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 < 0.0001), and further decreased with GL ingested prior to EX(p = 0.0254). RER was increased with GL(p < 0.0001) and EX(p = 0.0043), 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.

abstract: Obesity prevalence is high in the United States, in part due to increased fat storage following consumption of high fat/carbohydrate (sugar) foods. Following a meal, carbohydrate stimulates its own oxidation, while simultaneously suppressing fat oxidation, ultimately leading to fat storage. Aerobic exercise preceding a meal increases fat oxidation in the postprandial period, which may reduce fat storage. The ideal exercise prescription for optimal postprandial fat oxidation is unknown. The effect of low and moderate intensity continuous exercise (MIE) has been studied extensively, while the effects of high-intensity interval exercise (HIIE) on post-prandial substrate oxidation has not been examined. The purpose of this study was to compare the effects of MIE and HIIE on postprandial substrate oxidation after consumption of an isocaloric meal (2 glazed donuts; 520 kcal) in healthy adults. Ten subjects (8 males, 2 females; age=24yr, BMI=24 kg/m2) completed three conditions in random order: 1) no exercise control; 2) MIE: cycling at 60-75%HRmax; 3) HIIE: cycling at 90-95%HRmax. The duration of each exercise bout was sufficient to expend approximately 520 kcal, the energy equivalent of the donuts, which were consumed 1 hour post-exercise. Immediately after consuming the donuts, pulmonary ventilation and gas exchange were measured breath-by-breath continuously and recorded (min-by-min) for 5 hours. Repeated measures analysis of covariance was used to compare the mean differences in outcome variables accounting for gender. Absolute postprandial fat oxidation (g/5 hours) was 17.3±5.4, 27.1±9.6 and 23±1.2 for control, MIE and HIIE trials respectively, with the postprandial fat oxidation significantly greater for the two exercise conditions compared to control. Relative to baseline values, both exercise conditions resulted in cumulative net postprandial fat oxidation significantly greater than control (control = -1.79±3.99g; MIE = 11.51±8.41g, HIIE= 9.51±5.20g). Therefore, results indicate that exercise most certainly increases postprandial fat oxidation, and that exercise type, either MIE or HIIE, is not as important as total energy expended. The fact that exercise of ~1 hour was required to oxidize the amount of fat in two donuts, that required only a few minutes to consume, highlights the challenges of using exercise for weight control in an obesogenic environment. / Dissertation/Thesis / Masters Thesis Nutrition 2018

Macronutrient metabolism plays an essential role in the overall health of an individual. Depending on a number of variables, for example, diet, fitness level, or metabolic disease state, protein, carbohydrate and fat have varying capacities to be oxidized and balanced. Further, when analyzing the oxidation of carbohydrate and fat in the skeletal muscle specifically, carbohydrate balance happens quite rapidly, while fat balance does not. The ability of skeletal muscle to adapt and respond to various nutrient states is critical to maintaining healthy metabolic function. Habitual high fat intake has been associated with reduced oxidative capacity, insulin resistance, increased gut permeability, inflammation, and other risk factors often preceding metabolic disease states. The disruption of gut function leads to gut permeability and increases endotoxins released into circulation. Endotoxins have been shown to play an important role in obesity-related whole body and tissue specific metabolic perturbations. Each of these disrupted metabolic processes is known to associate with obesity, metabolic syndrome and diabetes. To date, limited research has investigated the role of high fat diet on skeletal muscle substrate oxidation and its relationship to gut permeability and endotoxins. The purpose of this study was to determine the effects of an acute, five-day, isocaloric high fat diet (HFD) on skeletal muscle substrate metabolism in healthy non-obese humans. An additional purpose was to determine the effects of a HFD on gut permeability and blood endotoxins on healthy, non-obese, sedentary humans. Thirteen college age males were fed a control diet for two weeks, followed by five days of an isocaloric HFD. To assess the effects of a HFD on skeletal muscle metabolic adaptability and postprandial endotoxin levels, subjects underwent a high fat meal challenge before and after a HFD. Muscle biopsies were obtained; blood was collected; insulin sensitivity was assessed via intravenous glucose tolerance test; and intestinal permeability was assessed via the four-sugar probe test before and after the HFD. Postprandial glucose oxidation and fatty acid oxidation in skeletal muscle increased before the HFD intervention but was decreased after. Skeletal muscle in vitro assay of metabolic flexibility was significantly blunted following the HFD. Insulin sensitivity and intestinal permeability were not affected by HFD, but fasting endotoxin was significantly higher following the HFD. These findings demonstrate that in young, healthy males, following five days of an isocaloric high fat diet, skeletal muscle metabolic adaptation is robust. Additionally, increased fasting endotoxin independent of gut permeability changes are potentially a contributor to the inflammatory state that disrupts substrate oxidation. These findings suggest that even short-term changes in dietary fat consumption have profound effects on skeletal muscle substrate metabolism and fasting endotoxin levels, independent of positive energy balance and whole-body insulin sensitivity. / Ph. D.

Elevated postprandial triacylglycerol concentrations ([TAG]) are associated with the development and progression of atherosclerosis, and are established as an independent risk factor for future cardiovascular disease. Considering the majority of the daytime is spent in a postprandial state typically, and the paediatric origins of atherosclerosis are well established, lifestyle interventions including manipulations of exercise energy expenditure and dietary energy intake should be initiated early in life. Therefore, this thesis aimed to investigate the postprandial metabolic responses to different exercise and energy intake manipulations in boys and girls, with concentrations of circulating TAG representing the primary outcome of interest. To achieve this, a total of 60 healthy 11 to 13 year old boys and girls were recruited into five experimental studies. The first experimental study (Chapter 4) demonstrated that a single session of high-intensity interval running (HIIR) involving 10 x 1 min intervals at 100% maximal aerobic speed (MAS) resulted in a moderate reduction in postprandial plasma [TAG] in 11 to 12 year old boys. In the second experimental study (Chapter 5), immediate replacement of the moderate-intensity exercise-induced energy deficit negated the reduction in postprandial plasma [TAG] in 11 to 13 year old boys. Furthermore, an exercise-induced energy deficit was required to promote an increase in whole-body fat oxidation. The importance of the associated energy deficit was explored further in Chapter 6, which demonstrated that a moderate-intensity exercise-induced energy deficit elicited a greater reduction in postprandial plasma [TAG] than an isoenergetic diet-induced energy deficit in 11 to 13 year old girls (21% vs. 10% respectively). Chapter 7 compared the effect of 10 x 1 min interval runs at 100% MAS (HIIR) and 5 x 1 min interval runs at 100% MAS combined with a mild reduction in habitual energy intake by 0.82 MJ (195 kcal; HIIR-ER) on postprandial metabolism in 11 to 13 year old girls. Acute manipulations of low volume HIIR and ER reduced postprandial plasma [TAG] and increased resting whole-body fat oxidation, with the magnitude of effect marginally, although not meaningfully, greater following HIIR than HIIR-ER. The final experimental chapter (Chapter 8) compared directly healthy 11 to 13 year old boys and girls postprandial TAG responses to acute HIIR. Although postprandial plasma [TAG] was substantially lower in boys compared with girls, the magnitude of reduction following HIIR was similar between the sexes (11% vs. 10% respectively). Collectively, these studies demonstrate the efficacy of acute moderate- and high-intensity exercise, and to a lesser extent energy-intake restriction, to reduce postprandial plasma [TAG] and increase resting whole-body fat oxidation in boys and girls. Furthermore, the beneficial effect of exercise on postprandial metabolism appears dependent on the maintenance of the associated energy deficit. These lifestyle interventions have the potential to provide a practical, effective and engaging stimulus to promote a healthier cardiovascular risk profile in early adolescence.

Iron-substituted manganese superoxide dismutase (Fe(Mn)SOD) was produced using an in vivo preparation method. It’s an inactive enzyme in catalyzing superoxide radical dismutation owing to the mis-incorporation of Fe in the active site evolved to use Mn. To investigate the possible toxicity of human Fe(Mn)SOD proposed by Yamakura, we studied the properties of Fe(Mn)SOD upon H2O2 treatment and compared to that of FeSOD. It’s found that the responses to H2O2 treatment were different, including the changes of optical spectra, variations of active site coordination and secondary structures. Fe3+ reduction was not observed in Fe(Mn)SOD even H2O2 is believed to oxidize proteins via highly reactive intermediates including Fe and formed via Fe2+, which is true in FeSOD. What’s more, the activities of Fe(Mn)SOD and FeSOD were totally different in the ABTS assay or Amplex Red assay. These results indicated that the mechanism of peroxidase reaction of Fe(Mn)SOD is not identical to that of FeSOD.

Consistently, reports in the literature have identified that a sedentary lifestyle contributes to the progression of a range of chronic degenerative diseases. The measurement of energy expenditure and physical activity pattern in children is a challenge for all professionals interested in paediatric health and from a broader perspective, the public health fraternity charged with considering longer term health consequences of physical inactivity. The primary objective of this thesis was to identify a suitable indirect and objective measurement technique for the assessment of energy expenditure and physical activity pattern in children. The ideal characteristics of such a technique are that it should be reproducible and have been validated against a criterion reference method. To achieve this goal, a series of methodological studies were undertaken (Chapters II and III). This work was essential to increase accuracy during the individualised laboratory calibration process and further minimise prediction errors when analysing data from 7 days of monitoring under free-living conditions in the second part of the study (Chapters IV and V). In the first study to verify the combined effect of body position, apparatus and distraction on children's resting metabolic rate (RMR), experiments were carried out on 14 children aged 8-12 (mean age = 10.1 years ± 1.4). Each participant underwent 2 test sessions, one week apart under three different situations: a) using mouthpiece and nose-clip (MN) or facemask (FM); b) sitting (SEAT) or lying (LY) and c) TV viewing (TV) or no TV viewing. In the first session, following 20 min rest and watching TV, the following protocol was used: LY: 20 min - stabilisation; 10 min using MN and 10 min using FM. Body position was then changed to seated: 20 min stabilisation; 10 min using FM; 10 min using MN. In the second session, FM and MN order was changed and participants did not watch TV. Data were analysed according to the eight combinations among the three studied parameters. Repeated measures ANOVA indicated statistically significant differences for &VO2 (p=0.01) and RMR (p=0.02), with TVMNSEAT showing higher values than TVFMLY. Bland-Altman analysis showed a bias for &VO2, &VCO2, RQ and RMR between TVFMLY and TVMNSEAT of -17.8±14.5 ml.min-1, -8.8±14.5 ml. min-1, 0.03±0.05 and -115.2±101.9 kcal.d-1, respectively. There were no differences in RMR measurements due to body position and apparatus when each variable was isolated. Analyses of distraction in three of four combinations indicated no difference between TV and no TV. In summary, different parameter combinations can result in increased bias and variability and thereby reported differences among children's RMR measurement. The second study dealt with treadmill adaptation and determination of self-selected (SS) walking speed. Assessment of individual and group differences in metabolic energy expenditure using oxygen uptake requires that individuals are comfortable with, and can accommodate to, the equipment being utilised. In this study, a detailed proposal for an adaptation protocol based on the SS was developed. Experiments were carried out on 27 children aged 8-12 (mean age = 10.3±1.2 yr). Results from three treadmill tests following the adaptation protocol showed similar results for step length with no significant differences among tests and lower and no statistically significant variability within- and between-days. Additionally, no statistically significant differences between SS determined over-ground and on a treadmill were verified. These results suggest that SS speed determined over-ground is reproducible on a treadmill and the 10 min familiarisation protocol based on this speed provided sufficient exposure to achieve accommodation to the treadmill. The purpose of the third study was to verify within- and between-day repeatability and variability in children's oxygen uptake ( &VO2), gross economy (GE) [ &VO2 divided by speed] and heart rate (HR) during treadmill walking based on SS. 14 children (mean age = 10.2±1.4 yr) undertook 3 testing sessions over 2 days in which four walking speeds, including SS, were tested. Within- and between-day repeatability was assessed using the Bland and Altman method and coefficients of variability (CV) were determined for each child across exercise bouts and averaged to obtain a mean group CV value for &VO2, GE and HR per speed. Repeated measures ANOVA showed no statistically significant differences in within- or between-day CV for &VO2, GE or HR at any speed. Repeatability within and between-day for &VO2, GE and HR for all speeds was verified. These results suggest that submaximal &V O2 during treadmill walking is stable and reproducible at a range of speeds based on children's SS. In the fourth study, the objective was to establish the effect of walking speed on substrate oxidation during a treadmill protocol based on SS. Experiments were carried out on 12 girls aged 8-12 (mean age = 9.9±1.4 yr). Each participant underwent 2 test sessions, one week apart. Workloads on the treadmill included 2 speeds slower than SS (1.6 [V1] and 0.8 km.h-1 [V2] slower than SS), SS (V3), and a speed 0.8 km.h-1 faster than SS (V4). Indirect calorimetry from respired gas measurements enabled total fat (FO) and carbohydrate (CHO) oxidation rates to be calculated according to the non-protein respiratory quotient (Peronnet and Massicote, 1991) and percentage of CHO and FO calculations using equations from McGilvery and Goldstein (1983). Repeated measures ANOVA followed by a Tukey Post Hoc test (p< 0.05) was used to verify differences in CHO and FO rates among speeds. Paired T-test was used to verify differences in CHO and FO rates between tests per velocity. The reliability between-day was assessed using intraclass correlation coefficient (ICC). Results showed significant differences for CHO among all speeds, as well as significant differences for FO between V1 and V2 against V3 and V4 in both tests. Analyses between trials per velocity showed no significant substrate use differences as well as acceptable reliability. At the self-selected speed (V3) there was an accentuation in FO reduction as well as an increase in CHO oxidation. The purpose of the fifth study was to determine whether there were differences in substrate oxidation between girls (G) and women (W) during a treadmill protocol based on SS. Experiments were carried out on 12 G aged 8-12 (mean age = 9.9±1.4 yr) and 12 W aged 25-38 (mean age = 32.3±3.8 yr). The treadmill protocol included 6 min workloads followed by 5 min rest periods. Workloads included 2 speeds slower than SS (1.6 (V1) and 0.8 km.h-1 (V2) slower than SS), SS (V3), and a speed 0.8 km.h-1 faster than SS (V4). Total fat and carbohydrate (CHO) oxidation rates were calculated from indirect calorimetry according to the non-protein respiratory quotient. Repeated measures ANOVA followed by a Tukey Post Hoc test was used to verify intra-test differences in CHO and fat oxidation rates among speeds. Intergroup differences were analysed using paired T-test. Fat utilisation in W achieved a plateau at a relative velocity 0.8 km.h-1 slower than SS, but for G, fat utilisation increased until SS, and then stabilised upon reaching the higher velocity. CHO oxidation curves rose abruptly above V2 for W, while for G the acute increase occurred after SS (V3). Collectively, these results indicate that as walking intensity increases G are able to meet the energy demands of the work by increasing fat oxidation together with the increased CHO oxidation up to SS. In contrast for W, increasing CHO oxidation is associated with an early decrease in fat utilisation at a velocity slower than the self-selected speed. The sixth study dealt with validation of indirect techniques for the measurement of energy expenditure in free-living conditions against the DLW technique. Experiments were carried out on 19 children aged 8-12 (mean age = 10.3±1.0 yr). To indirectly predict energy expenditure 12 different procedures were used. Only one procedure, combining activity and heart rate (AHbranched), was based on a group equation, the others were based on individualised regression. Three of the individually-based techniques were able to accurately predict energy expenditure in free-living conditions. These tecniques were HRPAnetRMR using HRnet [HR exercise minus sleep HR (SHR)] against PAnet (measured PA exercise minus measured RMR) and upper and lower body equations corrected by RMR; HRPAnet4act using the same procedure but corrected by the mean resting &VO2 for 4 resting activities [(4act) = supine watching TV, sitting watching TV, sitting playing computer games and standing], and HRPALBnet4act using only lower body activities and corrected by 4act. HRPAnetRMR was only slightly more accurate than HRPAnet4act and HRPALBnet4act, but this technique is only adjusted by RMR whereas the other two are heavily dependent on more complex laboratory calibration. Bland and Altman (1986) analyses showed no significant differences between AHbranched predicted and measured TEE using the DLW technique. A SEE of 79 kcal.d-1 and a mean difference of 72 kcal.d-1, with a 95% CI ranging from -238 to 93.9 kcal.d-1 was found. In addition, no significant differences between predicted HRPAnetRMR and measured TEE using DLW were found, showing an SEE of 99 kcal.d-1 and a mean difference of -67 kcal.d-1, and a 95% CI ranging from -276.6 to 141.9 kcal.d-1. AHbranched and HRPAnetRMR were both valid and similarly suitable for the prediction of energy expenditure in children under free-living conditions. Significant associations between DLWAEE and the after-school time window indicated that this time window as an important discretionary period representative of children physical activity. However, the duration of the after-school time windows should be more carefully considered. Accelerometer data showed a better association between the largest after-school time window (3.5 hr) and measured TEE. The final study, completed with 19 children aged 8-12 (10.3±1.0 yr) highlighted, under laboratory conditions across a range of walking and running speeds, the inadequacy of the use of the standard MET in children. This traditional approach overestimates energy expenditure with an increased difference linearly related to speed increments. Minute-by-minute analyses of 7 days of free-living monitoring showed an average overestimation of 64 minutes per day for moderate-to-vigorousphysical- activity (MVPA) using the standard MET compared with the individually measured MET. For all intensities, these differences were statistically significant (p< 0.001). The second part of this study showed a variability of 20% in the average time spent at MVPA when comparing HR I 140 bpm and HR > 50%P &VO2 (P &VO2 = the highest &VO2 observed during an exercise test to exhaustion). Results of the current study compared to observations in the literature showed that HR I 140 bpm consistently estimates lower MVPA time than HR > 50%P &VO2. When these two PA indices were compared with individual and standard MET measured minute-byminute, statistically significant differences were verified among all of them at MPA, but no differences were verified at VPA, except between individual and standard METs. However, whether each one of the PA indices used are under- or overestimating time at MVPA is still debatable due to the lack of a gold standard. Finally, each index used in this study classified different numbers of participants as achieving the PA target of 60 min.d-1. The wide variability between indices when attempting to classify children who are achieving the recommended target is cause for great concern because habitually these indices are utilised as screening tools in paediatric and public health settings and used to guide behavioural interventions.