin E: Low ratio of fat to carbohydrate oxidation as predictor of weight gain: study of 24-h RQ. The American journal of physiology 1990, 259(5 Pt 1):E

115 Marra M, Scalfi L, Covino A, Esposito-Del Puente A, Contaldo F: Fasting respiratory quotient as a predictor of weight changes in non-obese women. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 1998, 22(6): Marra M, Scalfi L, Contaldo F, Pasanisi F: Fasting respiratory quotient as a predictor of long-term weight changes in non-obese women. Annals of nutrition & metabolism 2004, 48(3): Froidevaux F, Schutz Y, Christin L, Jequier E: Energy expenditure in obese women before and during weight loss, after refeeding, and in the weight-relapse period. The American journal of clinical nutrition 1993, 57(1): Valtuena S, Salas-Salvado J, Lorda PG: The respiratory quotient as a prognostic factor in weight-loss rebound. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 1997, 21(9): Ranneries C, Bulow J, Buemann B, Christensen NJ, Madsen J, Astrup A: Fat metabolism in formerly obese women. The American journal of physiology 1998, 274(1 Pt 1):E Astrup A, Buemann B, Christensen NJ, Toubro S: Failure to increase lipid oxidation in response to increasing dietary fat content in formerly obese women. The American journal of physiology 1994, 266(4 Pt 1):E Salmon J, Bauman A, Crawford D, Timperio A, Owen N: The association between television viewing and overweight among Australian adults participating in varying levels of leisure-time physical activity. Int J Obes Relat Metab Disord 2000, 24(5): Dolkas CB, Greenleaf JE: Insulin and glucose responses during bed rest with isotonic and isometric exercise. J Appl Physiol 1977, 43(6): Lipman RL, Raskin P, Love T, Triebwasser J, Lecocq FR, Schnure JJ: Glucose intolerance during decreased physical activity in man. Diabetes 1972, 21(2):

116 Lutwak L, Whedon GD: The effect of physical conditioning on glucose tolerance. Clin Res 1959, 143(7) Blanc S, Normand S, Pachiaudi C, Fortrat JO, Laville M, Gharib C: Fuel homeostasis during physical inactivity induced by bed rest. J Clin Endocrinol Metab 2000, 85(6): Galgani JE, Moro C, Ravussin E: Metabolic flexibility and insulin resistance. American journal of physiology Endocrinology and metabolism 2008, 295(5):E Sparks LM, Ukropcova B, Smith J, Pasarica M, Hymel D, Xie H, Bray GA, Miles JM, Smith SR: Relation of adipose tissue to metabolic flexibility. Diabetes research and clinical practice 2009, 83(1): Simon J, Young JL, Blood DK, Segal KR, Case RB, Gutin B: Plasma lactate and ventilation thresholds in trained and untrained cyclists. J Appl Physiol 1986, 60(3): Bell LM, Watts K, Siafarikas A, Thompson A, Ratnam N, Bulsara M, Finn J, O'Driscoll G, Green DJ, Jones TW et al: Exercise alone reduces insulin resistance in obese children independently of changes in body composition. The Journal of clinical endocrinology and metabolism 2007, 92(11): Zunquin G, Theunynck D, Sesboue B, Arhan P, Bougle D: Comparison of fat oxidation during exercise in lean and obese pubertal boys: clinical implications. British journal of sports medicine 2009, 43(11): Ledikwe JH, Blanck HM, Kettel Khan L, Serdula MK, Seymour JD, Tohill BC, Rolls BJ: Dietary energy density is associated with energy intake and weight status in US adults. The American journal of clinical nutrition 2006, 83(6): Long SJ, Hart K, Morgan LM: The ability of habitual exercise to influence appetite and food intake in response to high- and low-energy preloads in man. The British journal of nutrition 2002, 87(5):

117 Kelley DE: Skeletal muscle fat oxidation: timing and flexibility are everything. The Journal of clinical investigation 2005, 115(7): Riddell MC, Jamnik VK, Iscoe KE, Timmons BW, Gledhill N: Fat oxidation rate and the exercise intensity that elicits maximal fat oxidation decreases with pubertal status in young male subjects. Journal of applied physiology 2008, 105(2): Grant DB: Fasting serum insulin levels in childhood. Archives of disease in childhood 1967, 42(224): Harrell JS, McMurray RG, Baggett CD, Pennell ML, Pearce PF, Bangdiwala SI: Energy costs of physical activities in children and adolescents. Medicine and science in sports and exercise 2005, 37(2): Siegler RS, Lemaire P: Older and younger adults' strategy choices in multiplication: testing predictions of ASCM using the choice/no-choice method. J Exp Psychol Gen 1997, 126(1): Medicine ACoS: Metabolic Equations for Gross VO2 in Metric Units Bar-Or O: New and old in pediatric exercise physiology. Int J Sports Med 2000, 21 Suppl 2:S ; discussion S Noakes TD: Implications of exercise testing for prediction of athletic performance: a contemporary perspective. Med Sci Sports Exerc 1988, 20(4): Midgley AW, McNaughton LR, Carroll S: Effect of the VO2 time-averaging interval on the reproducibility of VO2max in healthy athletic subjects. Clin Physiol Funct Imaging 2007, 27(2): Okano AH, Altimari LR, Simoes HG, Moraes AC, Nakamura FY, Cyrino ES, Burini RC: Comparison between anaerobic threshold determined by ventilatory variables and blood lactate response in cyclists. Rev Bras Med Esporte 2006, 12(1).

118 Loat CE, Rhodes EC: Relationship between the lactate and ventilatory thresholds during prolonged exercise. Sports medicine 1993, 15(2): Davis JA, Vodak P, Wilmore JH, Vodak J, Kurtz P: Anaerobic threshold and maximal aerobic power for three modes of exercise. J Appl Physiol 1976, 41(4): Peronnet F, Thibault G, Rhodes EC, McKenzie DC: Correlation between ventilatory threshold and endurance capability in marathon runners. Med Sci Sports Exerc 1987, 19(6): Caiozzo VJ, Davis JA, Ellis JF, Azus JL, Vandagriff R, Prietto CA, McMaster WC: A comparison of gas exchange indices used to detect the anaerobic threshold. Journal of applied physiology 1982, 53(5): Beaver WL, Wasserman K, Whipp BJ: A new method for detecting anaerobic threshold by gas exchange. J Appl Physiol 1986, 60(6): Kroidl RF, Schwarz S, Lehnigk B: Kursbuch Spiroergometrie. Thieme Verlag Weir JB: New methods for calculating metabolic rate with special reference to protein metabolism. The Journal of physiology 1949, 109(1-2): Zunts N: Ueber die Beduetung der verschiedenen Naehrstoffe als Erzeuger der Muskelkraft. Arch Gesamte Physiol, Bonn, Ger: 1901, 83(557): Deurenberg P, Pieters JJ, Hautvast JG: The assessment of the body fat percentage by skinfold thickness measurements in childhood and young adolescence. The British journal of nutrition 1990, 63(2): Womersley J, Durnin JV: An experimental study on variability of measurements of skinfold thickness on young adults. Human biology 1973, 45(2): Brozek J: Body Composition: The relative amounts of fat, tissue, and water vary with age, sex, exercise, and nutritional state. Science 1961, 134(3483):

119 Mahon AD, Marjerrison AD, Lee JD, Woodruff ME, Hanna LE: Evaluating the prediction of maximal heart rate in children and adolescents. Research quarterly for exercise and sport 2010, 81(4): Robergs R, Landwehr R: The Surprising History of the "HRmax=220-age" Equation. Journal of Exercise Physiology 2002, 5: Bellissimo N, Desantadina MV, Pencharz PB, Berall GB, Thomas SG, Anderson GH: A comparison of short-term appetite and energy intakes in normal weight and obese boys following glucose and whey-protein drinks. International journal of obesity 2008, 32(2): Bellissimo N, Thomas SG, Goode RC, Anderson GH: Effect of short-duration physical activity and ventilation threshold on subjective appetite and short-term energy intake in boys. Appetite 2007, 49(3): Bellissimo N, Pencharz PB, Thomas SG, Anderson GH: Effect of television viewing at mealtime on food intake after a glucose preload in boys. Pediatric research 2007, 61(6): Welsman JR, Armstrong N, Nevill AM, Winter EM, Kirby BJ: Scaling peak VO2 for differences in body size. Medicine and science in sports and exercise 1996, 28(2): Wilmore JH, Costill DL: Physiology of Sport and Exercise. IL: Human Kinetics 2005, 3rd ed. Champaign Chu L, Riddell MC, Takken T, Timmons BW: Carbohydrate intake reduces fat oxidation during exercise in obese boys. European journal of applied physiology 2011, 111(12): Bircher S, Knechtle B: Relationship between Fat Oxidation and Lactate Threshold in Athletes and Obese Women and Men. Journal of Sports Science and Medicine 2004, 3:

120 Toledo FG, Watkins S, Kelley DE: Changes induced by physical activity and weight loss in the morphology of intermyofibrillar mitochondria in obese men and women. The Journal of clinical endocrinology and metabolism 2006, 91(8): Dodd CJ, Welsman JR, Armstrong N: Energy intake and appetite following exercise in lean and overweight girls. Appetite 2008, 51(3): Lluch A, King NA, Blundell JE: No energy compensation at the meal following exercise in dietary restrained and unrestrained women. The British journal of nutrition 2000, 84(2): Hubert P, King NA, Blundell JE: Uncoupling the effects of energy expenditure and energy intake: appetite response to short-term energy deficit induced by meal omission and physical activity. Appetite 1998, 31(1): King NA, Lluch A, Stubbs RJ, Blundell JE: High dose exercise does not increase hunger or energy intake in free living males. European journal of clinical nutrition 1997, 51(7): Lluch A, King NA, Blundell JE: Exercise in dietary restrained women: no effect on energy intake but change in hedonic ratings. European journal of clinical nutrition 1998, 52(4): Feinle C, O'Donovan D, Horowitz M: Carbohydrate and satiety. Nutrition reviews 2002, 60(6): Jentjens RL, Jeukendrup AE: High rates of exogenous carbohydrate oxidation from a mixture of glucose and fructose ingested during prolonged cycling exercise. The British journal of nutrition 2005, 93(4): Jentjens RL, Underwood K, Achten J, Currell K, Mann CH, Jeukendrup AE: Exogenous carbohydrate oxidation rates are elevated after combined ingestion of glucose and fructose during exercise in the heat. Journal of applied physiology 2006, 100(3):

121 King NA: The relationship between physical activity and food intake. The Proceedings of the Nutrition Society 1998, 57(1): King NA, Burley VJ, Blundell JE: Exercise-induced suppression of appetite: effects on food intake and implications for energy balance. European journal of clinical nutrition 1994, 48(10): S. V-O, Tiryaki-Sonmez G, Bugdayci G, Ozen G: The effects of exercise on food intake and hunger: Relationship with acylated ghrelin and leptin Journal of Sports Science and Medicine 2011, 10( ) Çolak R, Özçelik O: Effects of Progressively Increasing Work Rate Exercise on Body Substrate Utilisation. Turkish Journal of Endocrinology and Metabolism 2002, 2: Ramos-Jimenez A, Hernandez-Torres RP, Torres-Duran PV, Romero-Gonzalez J, Mascher D, Posadas-Romero C, Juarez-Oropeza MA: The Respiratory Exchange Ratio is Associated with Fitness Indicators Both in Trained and Untrained Men: A Possible Application for People with Reduced Exercise Tolerance. Clinical medicine Circulatory, respiratory and pulmonary medicine 2008, 2: Kostyak JC, Kris-Etherton P, Bagshaw D, DeLany JP, Farrell PA: Relative fat oxidation is higher in children than adults. Nutrition journal 2007, 6: Bell RD, MacDougall JD, Billeter R, Howald H: Muscle fiber types and morphometric analysis of skeletal msucle in six-year-old children. Medicine and science in sports and exercise 1980, 12(1): Berg A, Kim SS, Keul J: Skeletal muscle enzyme activities in healthy young subjects. International journal of sports medicine 1986, 7(4): Eriksson BO, Gollnick PD, Saltin B: Muscle metabolism and enzyme activities after training in boys years old. Acta physiologica Scandinavica 1973, 87(4):

122 Gollnick PD, Armstrong RB, Saltin B, Saubert CWt, Sembrowich WL, Shepherd RE: Effect of training on enzyme activity and fiber composition of human skeletal muscle. Journal of applied physiology 1973, 34(1): Eriksson BO, Karlsson J, Saltin B: Muscle metabolites during exercise in pubertal boys. Acta paediatrica Scandinavica Supplement 1971, 217: Mahon AD, Duncan GE, Howe CA, Del Corral P: Blood lactate and perceived exertion relative to ventilatory threshold: boys versus men. Medicine and science in sports and exercise 1997, 29(10): Horowitz JF, Mora-Rodriguez R, Byerley LO, Coyle EF: Lipolytic suppression following carbohydrate ingestion limits fat oxidation during exercise. The American journal of physiology 1997, 273(4 Pt 1):E Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E, Wolfe RR: Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. The American journal of physiology 1993, 265(3 Pt 1):E Mackay EM, Bergman HC: The rate of Absorption of Glucose from the Intestinal Tract. The Journal of biological chemistry 1933: Marques-Lopes I, Forga L, Martinez JA: Thermogenesis induced by a highcarbohydrate meal in fasted lean and overweight young men: insulin, body fat, and sympathetic nervous system involvement. Nutrition 2003, 19(1): Martins C, Morgan LM, Bloom SR, Robertson MD: Effects of exercise on gut peptides, energy intake and appetite. The Journal of endocrinology 2007, 193(2): Perez-Martin A, Dumortier M, Raynaud E, Brun JF, Fedou C, Bringer J, Mercier J: Balance of substrate oxidation during submaximal exercise in lean and obese people. Diabetes & metabolism 2001, 27(4 Pt 1):

123 Pittet P, Chappuis P, Acheson K, De Techtermann F, Jequier E: Thermic effect of glucose in obese subjects studied by direct and indirect calorimetry. The British journal of nutrition 1976, 35(2): Timmons BW, Bar-Or O, Riddell MC: Influence of age and pubertal status on substrate utilization during exercise with and without carbohydrate intake in healthy boys. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 2007, 32(3): Rowland TW, Auchinachie JA, Keenan TJ, Green GM: Physiologic responses to treadmill running in adult and prepubertal males. International journal of sports medicine 1987, 8(4): Keytel LR, Goedecke JH, Noakes TD, Hiiloskorpi H, Laukkanen R, van der Merwe L, Lambert EV: Prediction of energy expenditure from heart rate monitoring during submaximal exercise. Journal of sports sciences 2005, 23(3): Hilloskorpi H, Fogelholm M, Laukkanen R, Pasanen M, Oja P, Manttari A, Natri A: Factors affecting the relation between heart rate and energy expenditure during exercise. International journal of sports medicine 1999, 20(7): Bar-Or O: Pediatric Sports Medicine for the Practitioner. From Physiologic Principles to Clinical Applications. New York: Springer Verlag Dodd CJ, Welsman JR, Armstrong N: Energy intake and appetite following exercise in lean and overweight girls. Appetite 2008, 51(3): Moore MS, Dodd CJ, Welsman JR, Armstrong N: Short-term appetite and energy intake following imposed exercise in 9- to 10-year-old girls. Appetite 2004, 43: Lluch A, King NA, Blundell JE: No energy compensation at the meal following exercise in dietary restrained and unrestarined women. British Journal of Nutrition 2000, 84:7.

124 Hubert P, King NA, Blundell JE: Uncoupling the Effects of Energy Expenditure and Energy Intake: Appetite Response to Short-term Energy Deficit Induced by Meal Omission and Physical Activity. Appetite 1998, 31: Lluch A, King NA, Blundell JE: Exercise in dietary restrained women: no effect on energy intake but change in hedonic ratings. European Journal of Clinical Nutrition 1998, 52: Stubbs RJ, Sepp A, Hughes DA, Johnstone AM, King N, Horgan G, Blundell JE: The effect of graded levels of exercise on energy intake and balance in free-living women. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 2002, 26(6): Stubbs RJ, Sepp A, Hughes DA, Johnstone AM, Horgan GW, King N, Blundell J: The effect of graded levels of exercise on energy intake and balance in free-living men, consuming their normal diet. European journal of clinical nutrition 2002, 56(2): Yeomans MR, Symes T: Individual differences in the use of pleasantness and palatability ratings. Appetite 1999, 32(3): Yeomans MR: Taste, palatability and the control of appetite. The Proceedings of the Nutrition Society 1998, 57(4): Bobroff EM, Kissileff HR: Effects of changes in palatability on food intake and the cumulative food intake curve in man. Appetite 1986, 7(1): Stensel D: Exercise, appetite and appetite-regulating hormones: implications for food intake and weight control. Annals of nutrition & metabolism 2010, 57 Suppl 2: Broom DR, Batterham RL, King JA, Stensel DJ: Influence of resistance and aerobic exercise on hunger, circulating levels of acylated ghrelin, and peptide YY in healthy males. American journal of physiology Regulatory, integrative and comparative physiology 2009, 296(1):R29-35.

125 Broom DR, Stensel DJ, Bishop NC, Burns SF, Miyashita M: Exercise-induced suppression of acylated ghrelin in humans. Journal of applied physiology 2007, 102(6): Ueda SY, Yoshikawa T, Katsura Y, Usui T, Fujimoto S: Comparable effects of moderate intensity exercise on changes in anorectic gut hormone levels and energy intake to high intensity exercise. The Journal of endocrinology 2009, 203(3): Ueda SY, Yoshikawa T, Katsura Y, Usui T, Nakao H, Fujimoto S: Changes in gut hormone levels and negative energy balance during aerobic exercise in obese young males. The Journal of endocrinology 2009, 201(1): Blundell J: Hunger, appetite and satiety - constructs in search of identities. Nutrition and Lifestyles 1979: Stubbs RJ, Hughes DA, Johnstone AM, Rowley E, Reid C, Elia M, Stratton R, Delargy H, King N, Blundell JE: The use of visual analogue scales to assess motivation to eat in human subjects: a review of their reliability and validity with an evaluation of new hand-held computerized systems for temporal tracking of appetite ratings. The British journal of nutrition 2000, 84(4): Olsen A, Ritz C, Hartvig DL, Moller P: Comparison of sensory specific satiety and sensory specific desires to eat in children and adults. Appetite 2011, 57(1): Yeomans MR, Lee MD, Gray RW, French SJ: Effects of test-meal palatability on compensatory eating following disguised fat and carbohydrate preloads. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity 2001, 25(8): Warwick ZS, Hall WG, Pappas TN, Schiffman SS: Taste and smell sensations enhance the satiating effect of both a high-carbohydrate and a high-fat meal in humans. Physiology & behavior 1993, 53(3):

126 Imbeault P, Saint-Pierre S, Almeras N, Tremblay A: Acute effects of exercise on energy intake and feeding behaviour. The British journal of nutrition 1997, 77(4): Patel BP, Bellissimo N, Thomas SG, Hamilton JK, Anderson GH: Television viewing at mealtime reduces caloric compensation in peripubertal, but not postpubertal, girls. Pediatric research 2011, 70(5): Panahi S, Luhovyy BL, Liu TT, Akhavan T, El Khoury D, Goff HD, Harvey Anderson G: Energy and macronutrient content of familiar beverages interact with pre-meal intervals to determine later food intake, appetite and glycemic response in young adults. Appetite 2013, 60(1): Blundell JE, King NA: Physical activity and regulation of food intake: current evidence. Medicine & Science in Sports & Exercise 1999, 31(11):S573 - S Fitzsimons TJ, Le Magnen J: Eating as a regulatory control of drinking in the rat. Journal of comparative and physiological psychology 1969, 67(3): Engell D: Interdependency of food and water intake in humans. Appetite 1988, 10(2): McKiernan F, Houchins J, Mattes R: Relationships between human thirst, hunger, drinking, and feeding. Physiol & Behav 2008, 94: Obika LF, Idu FK, George GO, Ajayi OI, Mowoe RS: Thirst perception and drinking in euhydrate and dehydrate human subjects. Nigerian journal of physiological sciences : official publication of the Physiological Society of Nigeria 2009, 24(1): Silha JV, Krsek M, Skrha JV, Sucharda P, Nyomba BL, Murphy LJ: Plasma resistin, adiponectin and leptin levels in lean and obese subjects: correlations with insulin resistance. European journal of endocrinology / European Federation of Endocrine Societies 2003, 149(4): Bickel PE: Metabolic fuel selection: the importance of being flexible. The Journal of clinical investigation 2004, 114(11):

127 Buczkowska EO, Jarosz-Chobot P: [Insulin effect on metabolism in skeletal muscles and the role of muscles in regulation of glucose homeostasis]. Przeglad lekarski 2001, 58(7-8): Woods SC, Lutz TA, Geary N, Langhans W: Pancreatic signals controlling food intake; insulin, glucagon and amylin. Philosophical transactions of the Royal Society of London Series B, Biological sciences 2006, 361(1471): Speechly DP, Buffenstein R: Appetite dysfunction in obese males: evidence for role of hyperinsulinaemia in passive overconsumption with a high fat diet. European journal of clinical nutrition 2000, 54(3): Pettersen SA, Fredriksen PM, Ingjer E: The correlation between peak oxygen uptake (VO2peak) and running performance in children and adolescents. aspects of different units. Scandinavian journal of medicine & science in sports 2001, 11(4): Carstens MT, Goedecke JH, Dugas L, Evans J, Kroff J, Levitt NS, Lambert EV: Fasting substrate oxidation in relation to habitual dietary fat intake and insulin resistance in non-diabetic women: a case for metabolic flexibility? Nutrition & metabolism 2013, 10(1): Coyle EF, Jeukendrup AE, Oseto MC, Hodgkinson BJ, Zderic TW: Low-fat diet alters intramuscular substrates and reduces lipolysis and fat oxidation during exercise. American journal of physiology Endocrinology and metabolism 2001, 280(3):E Rynders CA, Angadi SS, Weltman NY, Gaesser GA, Weltman A: Oxygen uptake and ratings of perceived exertion at the lactate threshold and maximal fat oxidation rate in untrained adults. European journal of applied physiology 2011, 111(9): Schwartz JG, Phillips WT, Aghebat-Khairy B: Revision of the oral glucose tolerance test: a pilot study. Clinical chemistry 1990, 36(1): Foss MC, Aoki TT: Effects of glucose loads of 50 and 100 g on carbohydrate and lipid oxidation in normal human subjects. Brazilian journal of medical and biological

128 112 research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica [et al] 1988, 21(4): Beyer J, Ardine C: Convergent and Discriminant Validity of a Self-Report Measure of Pain Intensity for Children. Children's Health Care 1988, 16(4) Erickson CJ: Pain measurement in children: problems and directions. Journal of developmental and behavioral pediatrics : JDBP 1990, 11(3): ; discussion Demello JJ, Cureton KJ, Boineau RE, Singh MM: Ratings of perceived exertion at the lactate threshold in trained and untrained men and women. Medicine and science in sports and exercise 1987, 19(4): Johnson ML, Burke BS, Mayer J: Relative importance of inactivity and overeating in the energy balance of obese high school girls. The American journal of clinical nutrition 1956, 4(1): Burke LM, Collier GR, Hargreaves M: Muscle glycogen storage after prolonged exercise: effect of the glycemic index of carbohydrate feedings. Journal of applied physiology 1993, 75(2): Vollestad NK, Blom PC: Effect of varying exercise intensity on glycogen depletion in human muscle fibres. Acta physiologica Scandinavica 1985, 125(3): Jeukendrup AE: Modulation of carbohydrate and fat utilization by diet, exercise and environment. Biochemical Society transactions 2003, 31(Pt 6): Flatt JP: Carbohydrate balance and body-weight regulation. The Proceedings of the Nutrition Society 1996, 55(1B): Mayer J: Glucostatic mechanism of regulation of food intake. The New England journal of medicine 1953, 249(1):13-16.

129 Robertson W, Stewart-Brown S, Wilcock E, Oldfield M, Thorogood M: Utility of accelerometers to measure physical activity in children attending an obesity treatment intervention. Journal of obesity 2011, 2011.

130 APPENDENCIES Appendix 1 Supplemental Data Food Intake (Not adjusted for kg body-weight) Compared to CON, FI was lower with GL (p < ), but not affected by EX. FI was higher in men when compared to boys AGE (p = ). GL*EX showed trend for a significant interaction GL*EX (p = ). No significant interactions were found. 1 Food Intake (kcal) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 880 ± ± ± ± ± 32 Men 1174 ± ± ± ± ± 43 Pooled 1027 ± ± ± ± 54 1 Means ± SEM (kcal); n=30. ANOVA analysis (GL, p < ; EX, p = ; GL*EX, p = ; AGE, p = ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

131 Net Energy Balance (Not adjusted for kg body-weight) Compared to CON, NEB was decreased by EX (p < ). GL (p = ) showed a trend to increased NEB. NEB was higher in men when compared to boys AGE (p = ). No significant interactions were found. 1 Net energy balance (kcal) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 848 ± ± ± ± ± 33 Men 1114 ± ± ± ± ± 47 Pooled 981 ± ± ± ± 54 1 Values are means ± SEM (kcal); n=30. ANOVA analysis (GL, p = ; EX, p < ; GL*EX, p = ; AGE, p = ; AGE*EX, p = ; AGE*GL, p = ; AGE*EX*GL, p = ).

132 Energy Expenditure (Not adjusted for kg body-weight) Compared to CON, EE was increased by GL (p = ), decreased by EX (p < ). EE was higher in men when compared to boys AGE (p < ). There was a significant interaction for AGE*EX (p < 0.001). No other significant interactions were found (. AGE specific analysis showed that EX (p < ) increased and GL (p = ) showed a trend to lower FATOX in boys. No other significant interaction was found. In men, EX (p < ) increased and GL (p =0.0619) increased EE. No other significant interaction was found. 1 Energy Expenditure (kcal) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 2 43 ± 3 50 ± ± ± 9 98 ± 7 Men 3 60 ± 3 61 ± ± ± ± 14 Pooled 52 ± 3 55 ± ± ± 14 1 Values are means ± SEM (kcal); n=15. ANOVA analysis (GL, p = ; EX, p < ; GL*EX, p = ; AGE, p < ; AGE*EX, p < ; AGE*GL, p = ; AGE*EX*GL, p = ). 2 Values are means ± SEM; n=15. ANOVA analysis for boys (GL, p = ; EX, p < ; GL*EX, P = ). 3 Values are means ± SEM; n=15. ANOVA analysis for men (GL, p = ; EX, p < ; GL*EX, p = ).

133 Carbohydrate Oxidation (Not adjusted for kg body-weight) Compared to CON, CHOOX was increased by GL (p < ) and EX (p < ). A significant interaction was found for GL*EX (p = ), showing an additional increase in CHOOX. Men had higher CHOOX when compared to boys AGE (p < ). There was an interaction for AGE*EX (p < ). No other significant interactions were found. AGE specific analysis showed that EX (p < ) and GL (p = ) increased CHOOX in boys. There was a significant interaction for GL*EX (p = ) in boys. In men, EX (p < ) and GL (p < ) increased CHOOX. GL*EX (p = ) interaction showed a trend but did not reach significance. 1 Carbohydrate oxidation (kcal) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 22 ± 2 30 ± 3 77 ± ± 9 58 ± 5 Men 33 ± 3 43 ± ± ± ± 12 Pooled 27 ± 2 37 ± ± ± 14 1 Values are means ± SEM (kcal); n=30. ANOVA analysis (GL, p < ; EX, p < ; GL*EX, p = ; AGE, p < ; AGE*EX, p < ; AGE*GL, p = ; AGE*EX*GL, p = ). 2 Values are means ± SEM; n=15. ANOVA analysis for boys (GL, p = ; EX, p < ; GL*EX, p = ). 3 Values are means ± SEM; n=15. ANOVA analysis for men (GL, p < ; EX, p < ; GL*EX, p = ).

134 Fat Oxidation (Not adjusted for kg body-weight) Compared to CON, FATOX was decreased by GL (p = ) and increased with EX (p < ). FATOX was similar in boys and men. No other significant interactions were found. 1 Fat oxidation (kcal) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 22 ± 3 19 ± 4 71 ± 9 50 ± 8 40 ± 4 Men 27 ± 3 17 ± 2 71 ± 7 47 ± ± 4 Pooled 24 ± 2 18 ± 2 71 ± 5 48 ± 7 21 Values are means ± SEM (kcal); n=30. ANOVA analysis (GL, p = ; EX, p < ; GL*EX, p = ; DEV, p = ; DEV*EX, p = ; DEV*GL, p = ; DEV*EX*GL, p = )

135 Water Consumption (Not adjusted for kg body-weight) EX (p = ) showed a trend for water consumption to be increased. GL had no effect on water consumption. No significant interactions were found. 1 Water consumption (ml) in boys and men Activity: Sedentary Exercise Pooled Drink: Water Glucose Water Glucose Boys 206 ± ± ± ± ± 22 Men 232 ± ± ± ± ± 27 Pooled 219 ± ± ± ± 35 1 Values are means ± SEM (kcal); n=30. ANOVA analysis (GL, p = ; EX, p = ; GL*EX, p = ; DEV, p = ; DEV*EX, p = ; DEV*GL, p = ; DEV*EX*GL, p = ).

136 12.2. Appendix 1A BMI for Age Percentile Charts in Boys 120

137 12.3. Appendix 1B CDC BMI Chart for men 121

138 12.4. Appendix 2A Telephone Screening Questionnaire Boys 122

139 12.5. Appendix 2B Telephone Screening Questionnaire Men 123

140 12.6. Appendix 3A Screening Questionnaire Boys 124

141 125

142 126

143 127

144 128

145 129

146 130

147 12.7. Appendix 3B Screening Questionnaire Men 131

148 132

149 133

150 134

151 135

152 136

153 12.8. Appendix 4A Recruitment Letter Boys 137

154 12.9. Appendix 4B Recruitment Letter Men 138

155 Appendix 5A Study information sheet and consent form boys 139

156 140

157 141

158 142

159 143

160 Appendix 5B Study Information Sheet and Consent Form Boys 144

161 145

162 146

163 147

164 Appendix 6 Pizza Form 148

165 Appendix 7 Session Sheet 149

166 Appendix 8A Recruitment poster boys 150

167 Appendix 8B Recruitment poster men 151

168 Visual Analog Scale Questionnaires Appendix 9A VAS Motivation to Eat Boys

169 Appendix 9B VAS Physical Comfort Boys 153

170 Appendix 9C VAS Preload Sweetness Boys 154

171 Appendix 9D VAS Preload and Pizza Palatability Boys 155

172 Appendix 10A VAS Motivation to Eat Men 156

173 Appendix 10B Physical Comfort Men 157

174 Appendix 10C Energy/Fatigue and Stress Men 158

175 Appendix 10D Pizza and Preload Palatability Men 159

176 Appendix 11 Nutritional Information Pizza 3 - Cheese Pepperoni Deluxe