Orientadores: José Rodrigo Pauli, Dennys Esper Corrêa Cintra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-26T10:24:59Z (GMT). No. of bitstreams: 1 Rodrigues_BarbaradeAlmeida_M.pdf: 2531599 bytes, checksum: 8ab5a3a29ab7a93465c0db3ffb7c5f4e (MD5) Previous issue date: 2014 / Resumo: A sinalização da insulina no hipotálamo desempenha papel crucial na manutenção da massa corporal. Uma vez ativada, a proteína Akt aumenta a fosforilação do fator de transcrição FoxO1, que é um importante mediador da sinalização insulina no hipotálamo e estimula a transcrição de neuropeptídeos anorexigênicos. Nesse contexto, devido a capacidade de se ligar diretamente a proteína quinase B (Akt), a Mammalian Homolog of Drosophila Tribbles 3 (TRB3) torna-se uma importante molécula de regulação da sinalização da insulina. Em adição, a sinalização do estresse de retículo endoplasmático (estresse de RE) via proteína quinase de retículo endoplasmático (pPERK) e um fator de transcrição mediador de apoptose denominado CHOP, é considerado como mecanismo responsável pela super-expressão de TRB3 em roedores obesos e diabéticos. Por outro lado, o exercício físico é considerado uma das mais importantes estratégias não farmacológicas para a prevenção e/ou tratamento da obesidade. O exercício é capaz de proteger contra a inflamação, o estresse de RE e aumentar a sinalização da insulina no hipotálamo de roedores obesos. Entretanto, os efeitos do exercício agudo na modulação da TRB3 não foram descritos. O objetivo do presente estudo foi investigar o papel do exercício agudo de natação sobre a expressão da TRB3 e associação TRB3/Akt no hipotálamo de roedores obesos. Foram utilizados ratos wistar divididos em três grupos: Lean (controle magro), alimentados com uma ração padrão para roedores, DIO (alimentados com uma dieta rica em gordura), e DIO-EXE (submetidos a um protocolo de natação aguda de exercício). Para reforçar que o aumento da TRB3 está associada com o estresse de RE, e que uma sessão aguda de exercício é capaz de reverter esse aumento, usamos outros três grupos: controle (Lean) recebendo uma infusão de veículo intracerebroventricular (i.c.v.), magros que receberam i.c.v. de thapsigargina (indutor farmaológico de estresse de RE) e magros que receberam i.c.v. de thapsigargina e submetidos a um protocolo de exercício agudo de natação. O protocolo de exercício físico consistiu de uma sessão de natação, composto por dois períodos de três horas, com uma pausa de 45 minutos. Previamente a eutanásia dos animais, foram realizadas as análises da glicemia, insulinemia, sensibilidade à insulina e a mensuração da ingestão alimentar no período de 12h. Quatro horas após a última sessão de exercício, o tecido hipotalâmico foi removido para análise de proteínas de interesse através das técnicas de imunoprecipitação, imunoblot e de PCR em tempo real. Como resultado, verificou-se que o exercício reduziu os níveis de proteína TRB3 e a interação entre a proteína TRB3 e Akt, aumentou a fosforilação de FoxO1 e restaurou os efeitos anorexígenos de insulina no hipotálamo de ratos DIO-EXE. Além disso, nossos resultados revelaram que a i.c.v. de thapsigargina bloqueou os efeitos anorexígenos mediados por insulina em ratos magros, e o exercício físico restaurou a ação da insulina e a fome, assim, os efeitos supressivos do exercício agudo sobre os níveis de proteína TRB3 pode ser relacionada, no mínimo em parte, pela redução do estresse de RE em neurônios hipotalâmicos / Abstract: Hypothalamic insulin signalling plays an important role in the maintenance of body weight. Once activated, Akt increases the phosphorylation of FoxO1, that is an important mediator of insulin signalling in the hypothalamus and stimulates the transcription of the orexigenic neuropeptides. In this context, due to the capacity to bind directly to protein kinase B (Akt), Mammalian Homolog of Drosophila Tribbles 3 (TRB3) is emerging as an important player in the regulation of insulin signalling. In addition, endoplasmatic reticulum (ER) stress signaling (pPERK and CHOP pathway) has been considered the main mechanism responsible by TRB3 overexpression in obese and diabetic rodent. On the other hand, the exercise is considered one of the most important non-pharmacological strategies to prevent and/or treat obesity. The exercise is able to protect against inflammation and ER stress and to increase insulin signaling in the hypothalamus of obese rodents. However, the effects of acute exercise on TRB3 modulation were not described. Thus, the main aim of the present study was to investigate the role of an acute swimming exercise on the hypothalamic TRB3 expression and the TRB3/Akt association in obese rodents. Male Wistar rats were divided randomly into three groups: control (lean) - fed with a standard rodent chow -, DIO - fed with a high fat diet -, and DIO submitted to a swimming acute exercise protocol (DIO-EXE). In order to reinforce that the TRB3 increase is associated with ER stress and that an acute session of exercise is able to reverse this increase, we used other three groups: control (lean) receiving an intracerebroventricular (i.c.v.) infusion of vehicle, lean receiving an i.c.v. infusion of thapsigargin (an ER stress inducer), and lean receiving an i.c.v infusion of thapsigargin and performed a swimming acute exercise protocol. The physical exercise protocol consisted of a swimming acute session, composed of two periods of three-hours with a break of forty-five minutes. Previously the euthanasia of the animals were performed the analyses of the glycaemia and insulinemia serum, insulin sensitivity through of insulin tolerance test and food intake after i.c.v. insulin injection. Four hours after the last exercise session and anesthesia procedures, the hypothalamic tissue was removed for analysis of the interested proteins through of immunoblotting, immunoprecipitation and PCR real time techniques. In this study, we demonstrated that the exercise reduced the TRB3 protein levels and disrupted the interaction between TRB3 and Akt protein, increased the phosphorylation of Foxo1 and restored the anorexigenic effects of insulin in the hypothalamus of DIO rats. Additionally, our results revealed that intrahypothalamic infusion of thapsigargin blocked the anorexigenic effects mediated by insulin in lean rats and physical exercise restored the suppressive action of insulin on appetite, Thus, the suppressive effects of acute exercise on TRB3 protein levels may be related, at least in part, to the decrease of endoplasmatic reticulum stress in the hypothalamic cells / Mestrado / Metabolismo e Biologia Molecular / Mestra em Ciências da Nutrição e do Esporte e Metabolismo

A vitamina K é um co-fator importante no processo de coagulação sanguínea e recentemente estudos têm demonstrado os benefícios de sua ingestão sobre o metabolismo ósseo. Dessa forma, a atual recomendação de ingestão dietética para esta vitamina vem sendo questionada, pois se acredita que o requerimento da mesma para a função óssea é muito mais alto do que os necessários para manter a homeostase sanguínea. No Brasil, poucos estudos avaliaram a ingestão de vitamina K em populações saudáveis. Assim, o principal objetivo deste trabalho é avaliar a ingestão de vitamina K em adultos e idosos saudáveis. Foi realizado um estudo transversal com a participação de 173 indivíduos de ambos os sexos. A ingestão dietética de cada participante foi avaliada através da aplicação de três recordatórios 24h não consecutivos. As variabilidades intra e interpessoal foram avaliadas utilizando o software PC-SIDE. Para verificar qual grupo de alimentos que mais contribuiu para ingestão de vitamina K foi feita uma análise dos componentes principais. Os adultos representaram 49,7% da amostra e 65, 3% da população eram do gênero feminino. A média de ingestão de vitamina K na amostra foi de 110,7 µg/dia, com uma mediana de 99 µg/dia, os idosos foram os que apresentaram maiores valores de ingestão com uma mediana de 104 µg/ dia. A ingestão de vitamina K através do consumo de gorduras foi estatisticamente diferente entre as faixas etárias, sendo esta diferença mais acentuada entre adultos jovens e idosos (p=0, 006). O consumo de vitamina K entre adultos e idosos apresentou diferença estatística (p=0,00) e o consumo de hortaliças apresentou forte correlação com o consumo de vitamina K total. Logo, conclui-se que independentemente da faixa etária e do sexo, a ingestão de vitamina K foi insuficiente em quase metade da amostra, o que pode ocasionar um maior risco de inadequação dietética. Isso é preocupante, pois pode acarretar problemas ósseos no futuro e/ou agravar os já presentes quando se trata da população idosa. / Vitamin K is an important cofactor in the blood coagulation process and recent researches have demonstrated benefits of vitamin K intake on bone metabolism. Thus, a current dietary recommendation for this vitamin has been questioned; it is believed that the requirement of this vitamin for this bone function is much higher than that necessary to maintain blood homeostasis. In Brazil, few studies evaluated the intake of vitamin K in healthy populations. Therefore, the aim of this study is to evaluate the intake of vitamin K in healthy adults and the elderly. A cross-sectional study involving 173 subjects of both sexes was conducted. The dietary intake of each participant was evaluated by applying three non-consecutive 24h recalls. The within- to between-person variability was evaluated using the PC-SIDE software. To determine which food group most contributed to the intake of vitamin K, an analysis of the major components was performed. Adults represented 49.7% of the subjects and 65.3% of the populations were female. The mean intake of vitamin K in the sample was 110.7 mg/day and median of 99 mg/day; the elderly presented the highest values with median intake of 104 mg/day. Vitamin K intake by consuming fats differ statistically between age groups; this difference was more pronounced among young adults and the elderly (p=0.006). The intake of vitamin K among adults and the elderly showed a statistical difference (p=0.00) and vegetable intake showed a strong correlation with the total intake of vitamin K. It can thus be concluded that independently of age and gender, the intake of vitamin K was insufficient in nearly half of the subjects, which can cause an increased risk of dietary inadequacy. This is worrying because it could cause bone disorders in the future and/or exacerbate those already present when it comes to the elderly population.

Humans are known as homeothermic endotherms. To ensure thermic balance at rest when exposed to cold, they dispose of two main thermogenic processes: shivering thermogenesis (ST) and non-shivering thermogenesis (NST). ST consists of involuntary muscle contractions and NST represents the component of Hprod that is not ST. While ST is difficult to tolerate, it is not yet known which nutrients and how much are required to stimulate NST and lower ST in the cold. Whether or not food caloric intake has an impact on the relative contribution of ST and NST to total Hprod remains to be determined. Therefore, the purpose of this thesis was 1) to quantify the effects of ingesting two quantities (1507 vs 3015 kJ) of same relative compositions on cold-induced whole-body Hprod and 2) to establish the effects of these two quantities of food on the relative contribution of ST and NST to total Hprod. Five healthy male participants were exposed to a 3h mild cold, using a liquid conditioned suit with water flowing at 15°C (COLD) or 33 °C (CON) for a total of 4 trials. Thermal, metabolic and shivering responses were measured at baseline, before and after shake ingestion. Results demonstrated that Hprod and ST intensity increased in the cold, while no significant differences were found between the ingested shakes at two different caloric equivalents. In addition, ST intensity did not change, which confirmed that NST remained the same between the two conditions. Thus, knowing that the caloric intake will not maximize the thermogenic effects in the cold (i.e. improve the comfort of the individual), is it more advantageous to bring food or additional clothing, for any activity? Clearly, more research on the exact pathways of each processes in the cold with food consumption needs to be made. To that extent, the investigation of the effect of food quality on changes in the thermogenic processes during cold exposure strikes us as a fascinating area for future research.

Objetivo: Evaluar la asociación entre síndrome metabólico (SM) y carga glicémica (CG) de la ingesta alimentaria en niños y adolescentes obesos atendidos en consulta endocrinológica de un hospital de referencia pediátrica en Lima, Perú. Pacientes y método: Estudio transversal en niños y adolescentes obesos (IMC ≥ percentil 95) de 10 a 15 años. Se utilizó los criterios de la International Diabetes Federation (IDF) para la clasificación de SM. Se aplicó recordatorio de 24 horas y se calculó la CG total y por tiempos de comida. Se evaluó la asociación entre los terciles de CG (comparación contra el inferior) y SM usando razones de prevalencia ajustadas (RPa) por variables demográficas, familiares, actividad física y consumo total de carbohidratos en modelos de regresión de Poisson con varianza robusta. Resultados: De 273 niños y adolescentes obesos, 52,4% fueron varones, 94,9% fueron físicamente inactivos y el IMC promedio fue 27,2 kg/m2. La mediana de carga glicémica fue de 213,3 (p25-p75:164,8 – 287,35) y de 2275 kcal (p25-p75: 1851,05 – 3024) de ingesta calórica diaria, dado principalmente por carbohidratos (62%).La prevalencia de SM fue de 22,3%, los componentes más prevalentes fueron la obesidad abdominal (81,7%) y el HDL bajo (63,7%). Se encontró asociación entre el consumo elevado de CG y el riesgo a presentar SM (RPa: 4,5; IC95%:1,3-15,31). Conclusiones: Existe asociación entre el alto consumo de CG y la presencia de SM en niños y adolescentes con obesidad. / Objective: Evaluate the association between metabolic syndrome (MS) and glycemic load (GL) of the food intake in obese children and adolescents attended in endocrinology consultation of a pediatric referral hospital in Lima, Peru. Patients and methods: Cross-sectional study in obese (BMI ≥ 95 percentile) children and adolescents between 10 to 15 years old. MS was classified according to the International Diabetes Federation (IDF). 24- hours recall was applied and calculated the GL total and by meal. The association between the tertiles of GL (comparison against the lower) and MS was found using adjusted prevalence ratios (RPa) for demographic variables, family, physical activity and total carbohydrates intake by Poisson regression models with robust variance. Results: 273 obese children and adolescents were recruited, 52.4% were male and 94.9% were physically inactive and IMC average was 27, 2 kg/m2. The median of GL was 213, 3 (164, 8 – 287, 35: 122, 6) and 2275 kcal (1851, 05 – 3024: 1172, 9) for daily caloric intake, mainly due of carbohydrates (62%). The prevalence of MS was 22, 3%, the components more relevant were abdominal obesity (81,7%) and low HDL (63,7%). The association between a high consumption of CG and the risk having MS (Rpa: 4,5; IC95%: 1,3-15,31) Conclusions: There is an association between the high intake of GL and the presence of MS. / Tesis

La compréhension des mécanismes cérébraux régulant la prise alimentaire est devenue cruciale face à la pandémie mondiale d’obésité. L’ingestion d’aliments est sous le contrôle d’un large réseau de structures cérébrales qui régulent la balance énergétique en régulant l’apport de nourriture et la dépense énergétique. Etant donné l’importance de l’olfaction dans l’appréciation des aliments, il est primordial d’identifier des mécanismes précis liant olfaction et nutrition.Nous avons choisi d’étudier l’activité dans le Bulbe Olfactif principal (BO), la première structure cérébrale qui permet la représentation spatiotemporelle des odeurs. De façon intéressante, des récepteurs aux hormones anorexigènes et orexigènes sont exprimés à la fois dans l’hypothalamus et le BO. Toutefois, l’impact de la leptine (et de l’obésité) sur l’activité du BO in vivo demeure inconnu.Pour évaluer l’action de la leptine et de l’obésité sur la plasticité olfactive, nous avons choisi le modèle murin ob/ob. Nous avons d’abord décrit la répartition spatiale de l’activité dans le BO en réponse à une odeur alimentaire et à une odeur neutre chez ces souris en les comparant à des souris témoins grâce une technique de neuroimagerie fonctionnelle, l’Imagerie par Résonance Magnétique renforcée au Manganèse (MEMRI). Par le développement d’une méthode de comparaison statistique indépendant de l’utilisateur, le SPM (Statistical Parametric Mapping), nous avons mis en évidence une augmentation de l’activité spontanée et de l’activité induite par odeur alimentaire dans le BO des ob/ob. Cependant, l’activité induite par une odeur neutre n’était pas plus élevée. L’injection de leptine a pour résultat d’augmenter le signal MEMRI dans les couches profondes du BO et ne restaure que partiellement l’activité des ob/ob. En quantifiant la neurogenèse dans le BO par injections de BrdU, nous avons mis en évidence une survie accrue des cellules granulaires et périglomerulaires chez les ob/ob.Pour évaluer la dynamique temporelle de l’activité du BO, nous avons enregistré par électrophysiologie les variations de l’activité oscillatoire du potentiel de champ local dans les couches profondes du BO pendant l’apprentissage d’une tâche de discrimination olfactive, le Go/NoGo. L’apparition d’une oscillation lente dite bêta (15-40 Hz) pendant cet apprentissage est un marqueur fonctionnel de l’état du réseau olfactif. Elle est dépendante des retours centrifuges provenant du cortex olfactif, majoritairement du cortex piriforme. Si nous n’avons pas observé de différence importante dans la discrimination olfactive, nous avons en revanche mis en évidence un problème motivationnel chez les animaux ob/ob, qui n’effectuaient la tâche que si la récompense hydrique contenait du sucre. Nos enregistrements montrent une forte augmentation des oscillations bêta chez les souris obèses après apprentissage comparé aux souris témoins. / Olfaction makes possible both the detection and the processing of odors related to food location and palatability. Interestingly, receptors to anorexigen and orexigen hormones and neuropeptides found in the hypothalamus are also expressed in the main olfactory bulb (MOB), suggesting that feeding state has an impact on odor representation. However, neuronal mechanisms linking olfaction, food intake and metabolic disorders are still unclear. In this PhD thesis, we wanted to further understand mechanisms of the olfactory-feeding crosstalk and tested the effects of obesity on olfactory activity in the MOB. Leptin, a peptidergic hormone produced by adipocytes, is a major regulator of the energy balance and inhibits food intake. Ob/ob mice are deficient in leptin from birth and are widely used as a murine model of obesity. We used a functional neuroimaging technique, Manganese Enhanced Magnetic Resonance Imaging (MEMRI), in association with statistical parametric mapping (SPM) to monitor food odor-evoked spatial activity in the MOB of these mice. Using MEMRI, we found that odor-evoked signal is different, both in terms of intensity and localization between lean and ob/ob mice for the spontaneous activity and in response to food odor. However, this is not the case for a neutral odor. Moreover, leptin impacts both spontaneous activity and food odor-evoked signal intensity in both groups. Interestingly leptin strongly activates deep layers (mitral cell layer and mainly granule cell layer) of the MOB. To pinpoint what cellular/molecular mechanisms can be responsible for these changes in the spatial distribution of activity, we analyzed the effects of the lack of leptin on the composition and activity of the MOB cellular network.We quantified bulbar adult neurogenesis and found that 21 days after BrdU injections, a cell birth marker, ob/ob mice showed an increased number of both new periglomerular and granular cells as compared to control, suggesting that leptin regulates new neuron elimination. We also tested whether an inflammation could be occurring in the MOB. Using RTPCR of different neuronal and glial markers we did not observe strong signs of inflammation. Then we probed changes in local network activity in the deep layers of the MOB by recording local field potentials during a Go/NoGo odor discrimination task. We observed that the power of beta oscillations, the functional marker of the olfactory network between the MOB and the olfactory cortex, is way higher in the MOB of ob/ob mice compared to control mice.

Ghrelin analogs in food intake regulation Ghrelin, to date the only known peripherally produced and centrally acting peptide that stimulates food intake, is mainly synthesized in the stomach and acts through growth- hormone-secretagogue receptor (GHS-R). In addition to its orexigenic effect, ghrelin stimulates growth hormone (GH) release, gastric motility and acid secretion. The diverse functions of ghrelin raise the possibility of its clinical application for GH deficiency, eating disorders, and gastrointestinal diseases. Ghrelin agonists could be a promising therapeutics in cachexia occurring at cancer or chronic inflammatory diseases. Octanoylation of Ser3 is crucial for preservation of ghrelin activity. In this study, biological properties (binding to GHS-R, food intake stimulation in mice) of full length and shorter ghrelin analogues with octanoic acid coupled to diaminopropionic acid (Dpr) replacing Ser3 or without octanoic acid were followed. This substitution resulted in a prolonged stability and orexigenic effect of above mentioned ghrelin analogues. Importance of N-terminal part of ghrelin and octanoylation peptide was also confirmed.

Prolactin-releasing peptide (PrRP) is a member of the family of RF-amide peptides. These peptides have typical C-terminal sequence -Arg-Phe-NH2 and similar biological effects. PrRP was discovered as an endogenous ligand of an orphan receptor GPR10 while searching for a factor responsible for a prolactin secretion. This effect was not later confirmed and nowadays, PrRP is mainly considered as an anorexigenic peptide. This is supported by a fact that PrRP and GPR10 deficient mice suffer from hyperphagia and late-onset obesity. Besides GPR10, PrRP is bound to NPFF2 receptor whose endogenous ligand is neuropeptide FF (NPFF). In this study, the PrRP's analogues modified at the N-terminus with fatty acids of different lenghts were tested in vitro on binding and activation MAPK/ERK1/2 signalling pathway. In in vivo experiments on food intake, the central anorexigenic effects of lipidized PrRP-analogues were tested provided their crossing blood brain barrier. Binding studies showed that all analogues bound to rat pituitary RC-4B/C cells with high affinity, analogues containing fatty acid with Ki of one order of magnitude lower than native PrRP. High affinity was also confirmed for binding to cells overexpressing GPR10 receptor and cell membranes with overexpressed NPFF2 receptor. All tested analogues...

Traditional dogma is that men have a higher metabolism compared to women. Because of an increased obesity rate in women, the purpose of this study was to test the hypothesis that compared to males, females will gain more body weight per gram of food consumed. Methods: To test our hypothesis male and female mice were fed either a high or low-fat diet for 20-weeks, while monitoring food intake and body weight. At the end of the 20 week feeding plan, a final weight on the mice was taken, as well as a measurement from the nose to rump of the mouse in order to calculate a BMI. Results: Male mice consistently consumed more food than female mice, regardless of the diet. As expected mice on the high-fat diet gained more weight than mice on the low-fat diet. Further, on the low-fat diet male mice gained more weight than the female mice. Interestingly, there was no difference between the amount of weight gain between male and female mice on the high fat. However, within the same diet, male mice gained more weight per gram of food consumed. On average, male mice gained approximately double the body weight per gram of food consumed compared with female mice (51 vs. 27 mg of body weight per gram of food consumed). Conclusions: We had hypothesized that increased food intake in male mice would primarily account for the differences in body weight between male and female mice. However, contrary to our hypothesis, the data shows that male mice gained more body weight per gram of food consumed. This finding provides a novel hypothesis to explain the mechanism by which, at least in rodents, females are more resistant to obesity. Future studies comparing metabolic rate and physical activity levels will need to be performed to explore this finding further.

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