published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Gut hormones are implicated in the regulation of energy balance. The studies in this thesis have examined the effects exercise on gut hormones (acylated ghrelin and peptide YY3-36), appetite and food intake, over extended durations. Sixty-nine young, healthy, predominantly Caucasian males were recruited to six studies. The age, height and body mass of the participants were: 22.4 ± 0.3 y, 1.80 ± 0.1 m, 76.2 ± 1.0 kg (mean ± SEM). In study one, 90 min of resistance exercise did not influence appetite or energy intake over 24 h of assessment, yet stimulated a latent preference for carbohydrate rich foods. Study two demonstrated that appetite was suppressed during 60 min of swimming but was elevated after consuming a post-exercise meal. Plasma acylated ghrelin was suppressed during swimming but was unaltered after. Energy/macronutrient intake remained unchanged. In study three, 60 min of brisk walking (45 ± 2% of max) did not influence appetite, energy/macronutrient intake or plasma concentrations of acylated ghrelin during an eight hour observation period. Study four showed that 90 min of treadmill running (69 ± 1% of max) transiently suppressed appetite and acylated ghrelin but did not influence these variables, or energy/macronutrient intake within 22.5 h after exercise. The findings of study five suggest that the suppression and subsequent rebound in plasma acylated ghrelin after exercise may be related to a delayed voluntary decision to eat after. Finally, study six showed that appetite, food intake and circulating concentrations of acylated ghrelin and peptide YY3-36 are responsive to acute deficits in energy induced by food restriction but are not sensitive to equivalent energy deficits induced by exercise. This thesis has shown that exercise transiently alters circulating levels of acylated ghrelin and peptide YY3-36 in directions expected to inhibit appetite however no changes are seen after exercise. Conversely, food restriction elicits marked compensatory changes in circulating acylated ghrelin and peptide YY3-36. This thesis also demonstrates that resistance exercise, brisk walking and running do not stimulate appetite or energy intake over defined periods, even when the energy expenditure elicited is high. Swimming appears to increase appetite in the latter hours after exercise.

Glucose is the primary fuel for the brain and also important for exercising muscle. The purpose of the thesis was to investigate the effects of the glycaemic index (GI) of mixed meals on appetite, cognitive performances and metabolic responses during intermittent exercise in recreationally active adults. Study one investigated whether a low GI (LGI) breakfast (GI = 42.5) could suppress appetite and reduce energy intake (EI) of 12 recreationally active females (28.2 ± 8.0 years) more than a high GI (HGI) breakfast (GI = 73.5). Area under the curve of the appetite score (AS AUC) following LGI breakfast was significantly greater than the HGI trial during the 60-min postprandial (pp) period (2568 ± 1027 vs. 2198 ± 821 mm∙min, p = 0.025). The HGI breakfast facilitated a stronger appetite suppressing effect up to eight hours post breakfast than the LGI trial (18834 ± 3906 vs. 21278 ± 3610 mm∙min, p = 0.028). The EI on the LGI trial day was significantly higher than on the pre-trial day (2,215 ± 576 vs. 1,748 ± 464 kcal, corrected p = 0.008). Fourteen recreationally active males (34.5 ± 8.9 years) in study two consumed the LGI (GI = 41.3) and HGI (GI = 74.3) breakfasts in the laboratory and then prescribed LGI and HGI meals in the free living environment. In line with study one, the AS AUC was significantly smaller following HGI than LGI breakfast over the 60-min pp period (2,989 ± 1,390 vs. 3,758 ± 1,290 mm∙min, p = 0.027). The HGI meals (GI = 76.9) suppressed appetite more than the LGI meals (GI = 39.6) over 12 hours on the trial day (35,454 ± 9,730 vs. 41,244 ± 8,829 mm∙min, p = 0.009) although energy balance was not different between trials. Study three investigated whether following a LGI breakfast (GI = 42.2) providing 1 g CHO kg-1 BM could result in a better vigilance and attention than a HGI breakfast (GI = 72.4), and reduced lunch EI in 16 recreationally active males (24.4 ± 3.6 years). A significant trial x time effect in the interference time of the Stroop Colour Word Task (SCWT) (p = 0.039) showed that the LGI breakfast maintained the attentional performance up to 90-min pp. Both high pre-task glucose concentration ([Glucose]) at 15-min pp and low pre-task [Glucose] at 105-min pp in the HGI trial were associated with unfavourable outcomes in vigilance in the Rapid Information Processing Task (RIPT). The LGI pre-task [Glucose] returning back to fasting level at 60-min pp was associated positively with the response time. The pre-lunch AS was a significant predictor of the lunch EI per fat free mass which explained 21% and 26% of variance in the LGI and HGI trials respectively. No significant difference was found in the ad libitum lunch EI between trials. Sixteen recreationally active males (27.8 ± 7.7 years) in study four consumed a LGI (GI = 42) and a HGI breakfast (GI = 72.8) providing 1.2 g CHO kg-1 BM consumed 60 minutes prior to intermittent running on two separate mornings. Better attentional performance at 150-min pp was found following LGI than HGI breakfast. The significant trial x time interaction in the SCWT (p = 0.045) showed the shortest interference time performed after the last exercise session in the LGI trial. The amounts of CHO and fat being oxidized were comparable between trials during three sessions of 16-min intermittent running with an average intensity of 65% V̇O2max. In conclusion, the pre-meal appetite sensation is more predictive of the subsequent meal EI than the pre-meal [Glucose]. The meal strategy for weight management in recreationally active adults may focus on greater appetite suppression by selecting HGI foods whilst maintaining healthy eating guidelines. Recreationally active males performing sports requiring high levels of vigilance and selective attention with low physical activity levels can benefit up to 60–90 min pp from the LGI breakfast. Their attentional performance can benefit from the LGI breakfast with moderate to high intermittent intensities in the late exercise period at 150–min pp. Recreationally active adults should consider the timing of meal consumption in relation to performing intermittent exercise, in order to maximize the advantages from the LGI or HGI breakfasts for cognitive performance or appetite suppression. They may be more liberal in pre-exercise food choices if substrate oxidation during intermittent running is only of their concern.

Current physical activity recommendations are being met by less than 21 % of children between 5-15 y. Recent Government initiatives are aiming to increase children’s participation in exercise. However, the effects on an imposed bout of exercise-induced energy expenditure (EE) on energy intake (EI) and appetite (hunger, fullness and prospective consumption) in normal weight children have received a limited research focus to date. Therefore, this thesis aimed to investigate how an imposed bout of exercise-induced energy expenditure (EE) on energy intake (EI) and appetite in normal weight children The first study investigated whether 17 habitually active girls were able to accurately increase their EI to match the EE following 60 min moderate intensity walking exercise. On average 17% of the EE was compensated for by an increase in EI. However, the ranged for EI change was -160% to +166% indicating large individual responses. The second study investigated whether 30 min of maximal sprint intermittent sprint cycling exercise would significantly alter EI or appetite in 13 boys and 13 girls. In the boys, hunger and prospective consumption were suppressed whilst fullness increased immediately following the exercise, whilst EI was significantly increased in response to the exercise condition. No significant changes to appetite or EI were observed in the girls. The third study investigated whether a mid-morning snack, moderate intensity cycling exercise (energy matched to snack) or both would alter EI or appetite in 20 boys and 18 girls. Irrespective of sex, hunger and prospective consumption were suppressed whilst fullness increased following the mid-morning snack, however this change in appetite did not alter EI as no significant differences were found between conditions. The fourth study investigated whether 99 recreational sports players (males/females, adults/children) were able to conceptualise their EE following 1 h habitual training into quantifiable amounts of food (chocolate) or drink (sports drink). Only 36 % of the EE from the exercise was met by the estimated amounts of food or drink. Age, sex nor sports participation significantly altered the participants’ accuracy of estimation. The fifth study investigated whether sex or dietary restraint impacted brain activation responses to visual food stimuli in 15 boys and 14 girls between a fed and fasted condition. Significant differences in brain activation were found between conditions, sexes and dietary restraint, potentially suggesting the differences observed in the previous experimental studies could be attributed to neurological alterations between participants. In conclusion, the findings presented demonstrate the changes in EI between young adolescents in response to an imposed bout of exercise are extremely variable. Whilst eating behaviours failed to correspond to the EI differences observed between participants, potentially brain activation differences may be responsible. The sex of the participant is more likely to impact EI and appetite following maximal sprint intensity exercise, more so than a bout of moderate intensity exercise. Future research should focus on determining what underpins the variable change in EI between participants following a bout of exercise.

Appetite control and satiety mechanisms help animals maintain energy homeostasis; however, these mechanisms can be misregulated, leading to overweight and obesity. Caenorhabditis elegans is an excellent model system to study appetite and satiety because of its conserved behavioral aspects of satiety and conserved molecular mechanisms. ASI senses nutrition and its activity is required for the behavioral state of satiety quiescence. The purpose of this thesis project was to elucidate the function of cGMP signaling in ASI by looking at behavioral effects from the pharmacological use of sildenafil (Viagra), a PDE inhibitor, and the effects on ASI activation from mutating guanylyl cyclase DAF-11. Sildenafil treatment increases satiety quiescence and decreases fat storage in a PDE-dependent manner. The daf-11 mutation decreased overall fluorescence intensity of ASI activation and the frequency at which ASI activated by about 50% compared to wild-type worms, suggesting that DAF-11 plays an important role in ASI to promote satiety.

Cross-sectional evidence associates breakfast omission with negative health outcomes. The present work aimed to examine if these cross-sectional associations have a causal component, by conducting randomised control trials in healthy humans. It was established using lean individuals that there are divergent hormonal responses to morning feeding and fasting, although increased energy intake at lunch following fasting incompletely compensated for breakfast intake. Hormonal and subjective appetite responses in the afternoon did not consistently provide evidence for increased hunger following fasting. In the same participants assigned to a 6-week free-living intervention of either 700 kcal pre 11:00 or fasting until 12:00 daily, it was found that energy intake was greater in those assigned breakfast consumption, but that physical activity was also greater than those fasting. Cardiovascular risk factors and measures of metabolic control were largely unaffected by either intervention. There was no adaptation of acute metabolic/hormonal responses to feeding following either intervention. In obese individuals, similar patterns of results were obtained for the hormonal and metabolic responses to acute feeding and fasting, but with no compensation for breakfast intake at lunch. Results from the free-living intervention demonstrated no difference in energy intake between groups or physical activity over the entire day, but greater energy expenditure during the morning in those consuming breakfast. Markers of cardiovascular health and metabolic control were generally not differently affected by either intervention. Neither intervention caused adaptation of the acute hormonal and metabolic responses to feeding. In summary, acute morning fasting does not cause complete compensation for breakfast intake at lunch, or result in greater hunger throughout the afternoon. Daily morning fasting does not affect acute responses to feeding or cause increased energy intake or weight gain relative to self-selected breakfast consumption, but seems to limit physical activity in lean, and to a lesser extent, in obese individuals.

Objetivos: Avaliar o efeito da menopausa sobre a composição corporal, a distribuição de gordura abdominal, o índice de massa corporal, a circunferência cintura, os percentuais de gordura androide, ginoide e a relação androide/ginoide, o consumo calórico total da alimentação diária e o nível de atividade física de mulheres climatéricas. Modelo: Estudo transversal com mulheres climatéricas recrutadas através de divulgação nas mídias eletrônica e impressa e realizado de março de 2014 a outubro de 2015. Local: Centro de Pesquisa Clínica do Hospital de Clínicas de Porto Alegre (CPC/HCPA), RS/Brasil. Amostra: A amostra foi constituída por mulheres na pré e pós-menopausa com idade entre 44 e 52 anos. Medidas de avaliação: Os instrumentos utilizados foram: Executive summary of the Stages of Reproductive Aging Workshop + 10 (STRAW +10, para a classificação de mulheres em relação ao estadiamento menopausal); Recordatório alimentar de 24 horas (para medir o consumo alimentar); um questionário semiestruturado sobre aspectos de saúde, hábitos de vida, familiares e parâmetros socioeconômicos; o Questionário Internacional de Atividade Física (IPAQ – versão curta, para a mensuração de atividade física da última semana); o Questionário de Avaliação da Menopausa (MRS, para quantificar a severidade dos sintomas da menopausa); avaliações antropométricas (estatura, peso, índice de massa corporal – IMC, circunferência abdominal e circunferência do quadril); absorciometria de raios-x de dupla energia (DEXA, para avaliação da composição corporal, estimativas de massa magra e gorda); e a Escala Visual Analógica de Apetite (para quantificação do nível de fome). Amostras sanguíneas foram coletadas para a análise de níveis de hormônios (estradiol e folículo estimulante – FSH) e parâmetros bioquímicos de metabolismo (colesterol total e frações – triglicerídeos, HDL, LDL – e glicemia de jejum). O banco de dados foi digitado e analisado no programa SPSS versão 18.0. Testes univariados (Teste t de Student e de Mann-Whitney) foram aplicados para comparações de médias/medianas entre os grupos, conforme normalidade da variável contínua pelo teste de Shapiro-Wilk. Análises de distribuições (Qui-quadrado com análises de valores residuais ajustados) foram aplicadas para comparações de frequências de variáveis categóricas entre os grupos. Correlações de Spearman foram aplicadas entre todas as variáveis analisadas. O nível de significância adotado para todas as análises foi fixado em 5%. Resultados: Avaliaram-se 114 mulheres, categorizadas em pré-menopausa (n=60), mediana de idade [Intervalo de Confiança – IC95%] de 47,5 [47,01–48,35 anos] e na pós-menopausa (n=54) com mediana de idade de 49 anos [48,29–49,56]. O tempo de pós-menopausa foi (mediana [95%IC] de 1,50[1,63–2,41] anos) e as mulheres na pré-menopausa classificadas como -3b segundo os critérios de STRAW+10. A maioria das participantes apresentava ensino médio ou superior (35,96% e 39,47%, respectivamente), era solteira ou sem parceiro (57,02%), não tabagista (97,37%) e não consumia álcool (57,89%). Quanto ao IMC, foram categorizadas como eutróficas (31,86%) ou obesas (40,71%), sem doença hipertensiva (98,25%), tireoidiana (97,37%) ou cardiovascular (100%). Em relação à atividade física, a maioria das mulheres apresentava nível ativo (51,75%). Na avaliação do apetite, as mulheres na pós-menopausa apresentaram escores maiores do que as prémenopáusicas (p=0,013). Níveis de colesterol total e de HDL foram maiores nas mulheres na pós-menopausa (p=0,040 e p≤0,0001, respectivamente). Não houve diferenças estatísticas entre os grupos quanto à massa corporal total, gordura androide e ginoide, conteúdo mineral ósseo, massa magra, consumo calórico, triglicerídeos e glicemia de jejum (p>0,05). Quanto aos sintomas climatéricos, as mulheres na pósmenopausa apresentaram mais queixas de fogachos e ressecamento vaginal (de moderado a extremamente severo) (p=0,056 e p=0,007, respectivamente) e significância marginal em relação aos problemas sexuais (p=0,086). O IMC, os triglicerídeos séricos e a glicemia de jejum foram positivamente correlacionados à circunferência da cintura, massa corporal, massa adiposa, massa magra e gorduras androides e ginoide. Colesterol HDL foi negativamente relacionado à circunferência da cintura, massa corporal, massa adiposa, massa magra e gordura androide. A escala visual analógica de apetite foi positivamente relacionada a humor deprimido, problemas sexuais e fogachos. Conclusões: Não houve diferenças estatisticamente significativas quanto à massa corporal total, gordura androide e ginoide, conteúdo mineral ósseo, massa magra, consumo calórico, triglicerídeos e glicemia de jejum, possivelmente, porque no início da pós-menopausa as possíveis modificações na composição corporal não sejam impactantes, o que permite considerar esse período como uma janela de oportunidade para intervenções precoces direcionadas ao estilo de vida, prevenindo-se agravos como perfil aterogênico e aumento do risco cardiovascular. / Objetives: To evaluate the effect of menopausal transition on body composition, abdominal fat distribution, body mass index, waist-hip circumference, percentages of android, gynoid and android/gynoid fats ratio, total daily caloric intake and the level of physical activity of climacteric women. Model: A cross-sectional study with climacteric women recruited by electronic and printed media and carried out from March 2014 to October 2015. Place: Clinical Research Center of Clinical Hospital of Porto Alegre (CPC/HCPA), RS/Brazil. Sample: The sample consisted of pre and postmenopausal women aged between 44 and 52 years. Measures of evaluation: The instruments used were: the Executive Summary of the Stages of Reproductive Aging Workshop + 10 (STRAW +10, for the classification of women in relation to menopausal staging); 24- hour food recall (to measure food consumption); a semi-structured questionnaire on aspects of health, life habits, family and socioeconomic parameters; the International Physical Activity Questionnaire (IPAQ – short version, for the measurement of physical activity of the last week); the Menopause Rating Scale (MRS, to quantify the severity of menopausal symptoms), anthropometric assessments (height, weight, body mass index – BMI, waist circumference and hip circumference); Dual-energy x-ray absorptiometry (DXA, to evaluate body composition, estimates of leand and fat mass); and the Visual Analogue Appetite Scale (to quantify the level of hunger). Blood samples were collected for the analysis of female hormone levels (estradiol and follicle-stimulating – FSH) and biochemical parameters of metabolism (total cholesterol and triglyceride fractions – HDL, LDL – and fasting glycemia). The database was entered and analyzed in SPSS version 18.0. Univariate tests (Student’s t test and Mann-Whitney test) were applied for comparisons of means/medians between groups, according to the normality of the continuous variable by the Shapiro-Wilk test. Distribution analyzes (Chi-Square with adjusted residual values) were applied for comparisons of frequencies of categorical variables between the groups. Spearman’s correlations were applied among all analyzed variables. The level of significance adopted for all analyzes was set at 5%. Results: A total of 114 women, pre-menopausal women (n=60) with median age [95% Confidence Interval – CI] of 47.5[47.01–48.35] years and postmenopausal women (n=54) with median age [95%CI] of 49.0[48.29–49.56]. Postmenopausal time median [95%CI] time was of 1.50[1.63–2.41] years and premenopausal women were classified as -3b, according to the STRAW + 10 criteria. The majority of participants had high school education (35.96% and 39.47%, respectively), was single or without partner (57.02%), non-smoker (97.37%) and did not consume alcohol (57.89%). Regarding BMI, they were categorized as either eutrophic (31.86%) or obese (40.71%), without hypertensive (98.25%), thyroid (97.37%) or cardiovascular (100.0%) diseases. In relation to physical activity, the majority of women had an active level (51.75%). In the evalution of appetite, postmenopausal women had higher scores than premenopausal women (p=0.013). Total cholesterol and HDL levels were higher in postmenopausal women (p=0.040 and p≤0.0001, respectively). There were no statistical differences between the groups regarding total body mass, android and gynoid fats, bone mineral content, lean mass, caloric intake, triglycerides and fasting glycemia (p>0.05). Considering the climacteric symptoms, postmenopausal women presented more complaints of hot flashes and vaginal dryness (moderate to extremely severe, p=0.056 and p=0.007, respectively) and marginal significance in relation to sexual problems (p=0.086). BMI, serum triglycerides, and fasting glycemia were positively correlated with waist circumference, body mass, adipose mass, lean mass, android and gynoid fats. HDL cholesterol was negatively related to waist circumference, body mass, adipose mass, lean mass and android fat. The visual analogue scale of appetite was positively related to depressive mood, sexual problems and hot flashes. Conclusions: There were no statistically significant differences in total body mass, android and gynoid fats, bone mineral content, lean mass, caloric intake, triglycerides and fasting glycemia, possible because at the the beginning of postmenopausal period the possible changes in body composition are not impacting, which allows us to consider this period as a time window of opportunity for early interventions directed to lifestyle, preventing ailments such as atherogenic profile.

The central nervous system (CNS) has a crucial role in the maintenance of energy homeostasis by orchestrating a plethora of signals from peripheral organs about the state of energy stores and the current energy intake needed to match energy expenditure. These signals converge into the hypothalamic regions and its complex local circuitry. CNS-derived cholecystokinin (CCK) is acting at central level to modulate energy balance by regulating the neuronal activity of hypothalamic neuronal populations that regulate food intake, energy storage and consumption. Moreover, our recent published work identifies CCK neurons as key integrators of the neuroendocrine negative feedback of glucocorticoids to the PVN. Glucose sensing neurons of the Ventromedial Hypothalamus (VMH) are integrating energy signals and are essential for mounting a counter-regulatory response and glucose homeostasis. VMH is also important in energy expenditure by regulating body weight and thermogenesis. CCK neurons are present in high density in the VMH.The source of endogenous CCK that acts on distinct neuronal components has not been elucidated. The research so far does not address the purpose of CCK neurons in the hypothalamus and their potential role in the network dynamics regarding energy homeostasis. In this study, we untangle the role of CCK neurons in the VMH nucleus by employing stereotactic intracranial delivery of adeno-associated viruses that result in cell-type specific chemogenetic inhibition or ablation of these neurons. Acute silencing of their neurotransmission with the cre-dependent AAV expression of the chemogenetic tool of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) increases their daily food intake due to increased meal numbers and eating frequency without meal size or meal duration being affected. CCK ablation by a newly generated double-recombinase-mediated Diphtheria Toxin Receptor (DTR) mouse line or AAV-DTA-mediated ablation resulted in hyperphagia, obesity and hyperglycaemia. We conclude that CCK^VMH neurons are implicated in the regulation of food intake, body weight and glucose homeostasis in the adult brain.

When most omnivores and herbivores become sodium depleted they engage in the motivated behavior of sodium appetite (AKA salt appetite), or the seeking out and ingestion of salty substances. Sodium appetite is associated with psychological processes that serve to enhance the incentive and rewarding value of salty substances in order to attract animals to salty substances and reinforce the ingestion of them. The experience of sodium depletion also produces long-lasting changes in behavior; one of the most apparent changes being a seemingly life-long increase in hypertonic salt intake which indicates sodium appetite is sensitized. Two neural circuits have been implicated in the sensitization of sodium appetite: 1) a forebrain neural circuit that regulates body fluid homeostasis, and 2) the mesolimbic dopamine system which mediates motivated behaviors. This dissertation has three aims that serve the overall purpose of providing a better understanding of the neurobiological mechanisms that mediate the sensitization of sodium appetite. The first aim is to develop a model of sodium depletion that is amenable to pharmacological manipulation in order to determine whether the -blockade of N-methyl-d-aspartate receptors, which are critical for neural plasticity, will prevent the sensitization of sodium appetite. The second aim is to determine whether sensitization is associated with relatively long-term molecular changes in forebrain areas that regulate body fluid homeostasis. The third aim is to identify how forebrain areas involved in body fluid homeostasis may connect to and influence activity in the mesolimbic dopamine system.

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