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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.

The role of gut hormones in the regulation of appetite and food intake is well established. The studies presented within this thesis have examined the effects of exercise and different environmental conditions on gut hormones (acylated ghrelin and total peptide YY), appetite and food intake. Forty-two young (mean ± SEM; 22.6 ± 0.4 y), healthy and generally lean (body mass index 23.7 ± 0.3 kg m2) males were recruited into four studies. In study one, 60 minutes of high intensity (70 % of O2 max) running and cycling exercise suppressed concentrations of the appetite-stimulating hormone acylated ghrelin to a similar extent. Study two revealed that after 60 minutes running in the heat (30 °C), hunger is lower in the pre-prandial period, and energy intake lower over the 7 h trial duration compared with a similar trial conducted in temperate (20 °C) conditions. Acylated ghrelin was suppressed during running in the temperate and hot environment but this did not appear to mediate the lower energy intake observed during the hot trial. In study three, energy intake tended to be higher after 60 minutes running in a cool environment (10 °C) compared with a temperate (20 °C) environment. During and shortly after running in the cold, perceived ratings of fullness and satisfaction were lower. Acylated ghrelin concentrations appeared to be suppressed to a lesser extent during running in the cold which could mediate the elevated energy intake observed at the first meal. However, energy intake was also higher at the second meal in the cold trial when acylated ghrelin concentrations were higher in the temperate trial. Study four showed that energy intake and acylated ghrelin concentrations were lower, and total PYY tended to be lower, in normobaric hypoxia suggesting a possible role for acylated ghrelin, but not PYY, in mediating the decrease in energy intake observed in hypoxia. This thesis confirms that exercise transiently suppresses acylated ghrelin concentrations regardless of the environmental conditions (temperature and altitude) exercise is performed in. The findings support anecdotal reports that appetite and energy intake are suppressed in the heat and stimulated in the cold. These responses may be partly mediated by acylated ghrelin immediately after running but other mechanisms are likely involved thereafter. Acute hypoxic exposure suppresses acylated ghrelin concentrations; an observation which may explain the decreased energy intake in hypoxia.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A obesidade é um dos maiores problemas de saúde pública que cresce em todo o mundo, resultante de um desequilíbrio entre ingestão alimentar e gasto energético. O aumento da adiposidade leva ao desenvolvimento de alterações funcionais. Pode-se dizer que a obesidade é o principal fator de risco para o desenvolvimento de doenças crônicas de maior prevalência como dislipidemias, doenças cardiovasculares e diabetes do tipo 2, acarretando na redução da qualidade e expectativa de vida. A Grelina é um hormônio sintetizado pelo estômago, que atua em diferentes tecidos através de um receptor específico (GHS-R1a), incluindo hipotálamo e tecido adiposo. A grelina tem uma ação direta sobre a regulação hipotalâmica da ingestão alimentar, induzindo um efeito orexígeno. Por outro lado, a grelina também modula o armazenamento de energia nos adipócitos. Esta dupla ação sugere que este hormônio pode atuar como uma ligação entre o sistema nervoso central e mecanismos periféricos. Portanto, considerando que a hiperalimentação neonatal induz obesidade na idade adulta por mecanismos desconhecidos, neste estudo foram pesquisados os efeitos da hiperalimentação no início da vida sobre o desenvolvimento da obesidade e, em particular, a sinalização da grelina no tecido adiposo em ratos jovens e adultos. Foram utilizados camundongos Swiss hiperalimentados através do modelo de redução da ninhada. Para induzir a hiperalimentação as ninhadas foram reduzidas a 3 filhotes machos por lactante no 30 dia de vida pós-natal. As ninhadas controles foram ajustadas em 9 filhotes por lactante. Foram avaliados parâmetros antropométricos como: massa corporal e massa do tecido adiposo visceral. A glicemia de jejum foi avaliada utilizando glicosímetro e fitas teste. A análise do conteúdo das proteínas envolvidas na via de sinalização da grelina foram detectadas pelo método de Western Blotting. Os grupos controle (C) e hiperalimentado (H) foram estudados aos 21 e 180 dias de vida. Os dados demonstram que a hipernutrição no início da vida induz um aumento significativo no peso corporal dos camundongos jovens, começando aos 10 dias, e este aumento de peso persistiu até à idade adulta (180 dias de idade). A glicemia e o peso da gordura visceral foram significativamente maiores no grupo hiperalimentado aos 21 e 180 dias, quando comparado com o grupo controle. Os níveis plasmáticos de grelina acilada apresentaram uma redução de 70% nos animais jovens e 49% adultos obesos. Além disso, no tecido adiposo branco, observamos um maior conteúdo (242%) do receptor de grelina (GHSR1a) nos animais hiperalimentados com 21 dias, e este aumento foi associado à modulação positiva do conteúdo e fosforilação de proteínas envolvidas no estoque e utilização de energia celular, tais como AKT, PI3K, AMPK, GLUT-4, e CPT1. No entanto, ao chegar à idade adulta os animais hiperalimentados não apresentaram diferença significativa no conteúdo de GHS-R1a e das proteínas AKT, PI3K, AMPK, GLUT-4, e CPT1. O conteúdo de PPARɣ foi menor no grupo obeso aos 21e 180 dias. Basicamente, mostramos que o metabolismo do tecido adiposo está alterado na obesidade adquirida no início da vida e, provavelmente, devido a essa modificação, ocorre um novo padrão da via de sinalização da grelina. / Obesity is a major public health problem that is growing worldwide, due to an imbalance between food intake and energy expenditure is a major risk factor for the development of most prevalent chronic diseases as dyslipidemias, heart disease and type 2 diabetes, resulting in reduced quality and life expectancy. Ghrelin is a hormone synthesized by the stomach that acts in different tissues via a specific receptor (GHS-R1a), including hypothalamus and adipose tissue. For instance, recent reports have shown that ghrelin has a direct action on hypothalamic regulation of food intake mainly inducing an orexigenic effect. On the other hand, ghrelin also modulates energy stores and expenditure in the adipocytes. This dual action has suggested that this hormone may act as a link between the central nervous system and peripheral mechanisms. Furthermore, concerning nutritional disorders, it has been suggested that obesity may be considered an impairment of the above cited link. Therefore, considering that neonatal overfeeding induces obesity in adulthood by unknown mechanisms, in this study we examined the effects of early life overnutrition on the development of obesity and in particular on adipose tissue ghrelin signaling in young mice. Our data demonstrated that overnutrition during early life induces a significant increase in body weight of young mice, starting at 10 days, and this increase in weight persisted until adulthood (180 days of age). In these animals, blood glucose and visceral fat weight were found higher at 21 and 180 days when compared to the control group. Acylated ghrelin circulating levels were found lower in the young obese pups and adult obese mice. In addition, in white adipose tissue ghrelin receptor (GHS-R1a) expression increased and was associated to positive modulation of content and phosphorylation of proteins involved in cell energy store and use as AKT, PI3K, AMPK, GLUT-4, and CPT1. However adulthood overfeeding animals showed no significant difference in the content of GHS-R1a and protein AKT, PI3K, AMPK, GLUT-4, and CPT1. PPARγ content decreased in obese group at 21 and 180 days. Basically, we showed that adipose tissue metabolism is altered in early life acquired obesity and probably due to such modification a new pattern of ghrelin signaling pathway takes place.

The research presented in this thesis relates to the gastrointestinal control of appetite and some of the consequences of nutritional frailty, namely postprandial hypotension and vitamin D insufficiency. Undernutrition and its consequences are increasingly common problems in an ageing population, and improved management is dependent on an understanding of the factors which are involved in the control of appetite, and the physiological decline of appetite with increasing age termed ‘the anorexia of ageing’. The role of the gastrointestinal hormone ghrelin was specifically evaluated, in relation to the effects of age and nutrient digestion on circulating ghrelin concentrations (Chapters 6 and 7). The effect of fat digestion on the postprandial blood pressure response in healthy older subjects was evaluated in the study reported in Chapter 8. In addition, the results of some intervention studies are described in Chapters 9 and 10, the former study relating to nutritional supplementation as a strategy to increase energy intake, and the latter study to the effects of vitamin D replacement therapy on glucose and insulin metabolism. Whilst plasma ghrelin concentrations are less in older than young rodents, the consequences of healthy ageing on circulating plasma ghrelin concentrations in humans are unclear. The variations in fasting ghrelin concentrations over a sixty year age range were evaluated in healthy young and older subjects (Chapter 6). Plasma ghrelin concentrations were higher in females than males, but did not correlate with age, and were inversely related to body mass index. Ghrelin was independently, and inversely, related to total body skeletal muscle mass, but not to any other body composition variable. Strategies for increasing muscle mass, through resistance exercises, may, accordingly, aid in abolishing the compensatory rise in ghrelin concentrations seen with undernutrition and weight loss. Plasma ghrelin concentrations increase before, and decrease to trough levels within one hour of ingestion of a meal. Macronutrients differ in their ability to suppress ghrelin, being earlier and more pronounced after carbohydrate, and relatively delayed after fat or protein, ingestion. The role of carbohydrate and fat digestion in the suppression of plasma ghrelin concentrations was investigated in healthy young adults (Chapter 7). The suppression of ghrelin concentrations following a sucrose drink was attenuated by acarbose, which slows small intestinal carbohydrate absorption. Ghrelin concentrations were also suppressed after consumption of a fat-enriched drink, however addition of orlistat, which reduces fat digestion and absorption, attenuated the fall in plasma ghrelin. Thus, nutrient digestion is required, in addition to exposure of the small intestine to nutrients, for suppression of ghrelin. Postprandial hypotension describes a significant fall in blood pressure occurring up to two hours after a meal. The magnitude of the fall in postprandial blood pressure depends, in part, on the macronutrient composition of a meal, and the effects are particularly discernable in older adults. Although carbohydrates are particularly potent in reducing postprandial blood pressure in older adults, fat ingestion appears to have comparable, but delayed effects. The role of fat digestion in modifying the blood pressure responses was evaluated in healthy older adults (Chapter 8). There was a fall in blood pressure after ingestion of a high-fat drink. Orlistat, a lipase inhibitor which reduces intestinal fat absorption, potentiated the fall in postprandial blood pressure after a fat-enriched drink. Gastrointestinal function and appetite can be modulated by dietary manipulation of the macronutrient composition of an individual’s diet. The intervention study described in Chapter 9 evaluated the effects of two weeks of dietary fat supplementation on the sensitivity to the satiating effects of intravenous cholecystokinin-8 in healthy older subjects. No differences were observed in fasting, or postprandial plasma cholecystokinin concentrations after the dietary supplementation period compared to regular diet. There were also no differences in spontaneous energy intake at a buffet meal in response to exogenously administered cholecystokinin between the two diet periods. Vitamin D deficiency is common, as is type 2 diabetes, and the two conditions may be linked. There is mounting evidence linking vitamin D deficiency with abnormalities of glucose and insulin metabolism. The effects of vitamin D therapy in healthy young and older adults with low vitamin D concentrations in the setting of normal or impaired glucose tolerance were evaluated (Chapter 10). Vitamin D therapy, which normalised serum 25-hydroxyvitamin D concentrations in these individuals, did not alter glucose or insulin concentrations or insulin sensitivity during an oral glucose tolerance test. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339020 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

In den nächsten Jahren wird weltweit ein starker Anstieg des Anteils Übergewichtiger an der Bevölkerung zu verzeichnen sein. Daraus resultiert auch ein Anstieg der Adipositas assoziierten Krankheiten und der damit verbundenen medizinischen und volkswirtschaftlichen Konsequenzen. Im Hinblick darauf sind Untersuchungen zur Klärung der Regulation des Körpergewichtes und der Entstehung von Übergewicht von grundlegender Bedeutung. Die vorliegende Promotionsarbeit liefert einen Beitrag zur Erforschung dieser Regulationsmechanismen des Körpergewichts, die aus einem komplexen Netzwerk hormoneller Signalwege bestehen. Dem Peptidhormon Obestatin, das wahrscheinlich an den Rezeptor GPR39 bindet, wurde eine gewichtsreduzierende Wirkung zugeschrieben. Ziel dieser Arbeit war es deshalb, die Wirkungsweise dieses Hormons in Bezug auf die Proliferation und die Differenzierung im (Prä-)Adipozytenmodell 3T3-L1 näher zu untersuchen. Der Einfluss von Obestatin in Abhängigkeit von seiner Konzentration auf diese murinen, embryonalen Zellen wurde zum einen mittels colorimetrischem Proliferationstest untersucht. Zum anderen wurde die Aktivität der MAP-Kinase ERK1/2, Schlüsselenym der Zellproliferation und Zelldifferenzierung, während der Proliferation mittels in situ ELISA und SDS-Page/Western Blot bestimmt. Der Einfluss von Obestatin auf die Differenzierung der 3T3-L1 Zellen wurde anhand ihres Differenzierungsgrad mit Hilfe des lipophilen Farbstoffes AdipoRed evaluiert. Darüberhinaus erfolgte mittels quantitativer PCR die Bestimmung der Expression des Obestatinrezeptor-Gens GPR39 in humanem Fettgewebe. Ausgehend von der Annahme, dass durch eine autogene Regulation die Rezeptorenanzahl in Abhängigkeit vom Körpergewicht variiert wird, wurde der statistische Zusammenhang zwischen der Expression des hGPR39-Rezeptor-Gens und dem Alter bzw. Body-Mass-Index von Patienten untersucht, denen Proben von viszeralem oder subkutanem Fett entnommen wurden. In Zusammenschau aller Versuchsergebnisse, die in dieser Dissertationsarbeit erzielt wurden, kann davon ausgegangen werden, dass das hier verwendete Obestatin insgesamt keinen Effekt auf das Adipozytenmodell 3T3-L1 besitzt, weil ein Substrat verwendet wurde, das wohl eine zu geringe Konzentration des Hormons Obestatin enthielt. Durch neue Forschungsergebnisse anderer Arbeitsgruppen muss vermutet werden, dass der tatsächliche Anteil von Obestatin in nicht speziell aufbereiteten Substraten zu gering ist, um mit den angewendeten Nachweisverfahren signifikante Effekte zu erzielen. Bei den Untersuchungen eines Zusammenhangs zwischen der Expression des hGPR39-Gens und dem BMI wurden die Ergebnisse der 12 Proben mithilfe der univarianten Varianzanalyse (SPSS) statistisch geprüft. Ohne Beachtung eines Ausreißer-Messwertes konnte dabei ein nahezu signifikanter, positiv linearer Zusammenhang zwischen der normierten Kopienzahl des Rezeptor-Gens und dem BMI gezeigt werden. Sollte durch zusätzliche Experimente dieser Zusammenhang Signifikanz erlangen, schiene es denkbar, dass Obestatin nicht – wie bisher angenommen – eine Verminderung der Gewichtszunahme bewirkt, sondern möglicherweise den adipösen Phänotyp und die damit verbundenen Begleit- und Folgekrankheiten befördert.

<p>Abstract</p><p>G-protein coupled receptors (GPCRs) are having the high medical importance since almost half of the medicinal drugs are designed as modulators of receptor molecules. Crystal structure or NMR structures of GPCRs are very difficult to determine because all GPCRs are typically bound to the cell membrane and thus their molecular activation mechanism is still unclear. The recent publication of the crystal structure of the 2-adrenoreceptor will provide new insights in the field of GPCR research.</p><p>Ghrelin is a peptide growth hormone which binds to the growth hormone secretagogue receptor (GHS-R) and stimulates the release of growth hormone. Based on the known ghrelin receptor binding core sequences wFwLL (upper letter and lower letter representative for L-form and D-form of the amino acids respectively), we prepared two novel peptide analogs with terminal S-(2-aminoethylsulfenyl) cysteine residues. These peptides were tested for their ability to suppress the binding of ghrelin to transfected COS7 cell-line (Kidney fibroblast line from the green African monkey) cells expressing the ghrelin wild-type receptor or certain mutants thereof. As a result we observed a significant reduction of the total number of binding sites accessible for ghrelin, which increased with the time the cells were incubated with our test compounds. This observations support our hypothesis that the peptides we tested form a covalent bond with free thiols located closely to the ligand binding-site of the receptor protein by disulfide thiol exchange which is an interesting target for development of anti-obesity drugs.</p>

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O excesso ou a privação de nutrientes em períodos específicos do desenvolvimento, tais como a lactação, estimulam alterações no metabolismo celular, por exemplo. Estas modificações perpetuam-se ao longo da vida e em conseqüência tornam o organismo mais suscetível ao aparecimento de patologias na idade adulta (Programação Metabólica). Estudamos a influência da grelina na secreção de insulina em camundongos Swiss de 120 dias submetidos à hiperalimentação na lactação. Para induzir a hiperalimentação as ninhadas foram reduzidas a 3 filhotes machos por lactante no 3o dia de vida pós-natal. As ninhadas controle foram ajustadas para 9 filhotes machos por lactante. Na idade adulta os animais hiperalimentados (AH) exibiram em comparação aos animais controle (AC) um incremento de 20% no peso corporal, maior índice de Lee (1705,63 g/mm + 29,3 vs 1374,10 g/mm + 54,9; p< 0,001), elevação da gordura corporal (31,0% + 4,6 vs 21,5% + 3,6; p< 0,01), aumento da gordura retroperitoneal (0,79 g + 0,1 vs 0,44 g + 0,1; p< 0,001), hiperglicemia de jejum (151,83 mg/ dl + 8,3 vs 118,0 mg/ dl + 1,0; p< 0,001), hiperinsulinemia de jejum (54,06 UI/ml + 2,3 vs 19,28 UI/ml + 1,53; p< 0,001) e hipogrelinemia de jejum (98,64 pg/ml + 56,5 vs 201,14 pg/ml + 46,4; p< 0,05). Os AH apresentaram maior secreção de insulina in vitro em presença de glicose aos 10 minutos (209,66 UI/ml + 46,5; p< 0,05), 30 minutos (441,88 UI/ml + 30,2; p< 0,05) e 60 minutos (214,34 UI/ml + 29,8) em comparação aos AC, respectivamente 86,90 UI/ml + 9,5; 74,31 UI/ml + 7,7 vs 27,45 UI/ml + 6,1; p< 0,05. As ilhotas pancreáticas dos AH adultos demonstraram em relação aos AC diminuição do consumo de O2 (1,76 pmols O2/ s. ilhota-1 + 0,4 vs 4,85 pmols O2/ s. ilhota-1 + 1,5; p< 0,001) e elevação do conteúdo do receptor de grelina GHSR1A (3,05 % + 2,13 vs 0,95 % + 0,1; p< 0,05). A grelina acilada estimulou a secreção de insulina in vitro dos AC aos 30 minutos (controle com grelina: 208,50 UI/ml + 40,85 vs controle sem grelina: 74,31 UI/ml + 7,7; p< 0,05) e diminuiu a razão do controle respiratório (controle com grelina: 1,45 + 0,2 vs controle sem grelina: 2,51 + 0,7; p< 0,05). Nos AH, a grelina acilada elevou o conteúdo de GLUT2 nas ilhotas pancreáticas em relação aos AC (hiperalimentados com grelina: 2,07 % + 0,5 vs controle com grelina: 0,85 % + 0,4; p< 0,01); entretanto a grelina não foi capaz de estimular a secreção de insulina nestes animais. Concluímos que a hiperalimentação na lactação associou-se ao aumento da gordura corporal e elevou a secreção de insulina na fase tardia do desenvolvimento. A grelina acilada estimulou a secreção de insulina somente nos AC adultos. / Excess or lack of nutrients at specific times of development generates adaptive responses that can change the body causing the onset of chronic diseases in adulthood (Metabolic Programming). We studied the influence of the hormone ghrelin in insulin secretion of adult Swiss mice overfed during lactation. To induce early postnatal overnutrition, the litter size was reduced to 3 pups per litter at the 3rd day after birth. In the control group, the litter size was adjusted to 9 pups per litter. In adulthood, overfed group (OG) had an increase of 20% in body weight compared to control group (CG). OG had increased in Lee index (1705.63 g/mm + 29.3 vs 1374.10 g/mm + 54.9; p< 0.001), high body fat (31.0% + 4.6 vs 21.5% + 3.6; p< 0.01), and an elevated retroperitoneal fat (0.79 g + 0.1 vs 0.44 g + 0.1; p< 0.001), fasting hyperglycemia (151.83 mg/ dl + 8.3 vs 118.0 mg/ dl + 1.0; p< 0.001), high fasting insulinemia (54.06 UI/ml + 2.3 vs 19.28 UI/ml + 1.53; p< 0.001), and low fasting plasma ghrelin (98.64 pg/ml + 56.5 vs 201.14 pg/ml + 46.4; p< 0.05) compared to CG at 120 days. OG exhibited high insulin secretion in vitro at 10 minutes (209.66 UI/ml + 46.5), 30 minutes (441.88 UI/ml + 30.2), and 60 minutes (214.34 UI/ml + 29.8) compared to CG, respectively 86.90 UI/ml + 9.5; 74.31 UI/ml + 7.7 vs 27.45 UI/ml + 6.1; p< 0.05. Pancreatic islets from OG had a decrease of O2 consumption compared to CG (1.76 pmols O2/ s. islets-1 + 0.4 vs 4.85 pmols O2/ s. islets-1 + 1.5; p< 0.001) and an increased of GHSR1A content (3.05 % + 2.13 vs 0.95 % + 0.1; p< 0.05). Acylated ghrelin increased control groups insulin secretion in vitro at 30 minutes (CG with ghrelin: 208.50 UI/ml + 40.85 vs CG without ghrelin 74.31 UI/ml + 7,7; p< 0.05) and decreased the respiratory control ration (CG with ghrelin: 1.45 + 0.2 vs CG without ghrelin: 2.51 + 0.7; p< 0.05). Also acylated ghrelin increased the GLUT2 content of pancreatic islets from OG compared to CG, respectively 2.07 % + 0.5 vs 0.85 % + 0.4; p< 0.01; however acylated ghrelin was not able to stimulate insulin secretion in OG. We conclude that the overnutrition during lactation is associated with increased body fat percentage and changes in pancreatic &#946; cells in the late stage of development. Acylated ghrelin stimulated insulin secretion only in adult CG.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O excesso ou a privação de nutrientes em períodos específicos do desenvolvimento, tais como a lactação, estimulam alterações no metabolismo celular, por exemplo. Estas modificações perpetuam-se ao longo da vida e em conseqüência tornam o organismo mais suscetível ao aparecimento de patologias na idade adulta (Programação Metabólica). Estudamos a influência da grelina na secreção de insulina em camundongos Swiss de 120 dias submetidos à hiperalimentação na lactação. Para induzir a hiperalimentação as ninhadas foram reduzidas a 3 filhotes machos por lactante no 3o dia de vida pós-natal. As ninhadas controle foram ajustadas para 9 filhotes machos por lactante. Na idade adulta os animais hiperalimentados (AH) exibiram em comparação aos animais controle (AC) um incremento de 20% no peso corporal, maior índice de Lee (1705,63 g/mm + 29,3 vs 1374,10 g/mm + 54,9; p< 0,001), elevação da gordura corporal (31,0% + 4,6 vs 21,5% + 3,6; p< 0,01), aumento da gordura retroperitoneal (0,79 g + 0,1 vs 0,44 g + 0,1; p< 0,001), hiperglicemia de jejum (151,83 mg/ dl + 8,3 vs 118,0 mg/ dl + 1,0; p< 0,001), hiperinsulinemia de jejum (54,06 UI/ml + 2,3 vs 19,28 UI/ml + 1,53; p< 0,001) e hipogrelinemia de jejum (98,64 pg/ml + 56,5 vs 201,14 pg/ml + 46,4; p< 0,05). Os AH apresentaram maior secreção de insulina in vitro em presença de glicose aos 10 minutos (209,66 UI/ml + 46,5; p< 0,05), 30 minutos (441,88 UI/ml + 30,2; p< 0,05) e 60 minutos (214,34 UI/ml + 29,8) em comparação aos AC, respectivamente 86,90 UI/ml + 9,5; 74,31 UI/ml + 7,7 vs 27,45 UI/ml + 6,1; p< 0,05. As ilhotas pancreáticas dos AH adultos demonstraram em relação aos AC diminuição do consumo de O2 (1,76 pmols O2/ s. ilhota-1 + 0,4 vs 4,85 pmols O2/ s. ilhota-1 + 1,5; p< 0,001) e elevação do conteúdo do receptor de grelina GHSR1A (3,05 % + 2,13 vs 0,95 % + 0,1; p< 0,05). A grelina acilada estimulou a secreção de insulina in vitro dos AC aos 30 minutos (controle com grelina: 208,50 UI/ml + 40,85 vs controle sem grelina: 74,31 UI/ml + 7,7; p< 0,05) e diminuiu a razão do controle respiratório (controle com grelina: 1,45 + 0,2 vs controle sem grelina: 2,51 + 0,7; p< 0,05). Nos AH, a grelina acilada elevou o conteúdo de GLUT2 nas ilhotas pancreáticas em relação aos AC (hiperalimentados com grelina: 2,07 % + 0,5 vs controle com grelina: 0,85 % + 0,4; p< 0,01); entretanto a grelina não foi capaz de estimular a secreção de insulina nestes animais. Concluímos que a hiperalimentação na lactação associou-se ao aumento da gordura corporal e elevou a secreção de insulina na fase tardia do desenvolvimento. A grelina acilada estimulou a secreção de insulina somente nos AC adultos. / Excess or lack of nutrients at specific times of development generates adaptive responses that can change the body causing the onset of chronic diseases in adulthood (Metabolic Programming). We studied the influence of the hormone ghrelin in insulin secretion of adult Swiss mice overfed during lactation. To induce early postnatal overnutrition, the litter size was reduced to 3 pups per litter at the 3rd day after birth. In the control group, the litter size was adjusted to 9 pups per litter. In adulthood, overfed group (OG) had an increase of 20% in body weight compared to control group (CG). OG had increased in Lee index (1705.63 g/mm + 29.3 vs 1374.10 g/mm + 54.9; p< 0.001), high body fat (31.0% + 4.6 vs 21.5% + 3.6; p< 0.01), and an elevated retroperitoneal fat (0.79 g + 0.1 vs 0.44 g + 0.1; p< 0.001), fasting hyperglycemia (151.83 mg/ dl + 8.3 vs 118.0 mg/ dl + 1.0; p< 0.001), high fasting insulinemia (54.06 UI/ml + 2.3 vs 19.28 UI/ml + 1.53; p< 0.001), and low fasting plasma ghrelin (98.64 pg/ml + 56.5 vs 201.14 pg/ml + 46.4; p< 0.05) compared to CG at 120 days. OG exhibited high insulin secretion in vitro at 10 minutes (209.66 UI/ml + 46.5), 30 minutes (441.88 UI/ml + 30.2), and 60 minutes (214.34 UI/ml + 29.8) compared to CG, respectively 86.90 UI/ml + 9.5; 74.31 UI/ml + 7.7 vs 27.45 UI/ml + 6.1; p< 0.05. Pancreatic islets from OG had a decrease of O2 consumption compared to CG (1.76 pmols O2/ s. islets-1 + 0.4 vs 4.85 pmols O2/ s. islets-1 + 1.5; p< 0.001) and an increased of GHSR1A content (3.05 % + 2.13 vs 0.95 % + 0.1; p< 0.05). Acylated ghrelin increased control groups insulin secretion in vitro at 30 minutes (CG with ghrelin: 208.50 UI/ml + 40.85 vs CG without ghrelin 74.31 UI/ml + 7,7; p< 0.05) and decreased the respiratory control ration (CG with ghrelin: 1.45 + 0.2 vs CG without ghrelin: 2.51 + 0.7; p< 0.05). Also acylated ghrelin increased the GLUT2 content of pancreatic islets from OG compared to CG, respectively 2.07 % + 0.5 vs 0.85 % + 0.4; p< 0.01; however acylated ghrelin was not able to stimulate insulin secretion in OG. We conclude that the overnutrition during lactation is associated with increased body fat percentage and changes in pancreatic &#946; cells in the late stage of development. Acylated ghrelin stimulated insulin secretion only in adult CG.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A obesidade é um dos maiores problemas de saúde pública que cresce em todo o mundo, resultante de um desequilíbrio entre ingestão alimentar e gasto energético. O aumento da adiposidade leva ao desenvolvimento de alterações funcionais. Pode-se dizer que a obesidade é o principal fator de risco para o desenvolvimento de doenças crônicas de maior prevalência como dislipidemias, doenças cardiovasculares e diabetes do tipo 2, acarretando na redução da qualidade e expectativa de vida. A Grelina é um hormônio sintetizado pelo estômago, que atua em diferentes tecidos através de um receptor específico (GHS-R1a), incluindo hipotálamo e tecido adiposo. A grelina tem uma ação direta sobre a regulação hipotalâmica da ingestão alimentar, induzindo um efeito orexígeno. Por outro lado, a grelina também modula o armazenamento de energia nos adipócitos. Esta dupla ação sugere que este hormônio pode atuar como uma ligação entre o sistema nervoso central e mecanismos periféricos. Portanto, considerando que a hiperalimentação neonatal induz obesidade na idade adulta por mecanismos desconhecidos, neste estudo foram pesquisados os efeitos da hiperalimentação no início da vida sobre o desenvolvimento da obesidade e, em particular, a sinalização da grelina no tecido adiposo em ratos jovens e adultos. Foram utilizados camundongos Swiss hiperalimentados através do modelo de redução da ninhada. Para induzir a hiperalimentação as ninhadas foram reduzidas a 3 filhotes machos por lactante no 30 dia de vida pós-natal. As ninhadas controles foram ajustadas em 9 filhotes por lactante. Foram avaliados parâmetros antropométricos como: massa corporal e massa do tecido adiposo visceral. A glicemia de jejum foi avaliada utilizando glicosímetro e fitas teste. A análise do conteúdo das proteínas envolvidas na via de sinalização da grelina foram detectadas pelo método de Western Blotting. Os grupos controle (C) e hiperalimentado (H) foram estudados aos 21 e 180 dias de vida. Os dados demonstram que a hipernutrição no início da vida induz um aumento significativo no peso corporal dos camundongos jovens, começando aos 10 dias, e este aumento de peso persistiu até à idade adulta (180 dias de idade). A glicemia e o peso da gordura visceral foram significativamente maiores no grupo hiperalimentado aos 21 e 180 dias, quando comparado com o grupo controle. Os níveis plasmáticos de grelina acilada apresentaram uma redução de 70% nos animais jovens e 49% adultos obesos. Além disso, no tecido adiposo branco, observamos um maior conteúdo (242%) do receptor de grelina (GHSR1a) nos animais hiperalimentados com 21 dias, e este aumento foi associado à modulação positiva do conteúdo e fosforilação de proteínas envolvidas no estoque e utilização de energia celular, tais como AKT, PI3K, AMPK, GLUT-4, e CPT1. No entanto, ao chegar à idade adulta os animais hiperalimentados não apresentaram diferença significativa no conteúdo de GHS-R1a e das proteínas AKT, PI3K, AMPK, GLUT-4, e CPT1. O conteúdo de PPAR&#611; foi menor no grupo obeso aos 21e 180 dias. Basicamente, mostramos que o metabolismo do tecido adiposo está alterado na obesidade adquirida no início da vida e, provavelmente, devido a essa modificação, ocorre um novo padrão da via de sinalização da grelina. / Obesity is a major public health problem that is growing worldwide, due to an imbalance between food intake and energy expenditure is a major risk factor for the development of most prevalent chronic diseases as dyslipidemias, heart disease and type 2 diabetes, resulting in reduced quality and life expectancy. Ghrelin is a hormone synthesized by the stomach that acts in different tissues via a specific receptor (GHS-R1a), including hypothalamus and adipose tissue. For instance, recent reports have shown that ghrelin has a direct action on hypothalamic regulation of food intake mainly inducing an orexigenic effect. On the other hand, ghrelin also modulates energy stores and expenditure in the adipocytes. This dual action has suggested that this hormone may act as a link between the central nervous system and peripheral mechanisms. Furthermore, concerning nutritional disorders, it has been suggested that obesity may be considered an impairment of the above cited link. Therefore, considering that neonatal overfeeding induces obesity in adulthood by unknown mechanisms, in this study we examined the effects of early life overnutrition on the development of obesity and in particular on adipose tissue ghrelin signaling in young mice. Our data demonstrated that overnutrition during early life induces a significant increase in body weight of young mice, starting at 10 days, and this increase in weight persisted until adulthood (180 days of age). In these animals, blood glucose and visceral fat weight were found higher at 21 and 180 days when compared to the control group. Acylated ghrelin circulating levels were found lower in the young obese pups and adult obese mice. In addition, in white adipose tissue ghrelin receptor (GHS-R1a) expression increased and was associated to positive modulation of content and phosphorylation of proteins involved in cell energy store and use as AKT, PI3K, AMPK, GLUT-4, and CPT1. However adulthood overfeeding animals showed no significant difference in the content of GHS-R1a and protein AKT, PI3K, AMPK, GLUT-4, and CPT1. PPAR&#947; content decreased in obese group at 21 and 180 days. Basically, we showed that adipose tissue metabolism is altered in early life acquired obesity and probably due to such modification a new pattern of ghrelin signaling pathway takes place.