Les oxydants infusés avec la nutrition parentéral (NP) néonatale induisent une modification du métabolisme des lipides et du glucose, donnant lieu à l’âge adulte à un phénotype de carence énergétique (faible poids, baisse de l’activité physique). L’hypothèse qu’une diète précoce riche en glucose prévient ces symptômes plus tard dans la vie, fut évalué chez le cobaye par un ANOVA en plan factoriel complet à deux facteurs (p < 0:05) : NP du jour 3 à 7, suivit d’une nourriture régulière (chow) (NP+) vs. chow à partir du 3ième jour (NP-), combiné avec une eau de consommation enrichie en glucose (G+) ou non (G-) à partir de la 3ième semaine. Les paramètres suivant ont été mesurés à l’âge de 9 semaine: taux de croissance, activité physique, activité de phosphofructokinase-1 et glucokinase (GK), niveau hépatique de glucose-6-phosphate (G6P), glycogène, pyruvate et potentiel redox du glutathion, poids du foie, glycémie, tolérance au glucose, concentrations hépatiques et plasmatiques en triacylglycérides (TG) et cholestérol. Le groupe G+ (vs. G-) avait un taux de croissance plus bas, une activité de GK et une concentration en G6P plus élevée, et un potentiel redox plus bas (moins oxydé). Le niveau plasmatique de TG était moins élevé dans le groupe NP+ (vs. NP-). Les traitements n’eurent aucun effet sur les autres paramètres. Ces résultats suggèrent qu’indépendamment de la NP, une alimentation riche en glucose stimule la glycolyse et déplace l’état redox vers un statut plus réduit, mais ne surmonte pas les effets de la NP sur le phénotype physique de carence énergétique. / Neonatal exposure to oxidant molecules from total parenteral nutrition (TPN) alters future lipid and glucose metabolism, resulting in an energy deficient phenotype characterized by lower body weight and physical activity. Using a guinea-pig model, the hypothesis that early diet supplementation with glucose could overcome such symptoms at week 9 of age was tested in a two-factor full-factorial ANOVA design (p<0:05): TPN day 3-7, chow thereafter (TPN+) vs: chow from day 3 (TPN-), combined with glucose-enriched diet from week 3 (G+) vs: plain chow throughout (G-). The growth rate, physical activity, phosphofructose kinase-1 and glucose kinase (GK) activities, glucose-6-phosphate (G6P), glycogen and pyruvate concentrations, relative liver weight, fasting blood glucose, glucose tolerance, hepatic and plasma triacylglyceride and cholesterol levels, individual glutathione levels and GSH/GSSG-based redox potential were determined at 9 weeks. Glucose supplementation (vs: the lack thereof) resulted in a lower growth rate, higher GK activity, and higher G6P concentration at week 9. Plasma triacylglycerides at week 9 were lower in TPN+ (vs: TPN-) subjects. Hepatic GSH=GSSG-derived redox potential shifted to a more reduced state in G+ (vs: G-) subjects. No other parameters showed significant differences. Independently of TPN, an early glucose-rich diet stimulated the glycolysis pathway, shifted the redox potential towards a more reduced status ; however, it did not overcome the effects of TPN on future physical and metabolic phenotype.

In einem Fütterungsversuch mit 15 weiblichen, genetisch identischen Zwillingspaaren wurde der anhaltende Effekt energetisch unterschiedlich konzentrierter Futterrationen auf Körper- und Blutmerkmale zwischen dem vierten und 21. Lebensmonat erfasst. Die gleichen Merkmale wurden an den Tieren auch während der Laktation erhoben, als die Tiere einheitlich gefüttert wurden. Zusätzlich wurde die Milchleistung untersucht. Während der Aufzucht wurden Körpergewicht, tägliche Gewichtszunahme, Rückenfettdicke und Widerristhöhe von der Fütterungsintensität signifikant beeinflusst. Körpergewicht und Rückenfettdicke zeigten vom siebenten bis 15. Lebensmonat die größten Unterschiede zwischen den Fütterungsgruppen. Im Gegensatz zum Körpergewicht, wurde der Fettansatz bis zum 21. Lebensmonat kaum gebremst. Für die Serumkonzentrationen von Insulin, Glukose und beta-Hydroybuttersäure und die Erythrozytenindizes MCV und MCH konnte ein signifikanter Fütterungseinfluss während der gesamten Aufzuchtphase nachgewiesen werden. Kortisol, Kreatinin, ASAT, GGT, GLDH, MCHC, Leukozytenzahl, Thrombozytenzahl reagierten auf den Fütterungsstimulus nur innerhalb bestimmter Altersabschnitte. Bis zum neunten Monat differierte der Insulinspiegel zwischen den Fütterungsgruppen kaum, ab dem 10. Lebensmonat aber sehr deutlich. Es kann daher ausgeschlossen werden, dass der Insulinspiegel im präpubertären Abschnitt die Entwicklung der späteren Milchleistung beeinflusste. Nach dem Abkalben war die intensiv gefütterte Gruppe stärkeren metabolischen Belastungen ausgesetzt und hatte eine geringere Milchleistung als die moderat gefütterte Gruppe. Offensichtlich wurde der Stoffwechsel durch die vorangegangene Fütterung geprägt, da der Fettansatz in der Intensivgruppe bei gleicher Fütterung früher einsetzte und auch intensiver erfolgte. Einige Kennwerte beim Jungtier korrelierten signifikant mit der späteren Milchleistung. Altersabhängige Veränderungen der Korrelationskoeffizienten weisen auf unterschiedlich sensible Phasen für die Prägung der späteren Milchleistung hin. / In a feeding trial with 15 pairs of genetically identical female twins, the effect of feeding intensity on body condition and blood parameters were investigated between the fourth and 21st month. The same traits were analysed on the cows during the first lactation when the animals were uniformly fed. In addition to these traits, the milk yield was investigated. During the rearing period; body weight, daily weight gain, back fat thickness, and withers height were significantly influenced by feeding. The largest differences between the feeding groups in body weight and back fat thickness were seen between the ages of seventh to 15th months. In contrast to body weight, back fat thickness hardly exceeded the 21st month between the groups. The serum concentrations of insulin, glucose, beta-Hydroxybutyric acid, and the erythrocyte indices MCV and MCH showed a significant feeding effect throughout the growing period. Cortisol, creatinine, Aspartate transaminase (AST), y-glutamyltransferase (GGT), Glutamate dehydrogenase (GDH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC) and platelet responded to the feeding stimulus only within certain ages. At age nine months, insulin levels were barely differed between the feeding groups but were distinct as from the 10th month. It can therefore be concluded that insulin levels at the pre-pubertal development affects the subsequent milk yield. After calving, the intensively fed group had more metabolic stress and had a lower milk yield than the moderately fed group. Obviously, the metabolism was programmed in the previous feeding period. There was an early onset and a more intensive fat deposition in the intensive group though; they had the same feeding level. Some traits in young animals were significantly correlated with subsequent milk yield. Age-dependent changes in the correlation coefficients suggest the fact that differences in sensible juvenile phases in traits could contribute to milk yield later.

Une des caractéristiques les plus connues de la programmation métabolique est qu’un événement commun physiopathologique à l'âge adulte obtenu indépendamment du stress nutritionnel au début de la vie. Cela a conduit à penser que les altérations métaboliques dûes au stress nutritionnel précoce pouvaient résulter de la programmation seulement d’un petit nombre de gènes qui agissent comme gardiens d'un réseau de gènes ou d'une voie de signalisation. Ici nous avons l’intention de tester cette hypothèse par l'analyse combinée du transcriptome et méthylome avec des échantillons de foie des rats nés de mères nourries avec une alimentation restreinte en protéines (MPR) ou carencée en donneur de méthyles (MDD) pendant la gestation et la lactation et comparer entre les 2 modèls. Au moment du sevrage, la progéniture MDD a été sacrifiée, tandis que la progéniture du groupe MPR a reçu une nourriture standard jusqu'à l'âge de 6 mois. Les rats à jours 21 nés de mères nourries avec un régime MDD ont 3.269 gènes surexprimé (P <0,0009) et 2.841 gènes sous-exprimés (P <0,0004) par rapport aux témoins. Les modifications de méthylation de l'ADN ont été trouvées dans les régions promotrices de 1.032 gènes. Les analyses fonctionnelles ont révélé que ces gènes sont principalement impliqués dans le métabolisme des lipides et du glucose, du système nerveux, la coagulation, le stress du réticulum endoplasmique et la fonction mitochondriale. Les master genes présentant des changements à la fois dans l'expression et la méthylation d'ADN sont limités à 266 gènes et ils sont principalement impliqués dans le système rénine-angiotensine, le métabolisme de la mitochondrie et de l'homéostasie phospholipide. La plupart de ces master genes participent à la Non Alcoholic Fatty Liver Disease (NAFLD). La restriction protéique maternelle (MPR) a entraîné une augmentation de la masse grasse abnominale, de l'hypertriglycéridémie, de l'hypercholestérolémie et un taux élevé d’acides gras par rapport aux témoins. 3.020 gènes sont surexprimés (P<0,0003) et 3.601 sous-exprimés (P<0,002) au niveau du transcriptome et 3.968 gènes modifiés au niveau du méthylome par rapport aux témoins. L'analyse fonctionnelle a indiqué que les gènes surexprimés sont principalement impliqués dans les voies métaboliques et les gènes sous-exprimés et différemment méthylés sont principalement impliqués dans des processus du développement. 998 master genes ont été trouvés, et léanalyse fonctionnelle de ces gènes a indiqué un effet significatif sur le développement des tissus, la régulation de la transcription et le métabolisme, et beaucoup d'entre eux sont associés à des maladies chroniques comme l'hypertension, l'obésité centrale et le diabète. L'expression des gènes et la méthylation de l'ADN du génome obtenus en utilisant ces modèles ont été comparés aux données de méthylome et de transcriptome précédemment obtenus à partir de foie des rats restreints en protéines et sacrifiés à la naissance. Cette analyse a révélé un ensemble commun de 46 gènes qui sont sur-exprimés et 42 gènes sous-exprimés dans les trois modèles de programmation métabolique par rapport aux animaux témoins. La plupart des gènes surexprimés sont impliqués dans la régulation de la fonction mitochondriale alors que les gènes sous-exprimés sont principalement impliquées dans la régulation de la prolifération cellulaire et l'expression des gènes. Nous avons identifié également un ensemble de 122 gènes dont les niveaux de méthylation ont été modifiés à la fois par une carence en donneurs de méthyle et une restriction protéique. Ces observations soutiennent l’hypothèse qu’un petit nombre de gènes essentiels sont à la base de la programmation de troubles métaboliques, indépendamment du stress nutritionnel / One of the most striking features of metabolic programming is that a common physiopathological output at adulthood is obtained irrespective to the nutritional insult during early life. This has suggested that the metabolic alterations due to early nutritional stress might result from the programming of only a small number of genes which act as gatekeepers of a fundamental gene network or signalling pathway. Here we aimed to test this hypothesis through the combined analysis of the transcriptome and methylome in rat liver samples derived from animals born to dams fed either a protein-restricted diet (MPR) or a methyl donor deficient (MDD) diet through gestation and lactation. At weaning, the offspring born to MDD dams were sacrificed whereas the pups from the MPR group were fed standard chow until the age of 6 months. 21-day-old rats born to mothers fed a MDD diet during gestation and lactation have 3,269 over-expressed (P<0.0009) and 2,841 under-expressed (P<0.0004) genes compared to controls. Modifications of DNA methylation were found in the promoter regions of 1,032 genes. Functional analyses revealed that these genes are mainly involved in glucose and lipid metabolism, nervous system, coagulation, endoplasmic reticulum stress and mitochondrial function. Master genes exhibiting changes in both gene expression and DNA methylation are limited to 266 genes and are mainly involved in the renin-angiotensin system, mitochondrion metabolism and phospholipid homeostasis. Most of these master genes participate in Non Alcoholic Fatty Liver Disease (NAFLD). Maternal protein restriction (MPR) resulted in increased fat mass, hypertriglyceridemia, hypercholesterolemia and high fatty acids compared to control. 3,020 genes were up-regulated (p < 0.0003) and 3,601 (p ? 0.002) down-regulated by MPR compared to controls. Modifications of DNA methylation was found in 3,968 genes. The functional analysis indicated that the overexpressed genes were mainly involved in metabolic pathways and the under-expressed and differentially methylated genes were mainly involved in physiological process. 998 master genes were found, functional analysis of these genes indicated a significant effect on tissue development, regulation of transcription and metabolism, and many of them are associated with chronic diseases such as hypertension, central obesity and diabetes. The genome-wide expression and DNA metylation results obtained using these models, were compared to previous methylome and transcriptome data obtained using liver from MPR pups sacrificed at birth. This analysis revealed a common set of 46 genes that were up regulated and 42 genes down regulated in the three models of metabolic programming compared to control animals. Most of the up regulated genes are involved in the regulation of mitochondrial function whereas the down-regulated genes are mainly involved in the regulation of cell proliferation and gene expression. We identified also a set of 122 genes whose methylation levels were changed both by methyl donor deficiency and protein-restriction. These observations sustain the hypothesis that a small set of core genes underlies the programming of metabolic disorders irrespective of the nutritional insult

Les oxydants infusés avec la nutrition parentéral (NP) néonatale induisent une modification du métabolisme des lipides et du glucose, donnant lieu à l’âge adulte à un phénotype de carence énergétique (faible poids, baisse de l’activité physique). L’hypothèse qu’une diète précoce riche en glucose prévient ces symptômes plus tard dans la vie, fut évalué chez le cobaye par un ANOVA en plan factoriel complet à deux facteurs (p < 0:05) : NP du jour 3 à 7, suivit d’une nourriture régulière (chow) (NP+) vs. chow à partir du 3ième jour (NP-), combiné avec une eau de consommation enrichie en glucose (G+) ou non (G-) à partir de la 3ième semaine. Les paramètres suivant ont été mesurés à l’âge de 9 semaine: taux de croissance, activité physique, activité de phosphofructokinase-1 et glucokinase (GK), niveau hépatique de glucose-6-phosphate (G6P), glycogène, pyruvate et potentiel redox du glutathion, poids du foie, glycémie, tolérance au glucose, concentrations hépatiques et plasmatiques en triacylglycérides (TG) et cholestérol. Le groupe G+ (vs. G-) avait un taux de croissance plus bas, une activité de GK et une concentration en G6P plus élevée, et un potentiel redox plus bas (moins oxydé). Le niveau plasmatique de TG était moins élevé dans le groupe NP+ (vs. NP-). Les traitements n’eurent aucun effet sur les autres paramètres. Ces résultats suggèrent qu’indépendamment de la NP, une alimentation riche en glucose stimule la glycolyse et déplace l’état redox vers un statut plus réduit, mais ne surmonte pas les effets de la NP sur le phénotype physique de carence énergétique. / Neonatal exposure to oxidant molecules from total parenteral nutrition (TPN) alters future lipid and glucose metabolism, resulting in an energy deficient phenotype characterized by lower body weight and physical activity. Using a guinea-pig model, the hypothesis that early diet supplementation with glucose could overcome such symptoms at week 9 of age was tested in a two-factor full-factorial ANOVA design (p<0:05): TPN day 3-7, chow thereafter (TPN+) vs: chow from day 3 (TPN-), combined with glucose-enriched diet from week 3 (G+) vs: plain chow throughout (G-). The growth rate, physical activity, phosphofructose kinase-1 and glucose kinase (GK) activities, glucose-6-phosphate (G6P), glycogen and pyruvate concentrations, relative liver weight, fasting blood glucose, glucose tolerance, hepatic and plasma triacylglyceride and cholesterol levels, individual glutathione levels and GSH/GSSG-based redox potential were determined at 9 weeks. Glucose supplementation (vs: the lack thereof) resulted in a lower growth rate, higher GK activity, and higher G6P concentration at week 9. Plasma triacylglycerides at week 9 were lower in TPN+ (vs: TPN-) subjects. Hepatic GSH=GSSG-derived redox potential shifted to a more reduced state in G+ (vs: G-) subjects. No other parameters showed significant differences. Independently of TPN, an early glucose-rich diet stimulated the glycolysis pathway, shifted the redox potential towards a more reduced status ; however, it did not overcome the effects of TPN on future physical and metabolic phenotype.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O cálcio tem se mostrado útil na regulação do metabolismo energético, favorecendo a perda de peso. Visto que tanto o desmame precoce como a exposição materna à nicotina na lactação são fatores condicionantes para o desenvolvimento de obesidade, hiperleptinemia e resistência à insulina, além de outras alterações endócrinas na idade adulta, decidimos avaliar os possíveis efeitos da suplementação dietética com cálcio sobre as disfunções apresentadas pelos seguintes modelos experimentais: 1) Desmame precoce (DP): ratas lactantes foram envolvidas com atadura para interromper o acesso da prole ao leite nos 3 últimos dias da lactação. As proles das ratas controles tiveram livre acesso ao leite materno durante todo o período da lactação (21 dias). 2) Exposição materna à nicotina (N): Dois dias após o nascimento, ratas lactantes receberam implantes de minibombas osmóticas contendo solução de nicotina (6 mg/kg/dia, 14 dias) ou salina (C), nas mesmas condições. Aos 120 dias de idade, as proles de ambos os modelos de obesidade experimental receberam dieta padrão ou dieta suplementada com cálcio (10g de carbonato de cálcio/kg de ração). O sacrifício ocorreu aos 180 dias de idade. Os dados foram considerados significativos quando p<0,05. Corroborando dados anteriores do nosso grupo, as proles de ambos os modelos de programação (N e DP) apresentaram maior gordura corporal total e visceral, hiperleptinemia, resistência hipotalâmica a leptina e distúrbios na homeostase glicêmica. Além disto, verificamos que as proles N e DP também exibiram aumento dos níveis séricos de 25-hidroxivitamina D3. Todos essas alterações endócrino-metabólicas foram corrigidas pelo tratamento com suplementação com cálcio. Além disso, a prole DP aos 180 dias mostrou hiperfagia e hipertrigliceridemia, que também foram normalizados pela suplementação dietética com cálcio. A prole N apresentou hipotireoidismo, maior conteúdo de catecolaminas e maior expressão de tirosina hidroxilase (TH). A terapia com cálcio reverteu a disfunção adrenal, embora não tenha sido eficaz para normalizar a hipofunção tireoideana. Assim, a suplementação dietética com cálcio normalizou a maioria dos parâmetros da síndrome metabólica observadas nos dois modelos de plasticidade ontogenética. É possível que a redução da adiposidade central induzida pela terapia com cálcio, por si, tenha sido o principal mecanismo que resultou na melhora dos parâmetros estudados. Uma vez que a suplentação de cálcio reverteu as concentrações séricas de 25-hidroxivitamina D3 dos animais obesos, é possível que o efeito anti-obesidade do cálcio também ocorra via ação do calcitriol sobre o adipócito. / Calcium influences energy metabolism regulation and causes body weight loss. Since early weaning and maternal nicotine exposure during lactation programs for obesity, hyperleptinemia, insulin resistance and others endocrine dysfunctions, we studied the possible effect of dietary calcium supplementation on endocrine dysfunctions in these two experimental model of obesity: 1) Early Weaning (EW): lactating rats were involved with a bandage to interrupt the lactation during the last 3 days of standard lactation, and C (control) - dams whose pups had free access to milk during all lactation (21 days). 2) Maternal nicotine exposure (N): Two days after birth, it was implanted on the mothers, osmotic minipumps containing nicotine solution (6 mg/Kg/day, 14 days) or saline (C) in the same condition. At 120 days-old, all offspring received dietary calcium supplementation (10g of calcium carbonate/Kg of rat chow) or standard diet. Rats were killed at 180 days-old. Significant data were p<0.05. As expected, N and DP offspring showed higher visceral and total body fat mass, hyperleptinemia, hypothalamic leptin resistance and insulin resistance. In addition they presented higher serum 25-hydroxyvitamin D3. All these disturbances were corrected after calcium supplementation. Besides, adult EW offspring displayed hyperphagia and hypertriglyceridemia that was normalized with calcium therapy. Also, N offspring presented hypothyroidism, higher tyrosine hydroxylase expression and higher adrenal catecholamine content. Despite calcium treatment have normalized adrenal dysfunction, it did not reverse the hypothyroidism. So, dietary calcium supplementation seems to revert most of the metabolic syndrome parameters observed in our two developmental plasticity models. It is conceivable that the reduction in fat mass per se, induced by calcium therapy, is the main mechanism that improves all parameters. Because serum 25-hydroxyvitamin D3 levels were corrected by calcium supplementation in obese offspring, it is possible that the anti-obesity effect of calcium occurs through calcitriol action on the adipocyte.

O comportamento alimentar de uma espécie é determinado por um conjunto de características filogenéticas, ontogenéticas, e epigenéticas, e regulado por fatores internos e externos ao organismo. Os fenômenos naturais que regem a vida no nosso planeta são periódicos em sua maioria, e a oferta de alimentos não é exceção. Cada safra é seguida de uma entressafra, e este ritmo sincroniza diversos outros ritmos, exógenos e endógenos, capazes de determinar a sobrevivência de espécies. Uma das estratégias adaptativas mais primitivas e bem sucedidas na dinâmica oscilatória da natureza é o acúmulo de reservas. Nossa espécie, nos últimos 50 anos, vive uma situação de grande oferta de alimentos, período este extremamente pequeno, se visto sob a ordem de grandeza da evolução humana. Este fenômeno tem sido determinante na prevalência do depósito de energia e em decorrência, do surgimento da obesidade e suas consequentes patologias. O hipotálamo está intimamente associado à homeostase energética e ao comportamento alimentar. No núcleo arqueado hipotalâmico encontram-se populações neuronais orexigênicas e anorexigênicas, dentre as quais, as que expressam os neuroreceptores POMC, NPY e o substrato de receptor de insulina IRS2. A modificação da expressão destas proteínas tem sido associada à alterações do comportamento alimentar, bem como à impressão e programação metabólica, capazes de induzir obesidade em ratos adultos. A correlação desta circuitaria neuronal com o comportamento alimentar, porém, ainda não está suficientemente compreendida. A detecção do estado de fome-saciedade nos ratos, fundamental no estudo da neurofisiologia relacionada ao comportamento alimentar, vem sendo obtida via de regra, por meio de procedimentos complexos de observação comportamental. O presente estudo contribui para o conhecimento de padrões de alimentação determinados por condições nutricionais, e sua relação com a expressão neurofisiológica hipotalâmica dos neurônios POMC, NPY e IRS2. Utilizando o modelo de programação metabólica de Plagemann (1999) obtivemos animais com 25% de sobrepeso em relação aos animais controle, hiperfágicos, e com padrões de tamanho e ritmo circadiano de refeição, distintos. Apesar dos níveis hormonais elevados de leptina (>100%, p<0,001) e insulina (>90%, p<0,05) em relação ao grupo controle, estes animais apresentaram baixa expressão no estado de fome, e alta expressão, na saciedade, de NPY hipotalâmico, sugerindo que o POMC estaria mais comprometido, a longo prazo, com a regulação do ritmo alimentar. A hiperinsulinemia e hiperleptinemia plasmática associada à reduzida expressão de POMC e IRS2 no ARC, corroboraram esta conclusão. Demonstramos também padrões de alimentação distintos. O método de registro da alimentação, baseado no som da roída foi validado como excelente, pelos registros obtidos nos vídeos, e mostrou-se eficiente. Quando os estados de fome-saciedade foram discriminados nos grupos controle e sobrepeso, os resultados da expressão hipotalâmica dos neuroreceptores estudados se mostraram associados aos particulares padrões de alimentação. / The feeding behavior of a specie is determined by a group of phylogenetic, ontogenetic, and epigenetic features, and regulated by internal and external factors to the organism. The natural phenomena that govern life in our planet are mainly periodic, and the food stocks is not an exception. Each harvest is followed by a time between harvests, and this rhythm synchronizes other several exogenous and endogenous rhythms, capable of determining the survival of species. One of the most primitive strategies of adaptative evolution of species, and what usually happens regarding the oscillatory dynamics of nature, is the reserve accumulation. Our species, in the last 50 years, has been living a situation of great food offer, such period is extremely small if analysed under the greatness order of the human evolution. This phenomenon has been decisive in the prevalence of the energy deposit and, in consequence, determining the appearance of obesity and its consequent pathologies. The hypothalamus is intimately associated to the energy homeostasis and the feeding behavior. In the arcuate nucleous are orexigenic and anorexigenic neuronal populations, that express the neuroreceptors POMC, NPY and insulin receptor substratum IRS2. The modification of these proteins expression, has been associated to alterations of the feeding behavior, as well as to the metabolic imprinting and programming, capable to induce obesity in adult rats. The correlation of this neuronal circuits with the alimentary behavior, however, it is not yet sufficiently understood. The detection of the hunger-satiation state in the rat, crucial in the neurophysiology studies related to the alimentary behavior, has been obtained through complex procedures of behavioral observation. The present study contributed to the knowledge of certain feeding patterns for nutritional conditions, and its relationship with the neurophysiological expression of POMC, NPY and IRS2 neurons. Using the metabolic programming model of Plagemann (1999) animals with 25% of overweight in relation to the control animals were obtained, hyperphagics, and with different size patterns and meal circadian rhythm. In spite of the high hormonal levels of leptin (>100%, p < 0,001) and insulin (>90%, p < 0,05) in relation to the control groups, these animals presented low expression in the hunger state, and high expression in the satiation of hypothalamic NPY, suggesting that POMC would be more committed, in the long term, with the regulation of the feeding rhythm. The hyperinsulinemia and plasmatic hyperleptinemia associated to the reduced POMC and IRS2 expression in the ARC, corroborated this conclusion. We also demonstrated different feeding patterns. The feeding registration method, based on the gnaw sound was validated as excellent, when comparedto a gold pattern, the registrations obtained in the videos, and it were considered efficient. When the hunger-satiation states were discriminated in the control and overweight groups, the results of the hypothalamic neuroreceptors expression studied showed association to the feeding patterns.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A saudável interação entre o indivíduo e o meio depende do alinhamento entre a dinâmica fisiológica do primeiro e os periódicos movimentos da natureza. A interação entre tais ritmos por sua vez constitui-se em base e derivação do processo de evolução. O comprometimento de tal alinhamento representa um risco para a sobrevivência das espécies. Neste contexto, os organismos alinham seus ritmos fisiológicos a diferentes ciclos externos. Desta forma, ciclos endógenos são coordenados por relógios biológicos que determinam em nosso organismo, específicos ritmos em fase com a natureza, tais como ritmos circadianos (RC), cujo período aproxima-se de 24 horas. O peso corporal, a ingestão de alimentos e o consumo de energia são processos caracterizados pelo RC e a obesidade está associada a uma dessincronização deste processo. A modulação do RC é resultado da expressão dos clock gens CLOCK e BMAL1 que formam um heterodímero responsável pela transcrição gênica de Per1, Per2, Per3, Cry1 e Cry2. As proteínas codificadas por estes genes, uma vez sintetizadas, formam dímeros (PER-CRY) no citoplasma que, a partir de determinada concentração, retornam ao núcleo, bloqueando a ação do heterodímero CLOCK/BMAL1 na transcrição dos próprios genes, formando assim uma alça de retroalimentação negativa de transcrição e tradução. Estes genes asseguram a periodicidade e são significativamente expressos no núcleo supraquiasmático (SCN) do hipotálamo. Para estudar esse processo em camundongos normais e hiperalimentados, saciados e em estado de fome, foi utilizado um método de registro do comportamento alimentar baseado no som produzido pela alimentação dos animais, e a correlação destes estados metabólicos com a expressão de CLOCK, BMAL1, Per1, Per2, Per3, bem como das proteínas Cry1 e Cry2 no SCN, por análise de imagens obtidas em microscopia confocal. Camundongos suíços controle em estado de fome (CF) e saciados (CS) foram comparados com animais hiperalimentados com fome (HF) e saciados (HS). Nenhum grupo demonstrou diferença nos conteúdos CLOCK e BMAL1, indicando capacidade potencial para modular os ritmos biológicos. No entanto, as proteínas Per1, Per2, Per3 e Cry1 apresentaram menor expressão no grupo CS, mostrando uma diferença significativa quando comparados com o grupo CF (P<0,05), diferença esta não encontrada na comparação entre os grupos HF e HS. A quantidade de proteína Cry2 não foi diferente na mesma comparação. Os resultados do estudo indicaram que as alterações dos ritmos endógenos e exógenos, refletido pelo comportamento hiperfágico observado em camundongos hiperalimentados, pode ser devido a um defeito no mecanismo de feedback negativo associado ao dímero Cry-Per, que não bloqueia a transcrição de Per1 Per2, Per3 e Cry1 pelo heterodímero CLOCK-BMAL1. / The healthy interaction between the subject and the environment depends on the alignment between the physiological dynamics of the first one and the periodical movements of nature. The interaction between these rhythms in turn is based on the derivation and evolution process. The involvement of such an alignment is a risk to the survival of species. In this context the bodies line up their physiological rhythms to different external cycles. Thus, endogenous cycles are coordinated by biological clocks which determine in our organism specific rhythms in phase with the nature, such as Circadian Rhythms (CR) whose period is close to 24 hours. The body weight, the food intake and the energy consumption are processes characterized by the CR and the obesity is associated with a different timing of this process. The CR modulation is a result of the formulation of clock-gens CLOCK and BMAL1 who form an heterodimer responsible for the gene transcription of Per1, Per2, Per3, Cry1 e Cry2. The proteins encoded by these genes, once synthesized, form dimers (PER-CRY) in the cytoplasm that, depending on a given concentration, return to the core blocking the action of the CLOCK/BMAL1 heterodimer in the transcription of its own genes, thus forming a negative feedback loop of transcription and translation. These genes secure the periodicity and are significantly expressed in the hypothalamus suprachiasmatic nucleus. In order to study this process in regular, hyper-fed, hungry and satiated mice, we used a registration method of feeding behavior based on the sound produced by animal feeding and the relation between the metabolic states with the expression CLOCK, BMAL1, Per1, Per2, Per3, as well as the Cry1 and Cry2 proteins in the SCN, by analysis of images obtained in confocal microscopy. Control Swiss mice in state of hunger/ satiated were compared to hyper-fed animals in the same conditions. None of them showed difference in the CLOCK and BMAL1 contents, showing a potential capacity to modulate the biological rhythms. However, the Per1, Per2, Per3 and Cry1 proteins showed a minor expression in the CS group and a significant difference when compared to the CF group (P<0,05). This difference cant be found in the HF and HS groups. The results of the studies indicated that the endogenous and exogenous changes, reflected by the hyperphagic behavior observed in hyper-fed mice, may be due to a defect in the mechanism of negative feedback associated to the Cry-Per dimer, which has abolished the blocking mechanism of Per1 Per2, Per3 and Cry1 by the CLOCK-BMAL1 heterodimer.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O cálcio tem se mostrado útil na regulação do metabolismo energético, favorecendo a perda de peso. Visto que tanto o desmame precoce como a exposição materna à nicotina na lactação são fatores condicionantes para o desenvolvimento de obesidade, hiperleptinemia e resistência à insulina, além de outras alterações endócrinas na idade adulta, decidimos avaliar os possíveis efeitos da suplementação dietética com cálcio sobre as disfunções apresentadas pelos seguintes modelos experimentais: 1) Desmame precoce (DP): ratas lactantes foram envolvidas com atadura para interromper o acesso da prole ao leite nos 3 últimos dias da lactação. As proles das ratas controles tiveram livre acesso ao leite materno durante todo o período da lactação (21 dias). 2) Exposição materna à nicotina (N): Dois dias após o nascimento, ratas lactantes receberam implantes de minibombas osmóticas contendo solução de nicotina (6 mg/kg/dia, 14 dias) ou salina (C), nas mesmas condições. Aos 120 dias de idade, as proles de ambos os modelos de obesidade experimental receberam dieta padrão ou dieta suplementada com cálcio (10g de carbonato de cálcio/kg de ração). O sacrifício ocorreu aos 180 dias de idade. Os dados foram considerados significativos quando p<0,05. Corroborando dados anteriores do nosso grupo, as proles de ambos os modelos de programação (N e DP) apresentaram maior gordura corporal total e visceral, hiperleptinemia, resistência hipotalâmica a leptina e distúrbios na homeostase glicêmica. Além disto, verificamos que as proles N e DP também exibiram aumento dos níveis séricos de 25-hidroxivitamina D3. Todos essas alterações endócrino-metabólicas foram corrigidas pelo tratamento com suplementação com cálcio. Além disso, a prole DP aos 180 dias mostrou hiperfagia e hipertrigliceridemia, que também foram normalizados pela suplementação dietética com cálcio. A prole N apresentou hipotireoidismo, maior conteúdo de catecolaminas e maior expressão de tirosina hidroxilase (TH). A terapia com cálcio reverteu a disfunção adrenal, embora não tenha sido eficaz para normalizar a hipofunção tireoideana. Assim, a suplementação dietética com cálcio normalizou a maioria dos parâmetros da síndrome metabólica observadas nos dois modelos de plasticidade ontogenética. É possível que a redução da adiposidade central induzida pela terapia com cálcio, por si, tenha sido o principal mecanismo que resultou na melhora dos parâmetros estudados. Uma vez que a suplentação de cálcio reverteu as concentrações séricas de 25-hidroxivitamina D3 dos animais obesos, é possível que o efeito anti-obesidade do cálcio também ocorra via ação do calcitriol sobre o adipócito. / Calcium influences energy metabolism regulation and causes body weight loss. Since early weaning and maternal nicotine exposure during lactation programs for obesity, hyperleptinemia, insulin resistance and others endocrine dysfunctions, we studied the possible effect of dietary calcium supplementation on endocrine dysfunctions in these two experimental model of obesity: 1) Early Weaning (EW): lactating rats were involved with a bandage to interrupt the lactation during the last 3 days of standard lactation, and C (control) - dams whose pups had free access to milk during all lactation (21 days). 2) Maternal nicotine exposure (N): Two days after birth, it was implanted on the mothers, osmotic minipumps containing nicotine solution (6 mg/Kg/day, 14 days) or saline (C) in the same condition. At 120 days-old, all offspring received dietary calcium supplementation (10g of calcium carbonate/Kg of rat chow) or standard diet. Rats were killed at 180 days-old. Significant data were p<0.05. As expected, N and DP offspring showed higher visceral and total body fat mass, hyperleptinemia, hypothalamic leptin resistance and insulin resistance. In addition they presented higher serum 25-hydroxyvitamin D3. All these disturbances were corrected after calcium supplementation. Besides, adult EW offspring displayed hyperphagia and hypertriglyceridemia that was normalized with calcium therapy. Also, N offspring presented hypothyroidism, higher tyrosine hydroxylase expression and higher adrenal catecholamine content. Despite calcium treatment have normalized adrenal dysfunction, it did not reverse the hypothyroidism. So, dietary calcium supplementation seems to revert most of the metabolic syndrome parameters observed in our two developmental plasticity models. It is conceivable that the reduction in fat mass per se, induced by calcium therapy, is the main mechanism that improves all parameters. Because serum 25-hydroxyvitamin D3 levels were corrected by calcium supplementation in obese offspring, it is possible that the anti-obesity effect of calcium occurs through calcitriol action on the adipocyte.

O comportamento alimentar de uma espécie é determinado por um conjunto de características filogenéticas, ontogenéticas, e epigenéticas, e regulado por fatores internos e externos ao organismo. Os fenômenos naturais que regem a vida no nosso planeta são periódicos em sua maioria, e a oferta de alimentos não é exceção. Cada safra é seguida de uma entressafra, e este ritmo sincroniza diversos outros ritmos, exógenos e endógenos, capazes de determinar a sobrevivência de espécies. Uma das estratégias adaptativas mais primitivas e bem sucedidas na dinâmica oscilatória da natureza é o acúmulo de reservas. Nossa espécie, nos últimos 50 anos, vive uma situação de grande oferta de alimentos, período este extremamente pequeno, se visto sob a ordem de grandeza da evolução humana. Este fenômeno tem sido determinante na prevalência do depósito de energia e em decorrência, do surgimento da obesidade e suas consequentes patologias. O hipotálamo está intimamente associado à homeostase energética e ao comportamento alimentar. No núcleo arqueado hipotalâmico encontram-se populações neuronais orexigênicas e anorexigênicas, dentre as quais, as que expressam os neuroreceptores POMC, NPY e o substrato de receptor de insulina IRS2. A modificação da expressão destas proteínas tem sido associada à alterações do comportamento alimentar, bem como à impressão e programação metabólica, capazes de induzir obesidade em ratos adultos. A correlação desta circuitaria neuronal com o comportamento alimentar, porém, ainda não está suficientemente compreendida. A detecção do estado de fome-saciedade nos ratos, fundamental no estudo da neurofisiologia relacionada ao comportamento alimentar, vem sendo obtida via de regra, por meio de procedimentos complexos de observação comportamental. O presente estudo contribui para o conhecimento de padrões de alimentação determinados por condições nutricionais, e sua relação com a expressão neurofisiológica hipotalâmica dos neurônios POMC, NPY e IRS2. Utilizando o modelo de programação metabólica de Plagemann (1999) obtivemos animais com 25% de sobrepeso em relação aos animais controle, hiperfágicos, e com padrões de tamanho e ritmo circadiano de refeição, distintos. Apesar dos níveis hormonais elevados de leptina (>100%, p<0,001) e insulina (>90%, p<0,05) em relação ao grupo controle, estes animais apresentaram baixa expressão no estado de fome, e alta expressão, na saciedade, de NPY hipotalâmico, sugerindo que o POMC estaria mais comprometido, a longo prazo, com a regulação do ritmo alimentar. A hiperinsulinemia e hiperleptinemia plasmática associada à reduzida expressão de POMC e IRS2 no ARC, corroboraram esta conclusão. Demonstramos também padrões de alimentação distintos. O método de registro da alimentação, baseado no som da roída foi validado como excelente, pelos registros obtidos nos vídeos, e mostrou-se eficiente. Quando os estados de fome-saciedade foram discriminados nos grupos controle e sobrepeso, os resultados da expressão hipotalâmica dos neuroreceptores estudados se mostraram associados aos particulares padrões de alimentação. / The feeding behavior of a specie is determined by a group of phylogenetic, ontogenetic, and epigenetic features, and regulated by internal and external factors to the organism. The natural phenomena that govern life in our planet are mainly periodic, and the food stocks is not an exception. Each harvest is followed by a time between harvests, and this rhythm synchronizes other several exogenous and endogenous rhythms, capable of determining the survival of species. One of the most primitive strategies of adaptative evolution of species, and what usually happens regarding the oscillatory dynamics of nature, is the reserve accumulation. Our species, in the last 50 years, has been living a situation of great food offer, such period is extremely small if analysed under the greatness order of the human evolution. This phenomenon has been decisive in the prevalence of the energy deposit and, in consequence, determining the appearance of obesity and its consequent pathologies. The hypothalamus is intimately associated to the energy homeostasis and the feeding behavior. In the arcuate nucleous are orexigenic and anorexigenic neuronal populations, that express the neuroreceptors POMC, NPY and insulin receptor substratum IRS2. The modification of these proteins expression, has been associated to alterations of the feeding behavior, as well as to the metabolic imprinting and programming, capable to induce obesity in adult rats. The correlation of this neuronal circuits with the alimentary behavior, however, it is not yet sufficiently understood. The detection of the hunger-satiation state in the rat, crucial in the neurophysiology studies related to the alimentary behavior, has been obtained through complex procedures of behavioral observation. The present study contributed to the knowledge of certain feeding patterns for nutritional conditions, and its relationship with the neurophysiological expression of POMC, NPY and IRS2 neurons. Using the metabolic programming model of Plagemann (1999) animals with 25% of overweight in relation to the control animals were obtained, hyperphagics, and with different size patterns and meal circadian rhythm. In spite of the high hormonal levels of leptin (>100%, p < 0,001) and insulin (>90%, p < 0,05) in relation to the control groups, these animals presented low expression in the hunger state, and high expression in the satiation of hypothalamic NPY, suggesting that POMC would be more committed, in the long term, with the regulation of the feeding rhythm. The hyperinsulinemia and plasmatic hyperleptinemia associated to the reduced POMC and IRS2 expression in the ARC, corroborated this conclusion. We also demonstrated different feeding patterns. The feeding registration method, based on the gnaw sound was validated as excellent, when comparedto a gold pattern, the registrations obtained in the videos, and it were considered efficient. When the hunger-satiation states were discriminated in the control and overweight groups, the results of the hypothalamic neuroreceptors expression studied showed association to the feeding patterns.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A saudável interação entre o indivíduo e o meio depende do alinhamento entre a dinâmica fisiológica do primeiro e os periódicos movimentos da natureza. A interação entre tais ritmos por sua vez constitui-se em base e derivação do processo de evolução. O comprometimento de tal alinhamento representa um risco para a sobrevivência das espécies. Neste contexto, os organismos alinham seus ritmos fisiológicos a diferentes ciclos externos. Desta forma, ciclos endógenos são coordenados por relógios biológicos que determinam em nosso organismo, específicos ritmos em fase com a natureza, tais como ritmos circadianos (RC), cujo período aproxima-se de 24 horas. O peso corporal, a ingestão de alimentos e o consumo de energia são processos caracterizados pelo RC e a obesidade está associada a uma dessincronização deste processo. A modulação do RC é resultado da expressão dos clock gens CLOCK e BMAL1 que formam um heterodímero responsável pela transcrição gênica de Per1, Per2, Per3, Cry1 e Cry2. As proteínas codificadas por estes genes, uma vez sintetizadas, formam dímeros (PER-CRY) no citoplasma que, a partir de determinada concentração, retornam ao núcleo, bloqueando a ação do heterodímero CLOCK/BMAL1 na transcrição dos próprios genes, formando assim uma alça de retroalimentação negativa de transcrição e tradução. Estes genes asseguram a periodicidade e são significativamente expressos no núcleo supraquiasmático (SCN) do hipotálamo. Para estudar esse processo em camundongos normais e hiperalimentados, saciados e em estado de fome, foi utilizado um método de registro do comportamento alimentar baseado no som produzido pela alimentação dos animais, e a correlação destes estados metabólicos com a expressão de CLOCK, BMAL1, Per1, Per2, Per3, bem como das proteínas Cry1 e Cry2 no SCN, por análise de imagens obtidas em microscopia confocal. Camundongos suíços controle em estado de fome (CF) e saciados (CS) foram comparados com animais hiperalimentados com fome (HF) e saciados (HS). Nenhum grupo demonstrou diferença nos conteúdos CLOCK e BMAL1, indicando capacidade potencial para modular os ritmos biológicos. No entanto, as proteínas Per1, Per2, Per3 e Cry1 apresentaram menor expressão no grupo CS, mostrando uma diferença significativa quando comparados com o grupo CF (P<0,05), diferença esta não encontrada na comparação entre os grupos HF e HS. A quantidade de proteína Cry2 não foi diferente na mesma comparação. Os resultados do estudo indicaram que as alterações dos ritmos endógenos e exógenos, refletido pelo comportamento hiperfágico observado em camundongos hiperalimentados, pode ser devido a um defeito no mecanismo de feedback negativo associado ao dímero Cry-Per, que não bloqueia a transcrição de Per1 Per2, Per3 e Cry1 pelo heterodímero CLOCK-BMAL1. / The healthy interaction between the subject and the environment depends on the alignment between the physiological dynamics of the first one and the periodical movements of nature. The interaction between these rhythms in turn is based on the derivation and evolution process. The involvement of such an alignment is a risk to the survival of species. In this context the bodies line up their physiological rhythms to different external cycles. Thus, endogenous cycles are coordinated by biological clocks which determine in our organism specific rhythms in phase with the nature, such as Circadian Rhythms (CR) whose period is close to 24 hours. The body weight, the food intake and the energy consumption are processes characterized by the CR and the obesity is associated with a different timing of this process. The CR modulation is a result of the formulation of clock-gens CLOCK and BMAL1 who form an heterodimer responsible for the gene transcription of Per1, Per2, Per3, Cry1 e Cry2. The proteins encoded by these genes, once synthesized, form dimers (PER-CRY) in the cytoplasm that, depending on a given concentration, return to the core blocking the action of the CLOCK/BMAL1 heterodimer in the transcription of its own genes, thus forming a negative feedback loop of transcription and translation. These genes secure the periodicity and are significantly expressed in the hypothalamus suprachiasmatic nucleus. In order to study this process in regular, hyper-fed, hungry and satiated mice, we used a registration method of feeding behavior based on the sound produced by animal feeding and the relation between the metabolic states with the expression CLOCK, BMAL1, Per1, Per2, Per3, as well as the Cry1 and Cry2 proteins in the SCN, by analysis of images obtained in confocal microscopy. Control Swiss mice in state of hunger/ satiated were compared to hyper-fed animals in the same conditions. None of them showed difference in the CLOCK and BMAL1 contents, showing a potential capacity to modulate the biological rhythms. However, the Per1, Per2, Per3 and Cry1 proteins showed a minor expression in the CS group and a significant difference when compared to the CF group (P<0,05). This difference cant be found in the HF and HS groups. The results of the studies indicated that the endogenous and exogenous changes, reflected by the hyperphagic behavior observed in hyper-fed mice, may be due to a defect in the mechanism of negative feedback associated to the Cry-Per dimer, which has abolished the blocking mechanism of Per1 Per2, Per3 and Cry1 by the CLOCK-BMAL1 heterodimer.