Em nosso laboratório foi demonstrado que ratos submetidos à restrição crônica de sal na dieta apresentaram maior peso corpóreo (PC), menor sensibilidade à insulina e alterações pressóricas em comparação com ratos submetidos à sobrecarga crônica de sal na dieta. No presente estudo, o objetivo foi avaliar alguns mecanismos de obesidade e alterações hormonais associados à dieta hipossódica. Foram utilizados ratos Wistar machos submetidos à dieta hipo (HO: 0,15% NaCl), normo (NO: 1,27% NaCl) ou hipersódica (HR: 7,94% NaCl) desde o desmame até 12 semanas de idade. Nestes animais foram realizadas medidas de pressão arterial (PA), freqüência cardíaca (FC), consumo de ração, PC, perfil dos hormônios [leptina (LEP), GH, insulina (INSh - anticorpo anti-insulina humana), T3, T4 e TSH] e decaimento da 125I-insulina (DEC-INS). O consumo de ração foi avaliado durante sete dias consecutivos com um (1G) ou quatro (4G) ratos por gaiola de plástico e com um rato por gaiola metabólica (1GM). O PC também foi medido neste período. Glicemia (GLI) e insulinemia (INSr - anticorpo anti-insulina de rato) basais foram quantificadas antes da realização do estudo do DEC-INS. Para avaliar o DEC-INS, foi injetado 1mCi de 125I-insulina humana pelo cateter implantado na veia jugular, após 6 a 8 horas de jejum fisiológico. As amostras de sangue foram coletadas pelo cateter implantado na carótida a cada 30 segundos durante 2 minutos e depois a cada 1 minuto até 10 minutos de experimento. Os resultados obtidos demonstraram que ratos submetidos à dieta HR apresentaram maior PA em comparação a ratos em dieta NO e HO. Os ratos submetidos à dieta HO apresentaram menor FC cardíaca em relação ao grupo NO. A avaliação de ingestão alimentar revelou que ratos em dieta HR consumiram maiores quantidades de ração (1G e 4G) em comparação com ratos em dieta NO e HO. O grupo HO apresentou maior PC em comparação com os grupos de ratos NO e HR. Os ratos em dieta HR ou HO apresentaram níveis diminuídos de LEP em comparação com ratos submetidos à dieta NO. Os ratos que receberam dieta HO apresentaram níveis elevados de INSr em comparação com ratos em dieta HR e NO. Os ratos em dieta HR apresentaram níveis elevados de T4 total e níveis reduzidos de TSH em comparação com animais em dieta NO e HO. INSr e GLI basais foram maiores nos ratos em dieta HO do que nos ratos que receberam dieta HR e NO. O DEC-INS (decaimento exponencial) foi mais rápido no grupo HR demonstrado por meio de menor meia-vida da 125I-insulina. Os resultados sugerem que maior PC nos ratos sob restrição salina crônica é devido a maior eficiência metabólica (maior PC e menor consumo de dieta) que pode estar relacionada com alterações hormonais e com a dieta HO. Palavras-chave: Pressão arterial, sal, sódio, ingestão alimentar e perfil hormonal. / Previous studies from our laboratory have shown that chronic salt restriction decreases blood pressure, increases insulin resistance, and body weight (BW) in Wistar rats. The aim of this study was to evaluate some mechanisms of obesity and hormonal alterations associated with chronic salt restriction. Male Wistar rats were fed a low (LSD: 0,15% NaCl), normal (NSD: 1,27% NaCl), or high salt diet (HSD: 7,94% NaCl) from weaning. At the 12th week of age, tail-cuff blood pressure (TCBP), intra arterial blood pressure (BP), heart rate ((HR), food intake, BW, hormonal levels [leptin (LEP), growth hormone (GH), insulin (INSh - antibody anti-human insulin), T3, T4 and TSH] and 125I insulin decay study (DECAY-INS) were measured. To evaluate the food intake and body weight, each dietary group was divided in 3 subgroups, according to housing conditions: one (1C) or four (4C) rats per cage and one rat per metabolic cage (1MC) for daily food consumption and BW determinations during 7 days. Fasting plasma glucose (GLU) and insulin (INSr - antibody anti-rat insulin) were measured before the DECAY-INS. To evaluate the DECAY-INS, 1mCi human 125I-insulin was injected through the jugular catheter after 6-8 hours of food restriction. Blood samples were withdrawn through the carotid catheter every 30 sec during 2min, and in sequence, every 1 min for additional 10 minutes. 125Iinsulin was determined by RIA for human insulin. Rats on HSD had higher intra arterial BP and TCBP compared to rats on NSD and LSD. Heart rate was lower on LSD than on NSD. In all housing conditions, BW was higher on LSD than on NSD and HSD. Food intake was higher on HSD (1C and 4C) than on NSD and LSD. Plasma GH and LEP were higher on NSD than on the 68 other two groups. Plasma INSh was higher in LSD compared to HSD and NSD rats. Plasma total T4 was higher on HSD than on the NSD and LSD, TSH was lower on HSD than on NSD and HSD, and T3 was not different among all groups. Fasting GLU and INSr were higher on LSD compared to HSD and NSD rats. The exponential insulin decay was faster on HSD demonstrated by a lower 125I-INS half-life. These results suggest that obesity in rats on chronic LSD is due to a higher metabolic efficiency (higher body weight and lower diet consumption), that may be related to the hormonal consequences of LSD. Key Words: blood pressure, salt, sodium, food intake and hormonal profile.

INTRODUÇÃO: a ação dos hormônios contrarreguladores da insulina na cetoacidose diabética tem sido estudada desde a década 1970-80, e é sabido que seus níveis elevados, aumentando a resistência à insulina, têm papel importante na gênese da CAD. Leptina e ghrelina foram mais recentemente associadas à homeostase da glicose, no entanto, seu papel na CAD ainda é controverso. Os objetivos deste estudo foram: avaliar as alterações nas concentrações séricas de leptina e ghrelina presentes ao diagnóstico da CAD durante os primeiros três dias de seu tratamento e após a estabilização completa do quadro, as correlações com a insulina e outros contrarreguladores, comparando com indivíduos saudáveis. MÉTODOS: foram analisados 25 episódios de cetoacidose diabética em 22 pacientes admitidos no setor de emergência pediátrica de um hospital terciário em São Paulo, Brasil, entre março de 2010 e julho de 2013. Os episódios de cetoacidose foram manejados com reposição endovenosa de fluidos e eletrólitos e análogos de ação ultrarrápida de insulina subcutânea intermitente. Amostras para glicose, insulina, leptina, ghrelina, GH, cortisol e catecolaminas foram obtidas no momento da admissão (T0), durante o tratamento da cetoacidose (após 2, 4, 6, 12, 24 e 72 horas) e em um momento estável após a alta (TE). Os dados foram analisados utilizando-se os testes ANOVA ou Kruskal-Wallis para a comparação de variáveis contínuas durante o tratamento, Teste t de Student ou Mann Whitney para a comparação entre pacientes e grupo controle, e testes de Pearson ou Spearman para correlação entre as variáveis; p < 0.05 foi considerado significativo. RESULTADOS: observamos três fases distintas (a): o diagnóstico de CAD (T0) em que prevalecem hiperglicemia, insulinopenia e elevação de hormônios contrarreguladores; nesse momento, as concentrações de leptina foram menores que no grupo controle, provavelmente relacionadas à insuficiência de energia, estado hipercatabólico e elevação dos hormônios contrarreguladores; as concentrações de ghrelina foram menores que no grupo controle, apesar do hipercatabolismo, da hipoinsulinemia e da hiperglucagonemia, todas situações que fisiologicamente elevariam seus níveis, possivelmente devido à hiperglicemia marcante do momento; (b) durante o tratamento da CAD (T2 a T72): com redução gradual da glicemia até T24, elevação gradual da insulina, redução de glucagon, GH, cortisol e norepinefrina; nesse período, ocorreu elevação gradual da leptina após o início do tratamento com insulina, que atingiu níveis comparáveis ao GC no T72; redução da ghrelina (T4 menor que T72), provavelmente inibida pela hiperglicemia e por doses suprafisiológicas de insulina; e (c) após a resolução da CAD (TE): com hiperinsulinização; GH, cortisol e norepinefrina comparáveis ao GC, glucagon reduzido em relação ao GC, possivelmente supresso pelos altos níveis de insulina; as concentrações de leptina foram maiores que em T0 e comparáveis ao GC; os níveis de ghrelina, comparáveis ao diagnóstico e durante o tratamento da CAD, ainda significativamente menores que no GC, provavelmente influenciados pela hiperglicemia, hiperinsulinemia e baixos níveis de glucagon. CONCLUSÕES: as concentrações de leptina diminuídas ao diagnóstico de CAD tornam-se semelhantes em pacientes com DM1 estáveis em relação a indivíduos saudáveis, podendo ser um marcador de hipercatabolismo. As concentrações de ghrelina permaneceram baixas durante todo o estudo em pacientes diabéticos, independentemente da descompensação / INTRODUCTION: The role of glucoregulatory hormones in diabetic ketoacidosis have been investigated since 1970-80s and the elevation of growth hormone, cortisol and norepinephrine reduce the sensitivity to insulin. Leptin and Ghrelin have more recently been shown to regulate glucose and insulin metabolism; however, their functions in DKA are still controversial. The aims of this study were to analyze leptin, ghrelin and their relationships with other glucoregulatory hormones on diagnosis of diabetic ketoacidosis, during the first 72 hours of treatment and after recovery compared with healthy subjects. METHODS: We examined 25 DKA episodes in 22 patients who were admitted to the pediatric emergency department of a tertiary hospital in São Paulo, Brazil, from March 2010 to July 2013. These episodes were managed with fluids and electrolytes replacement and intermittent subcutaneous fast-acting insulin analogues. Samples for blood glucose, insulin, leptin, ghrelin, GH, cortisol, and catecholamines were obtained on admission (T0), during treatment (after 2, 4, 6, 12, 24 and 72 hours) and after discharge (TS). The control group (CG) was comprised by 21 healthy subjects, who submitted a single blood sample. Data were analyzed by ANOVA or Kruskal-Wallis to compare continuous variables during treatment, student t-test or Mann Whitney for comparisons between patients and controls, and Pearson or Spearman correlations between variables; p < 0.05 was considered to be significant. RESULTS: we observed three distinct phases: (a) on diagnosis of DKA (T0) where hyperglycemia, insulinopenia, and elevated glucoregulatory hormones prevail; leptin concentrations were lower than CG at this moment, probably related to energy insufficiency, hypercatabolic state, and elevated glucoregulatory hormones; ghrelin concentrations were lower than CG at this moment, despite hypercatabolism, hypoinsulinemia and hyperglucagonemia, situations that physiologically would increase them, possibly related to marked hyperglycemia at T0; (b) during DKA treatment (T2 to T72): with gradual reduction of blood glucose until T24, gradual rise of insulin; reduction of glucagon, GH, cortisol and norepinephrine. Leptin levels rises gradually after the start of insulin treatment and is comparable to control group at T72; reduction of ghrelin (T4 lower than T72), possibly inhibited by hyperglycemia and supraphysiological doses of insulin, all lower than CG; and (c) After DKA (TS), in an outpatient setting: with marked hyperinsulinization, GH, cortisol and norepinephrine were comparable to CG. Glucagon was lower than CG, possibly suppressed by high insulin levels; leptin was higher than T0 and comparable to CG; ghrelin levels were comparable to all samples during DKA treatment, and still significantly lower than CG, probably influenced by hyperglycemia, hyperinsulinemia and low glucagon levels. CONCLUSIONS: Low leptin levels were a marker of hypercatabolic state, with normalization of its concentrations with DKA resolution. Ghrelin was low in diabetic patients independent of metabolic derangements

Avaliamos o efeito do treinamento físico intenso e da restrição alimentar no eixo reprodutor e na expressão do RNAm do MCH em ratas fêmeas. Utilizamos ratas fêmeas sedentárias alimentadas (SA), sedentárias com restrição alimentar, treinadas alimentadas (TA) e treinadas com restrição alimentar. O grupo TR entrou em anestro a partir da quarta semana de experimento. Os grupos restritos apresentaram redução de peso corporal, ovários, úteros e tecido adiposo branco, comparados aos alimentados. Houve redução sérica de leptina nos grupos SR e TR. Os grupos TA, SR e TR apresentaram cistos ovarianos. A expressão do RNAm do ppMCH na área incerto-hipotalâmica (IHy) foi menor nos animais TR, SR e nos animais alimentados em proestro, e na área hipotalâmica lateral (LHA) foi maior nos animais em restrição alimentar e nos animais alimentados em proestro. Conclui-se que a leptina exerce forte influencia no eixo reprodutor feminino, tal ação parece ter envolvimento do MCH, sobretudo o proveniente da IHy, uma vez que o MCH da LHA tem relação com o estímulo a busca de alimento. / We evaluated the effect of intense physical training and food restriction on the reproductive axis and MCH mRNA expression in female rats. We used female rats fed sedentary (SF), food-restricted sedentary, trained fed (TF) and trained with food restriction. The group entered anestrus TR from the fourth week of the experiment. The restricted groups had reduced body weight, ovary, uterus and white adipose tissue, compared to the fed. There was a reduction of serum leptin in the SR and TR groups. The TF groups, SR and TR had ovarian cysts. The expression of mRNA ppMCH incerto-hypothalamic area (IHy) was lower in TR animals, SR and animals fed on proestrus, and the lateral hypothalamic area (LHA) were higher in animals on restricted food and animal feeding in proestrus. It is concluded that leptin exerts a strong influence on female reproductive axis, such action appears to have involvement of MCH, especially from the IHy, since the MCH of the LHA is related to the motivational behavior associated to the search for food.

L’hypertension artérielle pulmonaire (HTAP) est une maladie grave caractérisée par une obstruction progressive des artères pulmonaires de petit calibre, conduisant à une augmentation des résistances vasculaires pulmonaires et, à terme, à une défaillance cardiaque droite et au décès du patient. La vasoconstriction, le remodelage vasculaire et la dysfonction endothéliale pulmonaire sont autant de facteurs qui contribuent au développement et à la progression de la maladie. Plusieurs arguments sont également en faveur d’une hypothèse de désordres immunologiques, voire autoimmuns, dans la physiopathologie de l’HTAP. Malgré ces données, le lien entre endothélium pulmonaire et système immunitaire dans cette maladie restent peu connus. Ce travail de thèse a donc eu pour objectif d’étudier et mieux comprendre la nature et les conséquences d’une communication aberrante entre cellules endothéliales pulmonaires et système immunitaire dans la pathogénèse de l‘HTAP, afin d’identifier de nouvelles cibles thérapeutiques. Pour cela, nous avons analysé le rôle de la dysfonction endothéliale dans la régulation de deux processus de la dysfonction immunitaire : l’autoimmunité pour la réponse adaptative d’une part, et la sécrétion de cytokines en ce qui concerne la réponse innée d’autre part. A travers ce travail de thèse, nous avons mis en évidence l’existence d’une communication aberrante entre endothélium pulmonaire et système immunitaire dans l’HTAP et montré que l‘endothélium pulmonaire jouait un rôle primordial dans le contrôle des réponses adaptatives, en régulant la fonction des lymphocytes T régulateurs via la leptine, et dans la participation active à la réponse innée, en acquérant un phénotype pro-inflammatoire. Cette meilleure compréhension du rôle de la dysfonction endothéliale dans la dérégulation du système immunitaire présente dans l’HTAP pourrait aider au développement de nouvelles stratégies thérapeutiques dans cette maladie. / Pulmonary arterial hypertension (PAH) is a severe disease characterized by a progressive obstruction of small pulmonary arteries, leading to an increase in pulmonary vascular resistance and ultimately right heart failure and death. Vasoconstriction, vascular remodeling and pulmonary endothelial dysfunction contribute to the disease development and progression. Increasing evidence are also suggesting the importance of immune disorders, such as autoimmunity, in PAH pathophysiology. Despite these data, the link between pulmonary endothelium and immune system is still unclear. The objective of this work was to investigate and elucidate the nature and the consequences of an aberrant communication between pulmonary endothelial cells and immune system in PAH pathogenesis, in order to identify new therapeutical targets. Therefore, we analyzed the role of endothelial dysfunction in the control of two types of altered immune responses: autoimmunity for the adaptive response and cytokine secretion for the innate response. In this work, we highlighted the existence of an aberrant communication between pulmonary endothelium and immune system in PAH and showed that pulmonary endothelium played a key role in the control of adaptive responses, by regulating regulatory T lymphocyte function in a leptin-dependent manner, and by actively participating to the innate responses through a pro-inflammatory phenotype. A better understanding of the role of endothelial dysfunction in PAH immune system dysregulation may help to the development of new therapeutical strategies for this disease.

Introdução. A baixa capacidade aeróbia é um importante marcador de mau prognóstico e um forte preditor de risco de morte em pacientes encaminhados para o teste de esforço cardiopulmonar (TECP) por razões clínicas. O índice oxygen uptake efficiency slope (OUES), um novo índice de eficiência cardiorrespiratória, tem sido utilizado como marcador submáximo durante o TECP. Estudos recentes sugerem que a leptina pode desempenhar um papel importante na regulação da respiração e, consequentemente, o aumento dos níveis de leptina pode estar relacionado com a diminuição do OUES. No entanto, o impacto da síndrome metabólica (SMet) e da apneia obstrutiva do sono (AOS) no OUES é desconhecido. Objetivo. Investigar o comportamento do OUES em pacientes com SMet, associado ou não à AOS. Métodos. Foram estudados 73 pacientes com SMet (ATP-III), alocados em dois grupos de acordo com o índice de apneia/hipopneia (IAH) obtido na polissonografia noturna: SMet+AOS (IAH >= 15 eventos/hora, n=38, 49±1 anos, 33±0,6 kg/m2) e SMet-AOS (IAH < 15 eventos/hora, n=35, 46±1 anos, 31,8±0,6 kg/m2). Um grupo controle saudável (CS, n=20, 47±1 anos, 26,1±0,8 kg/m2), pareado por idade e gênero, foi também estudado. Os pacientes realizaram as seguintes avaliações: polissonografia; exames laboratoriais (glicemia, triglicérides, colesterol total e frações, leptina e proteína C-reativa); medidas antropométricas (altura, peso corporal, índice de massa corpórea, circunferência abdominal); composição corporal pela bioimpedância; medidas de pressão arterial; e TECP. Resultados. Ambos os grupos com SMet apresentaram prejuízo quando comparados ao grupo CS no peso, índice de massa corpórea e nos fatores de risco da SMet (circunferência abdominal, glicemia, triglicérides, HDL-c e pressão arterial sistólica e diastólica, P < 0,05). No TECP os grupos SMet+AOS e SMet-AOS apresentaram menores valores de consumo de oxigênio pico (VO2pico, 22,2±0,7; 21,7±0,9 e 28,0±1,1 ml/kg/min, respectivamente, Interação; P < 0,001) comparados com CS. Da mesma forma, os grupos SMet tiveram menores: VO2 no limiar anaeróbio (VO2LA), na relação VO2 e carga de trabalho (deltaVO2/deltaW) e no OUES (25,3±0,8; 25,0±0,9 e 31,1±1,2; Interação; P < 0.001) quando comparado com CS. Em análises posteriores, o OUES se correlacionou apenas com a massa gorda e com a leptina (R=-0,35; P=0,006). Conclusão. Independente da presença da AOS, pacientes com SMet apresentam diminuição da eficiência cardiorrespiratória. Os níveis elevados de leptina pode ser uma das explicações para essa diminuição nesses pacientes / Introduction. Low aerobic capacity is an important marker of poor prognosis and a strong predictor of risk of death in patients referred for cardiopulmonary exercise testing (CPET) for clinical reasons. The oxygen uptake efficiency slopes (OUES), a new cardiorespiratory efficiency index, has been used as a submaximal marker during CPET. Recent studies suggest that leptin may play an important role in regulating respiration, and consequently, increased levels of leptin may be related to decreased OUES. However, the impact of metabolic syndrome (MetS) and obstructive sleep apnea (OSA) in the OUES is unknown. Objective. To investigate the OUES in MetS patients with or without OSA. Methods. We studied 73 patients with MetS (ATP-III), allocated into two groups according to apnea/hypopnea index (AHI), assessed by nocturnal polysomnography: MetS+OSA (AHI >= 15 events/hour, n=38, 49±1 years, 33±0.6 kg/m2) and MetS-OSA (AHI < 15 events/hour, n=35, 46±1 years, 32±0.6 kg/m2). A healthy control group (CG, n=20, 47±1 years, 26.1±0.8 kg/m2), matched for age and gender, was also studied. The patients performed the following evaluations: polysomnography; Laboratory tests (glycemia, triglycerides, total cholesterol and fractions, leptin and C-reactive protein); Anthropometric measurements (height, body weight, body mass index, waist circumference); Body composition by bioimpedance; Blood pressure measurements; and CPET. Results. Both MetS groups had impairment in weight, body mass index, and MetS risk factors (waist circumference, glycemia, triglycerides, HDL-c and systolic and diastolic blood pressure, P < 0.05) compared with CG. MetS+OSA and MetS-OSA groups presented lower values of peak oxygen consumption (VO2peak, 22.2±0.7, 21.7±0.9 and 28.0±1.1 ml/kg/min, respectively; Interaction; P < 0.001) compared to CG. In the same way, MetS groups had lowest: VO2 at anaerobic threshold (VO2LA), ratio of VO2 and workload (deltaVO2/deltaW) and OUES (25.3±0.8, 25.0±0.9 and 31.1±1.2, Interaction; P < 0.001) compared with CG. In further analyzes, OUES correlated only with fat mass and leptin (R =-0.35, P =0.006). Conclusion. Regardless of the presence of OSA, MetS patients present decreased cardiorespiratory efficiency. Elevated levels of leptin may be one of the explanations for this decrease in these patients

Les maladies métaboliques telles que l’obésité ou le diabète de type 2 sont multifactorielles et multigéniques. Ces dernières décennies, la prévalence de ces pathologies notamment chez les enfants et les jeunes adultes a drastiquement augmenté, désignant ainsi l’environnement comme un élément-clé de ces pathologies. Ainsi, un environnement métabolique et/ou endocrinien déséquilibré pendant les périodes de gestation et/ou lactation prédispose la descendance à certaines pathologies à l’âge adulte. Ces phénotypes sont souvent associés, dans l’hypothalamus, structure impliquée dans la régulation de l’homéostasie énergétique, à des modifications de l’expression d’ARNm ou de protéines. Les microARNs (miARNs), régulateurs post-transcriptionnels majeurs, apparaissent donc comme des candidats intéressants pour l’exploration des mécanismes moléculaires sous-tendant les dysfonctionnements hypothalamiques. Le but du ce travail de thèse a été de caractériser le phénotype ainsi que le profil hypothalamique d’expression des miARNs chez des rats adultes nés et/ou allaités dans deux contextes différents de programmation métabolique. Dans un premier temps, nous avons étudié les conséquences à long-terme du blocage de la leptine postnatale, hormone contrôlant la mise en place du métabolisme et l’établissement de connexions hypothalamiques. Les rats traités avec un antagoniste de la leptine présentent un surpoids quel que soit le régime (normal ou hyperlipidique). Ces animaux présentent des signes de résistance à l’insuline dès le sevrage. Leur profil hypothalamique d’expression des microARNs est modifié notamment en ce qui concerne l’expression de certains miARNs associés à l’insulino-résistance périphérique. Dans un second temps nous avons étudié l’impact d’un régime maternel hyperlipidique. Ce régime maternel induit un moindre poids chez la descendance dès le deuxième jour postnatal. A l’âge adulte, les mâles présentent une sensibilité normale à l’insuline et à la leptine et ne sont pas prédisposés au surpoids lorsqu’ils sont soumis à un régime hyperlipidique. Les femelles présentent le même phénotype associé à une meilleure tolérance au glucose. Nous avons étudié le profil d’expression des miARNs dans les noyaux arqué et paraventriculaire de l’hypothalamus des mâles. L’expression de certains miARNs abondants est modulée chez les animaux nés de mères soumises au régime hyperlipidique. Les travaux de cette thèse ouvrent la voie à une étude systématique des profils hypothalamiques d’expression des miARNs dans un contexte de programmations métaboliques diverses. / Epidemiological studies have demonstrated that the incidence of metabolic diseases in adults such as hypertension, insulin resistance, obesity and the metabolic syndrome is markedly increased when maternal nutrition is altered at critical periods of foetal development. Numerous studies in humans and rodents have demonstrated the importance of the perinatal environment in metabolic programming. Thus, a metabolic or endocrine unbalanced environment predisposes offspring to various metabolic diseases in adulthood. These phenotypes are often associated with changes in mRNA or protein expression in the hypothalamus, a central structure involved in the regulation of energy homeostasis. In this context, microRNAs (miRNAs) appear as attractive candidates for exploration of the molecular mechanisms underlying hypothalamic dysfunction observed during metabolic programming. The aim of my project was to characterize the phenotype and the hypothalamic profile of miRNAs in the adult offspring in two contexts of metabolic programming. First, we studied the long-term consequences of early postnatal leptin blockade, a hormone described to be involved in the establishment of metabolism and hypothalamic connections. Rats treated with a leptin antagonist exhibit overweight (under chow and high-fat diet) and are also subjected to insulin resistance. As signs of insulin resistance appeared as soon as weaning, we analyzed at this age by microarray the hypothalamic miRNAs expression profile which reveals modification of hypothalamic miRNA expression pattern including miRNAs previously linked to peripheral insulin-résistance. In the second part of my thesis, we have demonstrated that a maternal high-fat diet induced a lower weight in the offspring from the second postnatal day. In adulthood, these animals exhibit similar insulin and leptin sensitivities as compared to controls and are not predisposed to overweight when exposed to a high fat diet. This phenotype is associated with changes in the miRNA expression profile in arcuate and paraventricular hypothalamic nuclei. In conclusion, we show for the first time that metabolic programming is associated with altered hypothalamic expression of miRNAs, which could contribute, at least partially, to the establishment of the offspring phenotype.

A hiperplasia prostática benigna (HPB) é a doença na qual a próstata demonstra um crescimento anormal e sua prevalência aumenta com o envelhecimento. Leptina, a adipocina mais notável, tem um importante papel na regulação do sistema reprodutivo. Esse trabalho tem por fim avaliar o papel da leptina no tecido prostático humano, utilizando a cultura de tecido in vitro, avaliando a proliferação celular e a expressão dos genes do fator de crescimento do fibroblasto 2 (FGF2), da enzima aromatase e dos genes apoptóticos. De 2009 a 2011, amostras de tecido hiperplásico humano foram obtidas pela prostatectomia transvesical em quinze pacientes com próstatas de volume aumentadas. Cada amostra foi dividida em quatro partes simétricas, mantidas no meio RPMI suplementado com soro fetal bovino a 10%, e 1ng/mL de gentamicina, adicionados a 16 ng/mL de leptina (Leptina) ou não (Controle). Após três horas a expressão dos genes de FGF2, aromatase, Bax, Bcl-x e Bcl-2 foram avaliados por RT-PCR em tempo real. A proliferação celular foi avaliada por imunohistoquímica para PCNA. O tratamento com leptina levou a um aumento na expressão de Bax (C=0.40.1; L=0.90.2; p<0.05), enquanto as expressões de Bcl-2 (C=19.95.6; L=5.61.8; p< 0.05) e Bcl-x (C=0.20.06; L=0.070.02; p<0.05) foram significativamente reduzidas. Não houve alteração significativa na expressão de FGF2, enquanto a expressão da aromatase foi significativamente (C=1.90.6; L=0.40.1; p<0.04) reduzida. A leptina também levou a um aumento na proliferação celular (C=21.80.5; L=64.80.9; p<0.0001). Dessa forma, concluímos que a leptina tem um importante papel na manutenção do crescimento fisiológico da próstata, desde que estimula tanto a proliferação celular como a apoptose, com diminuição na expressão do gene da aromatase. / The benign prostate hyperplasia (BPH) is the disease in which the prostate shows an abnormal growth and its prevalence rise up with aging. Leptin, the most well characterized adipokine, has an important role regulating the reproductive system. This paper aimed to evaluate the leptin role in the human prostate tissue, using an in vitro tissue culture system, on the cellular proliferation and the expression of fibroblast growth factor 2, aromatase enzyme and apoptotic genes. From 2009 to 2011, hyperplasic prostate tissue samples obtained by transvesical prostatectomy in fifteen patients with enlarged prostates were used. Each sample was divided in four symmetric parts which were maintained in RPMI medium supplemented with 10% fetal bovine serum, 1 ng/mL of gentamicin and added with 16 ng/mL leptin (L) or not (C). After 3 hours gene expression of FGF2, aromatase, Bax, Bcl-x and Bcl-2 were evaluated by Real Time RT-PCR. Cellular proliferation was evaluated by imunohistochemistry for PCNA. The leptin treatment led to an increase in the expression of Bax (C=0.40.1; L=0.90.2; p<0.05) while Bcl-2 (C=19.95.6; L=5.61.8; p< 0.05) and Bcl-x (C=0.20.06; L=0.070.02; p<0.05) expressions were significantly reduced. There was no significant alteration in the FGF2 expression, while the aromatase expression was significantly (C=1.90.6; L=0.40.1; p<0.04) reduced. Leptin also resulted in an increase in cellular proliferation (C=21.80.5; L=64.80.9; p<0.0001). The conclusion is that leptin has an important role in maintaining the physiological growth of the prostate since it stimulates both cellular proliferation and apoptosis, with the decrement in the aromatase gene expression

La voie de signalisation mTORC1 hypothalamique (mammalian target of rapamycincomplexe 1) intègre les signaux hormonaux et nutritionnels. La disponibilité des nutrimentsmodule les espèces réactives derivées de l’oxygène (ROS) qui régulent l’activité des neuronesà propiomélanocortine (POMC). La modulation de la prise alimentaire induite par les ROSpourrait impliquer mTORC1.Des souris C57Bl6J et wild-type (WT) ou invalidées pour S6K1 (S6K1-KO), principaleprotéine cible de mTORC1, ou invalidées pour raptor, protéine clé de mTORC1,sélectivement au niveau des neurones anorexigènes POMC (POMC-raptor-KO) ont ététraitées par injections intracérébroventriculaires (ICV) d’H2O2 ou d’honokiol (piégeur deROS), uniques ou combinées avec un inhibiteur de mTOR (rapamycine) ou un activateur demTOR (leptine).L’H2O2 ICV induit une augmentation de l’activité hypothalamique mTORC1, de l’activationneuronale du noyau arqué, de l’expression des ROS dans les neurones POMC, associée à unediminution de la prise alimentaire et du poids. Cet effet anorexigène est diminué chez lessouris S6K1-KO, chez les C57Bl6J après administration de rapamycine, et chez les POMCraptor-KO.L’honokiol ICV bloque l’effet anorexigène de la leptine, suggérant que cet effet soitdépendant des ROS. La leptine ICV entraine une augmentation des ROS dans les neuronesPOMC des souris C57Bl6J et POMC-raptor-WT, mais pas chez les POMC-raptor-KO.Nos résultats montrent que la régulation de la prise alimentaire induite par les ROS nécessiteune voie mTORC1 fonctionnelle et que l’effet anorexigène de la leptine nécessite uneaugmentation de ROS, mTORC1 dépendante, au niveau des neurones POMC. / The mechanistic target of rapamycin complex 1 (mTORC1) pathway is an importanthypothalamic integrator of nutrients and hormones. Nutrient availability also affects thereactive oxygen species (ROS) in propiomelanocortin (POMC) neurons and regulatesneuronal activity. We hypothesize that modulation of mTORC1 activity mediates ROS effectson food intake.To this purpose, C57Bl6J mice or WT mice and their KO littermates either deficient for themTORC1 downstream target S6K1 or for the mTORC1 component raptor specifically inPOMC neurons (POMC-raptor-KO) were treated with an intracerebroventricular (ICV)injection of the ROS producer H2O2 or the ROS scavenger honokiol, alone or in combinationwith the mTOR inhibitor rapamycin or the mTOR activator leptin.ICV H2O2 induced phosphorylation of S6K1 within the hypothalamus, increased expressionof c-fos, a marker of neuronal activity, in the arcuate nucleus and increased ROS in POMCneurons. These effects were associated with a significant decrease in food intake. Theanorexigenic effect of ICV H2O2 was not seen in S6K1-KO mice, in C57Bl6J mice cotreatedwith rapamycin (an mTOR inhibitor) and in POMC-raptor-KO mice.Similarly, ICV honokiol administration combined with a leptin injection blunted theanorexigenic effect of leptin, suggesting that leptin requires ROS formation to reduce FI. ICVadministration of leptin increased ROS in POMC neurons in C57Bl6J and POMC-raptor-WTmice, but not in POMC-raptor-KO mice.Our results demonstrate that ROS modulators require a functional mTORC1 pathway toregulate food intake and that leptin needs an mTORC1-dependent increase in ROS levels inPOMC neurons to decrease food intake.

Avaliamos o efeito do treinamento físico intenso e da restrição alimentar no eixo reprodutor e na expressão do RNAm do MCH em ratas fêmeas. Utilizamos ratas fêmeas sedentárias alimentadas (SA), sedentárias com restrição alimentar, treinadas alimentadas (TA) e treinadas com restrição alimentar. O grupo TR entrou em anestro a partir da quarta semana de experimento. Os grupos restritos apresentaram redução de peso corporal, ovários, úteros e tecido adiposo branco, comparados aos alimentados. Houve redução sérica de leptina nos grupos SR e TR. Os grupos TA, SR e TR apresentaram cistos ovarianos. A expressão do RNAm do ppMCH na área incerto-hipotalâmica (IHy) foi menor nos animais TR, SR e nos animais alimentados em proestro, e na área hipotalâmica lateral (LHA) foi maior nos animais em restrição alimentar e nos animais alimentados em proestro. Conclui-se que a leptina exerce forte influencia no eixo reprodutor feminino, tal ação parece ter envolvimento do MCH, sobretudo o proveniente da IHy, uma vez que o MCH da LHA tem relação com o estímulo a busca de alimento. / We evaluated the effect of intense physical training and food restriction on the reproductive axis and MCH mRNA expression in female rats. We used female rats fed sedentary (SF), food-restricted sedentary, trained fed (TF) and trained with food restriction. The group entered anestrus TR from the fourth week of the experiment. The restricted groups had reduced body weight, ovary, uterus and white adipose tissue, compared to the fed. There was a reduction of serum leptin in the SR and TR groups. The TF groups, SR and TR had ovarian cysts. The expression of mRNA ppMCH incerto-hypothalamic area (IHy) was lower in TR animals, SR and animals fed on proestrus, and the lateral hypothalamic area (LHA) were higher in animals on restricted food and animal feeding in proestrus. It is concluded that leptin exerts a strong influence on female reproductive axis, such action appears to have involvement of MCH, especially from the IHy, since the MCH of the LHA is related to the motivational behavior associated to the search for food.

INTRODUÇÃO: a ação dos hormônios contrarreguladores da insulina na cetoacidose diabética tem sido estudada desde a década 1970-80, e é sabido que seus níveis elevados, aumentando a resistência à insulina, têm papel importante na gênese da CAD. Leptina e ghrelina foram mais recentemente associadas à homeostase da glicose, no entanto, seu papel na CAD ainda é controverso. Os objetivos deste estudo foram: avaliar as alterações nas concentrações séricas de leptina e ghrelina presentes ao diagnóstico da CAD durante os primeiros três dias de seu tratamento e após a estabilização completa do quadro, as correlações com a insulina e outros contrarreguladores, comparando com indivíduos saudáveis. MÉTODOS: foram analisados 25 episódios de cetoacidose diabética em 22 pacientes admitidos no setor de emergência pediátrica de um hospital terciário em São Paulo, Brasil, entre março de 2010 e julho de 2013. Os episódios de cetoacidose foram manejados com reposição endovenosa de fluidos e eletrólitos e análogos de ação ultrarrápida de insulina subcutânea intermitente. Amostras para glicose, insulina, leptina, ghrelina, GH, cortisol e catecolaminas foram obtidas no momento da admissão (T0), durante o tratamento da cetoacidose (após 2, 4, 6, 12, 24 e 72 horas) e em um momento estável após a alta (TE). Os dados foram analisados utilizando-se os testes ANOVA ou Kruskal-Wallis para a comparação de variáveis contínuas durante o tratamento, Teste t de Student ou Mann Whitney para a comparação entre pacientes e grupo controle, e testes de Pearson ou Spearman para correlação entre as variáveis; p < 0.05 foi considerado significativo. RESULTADOS: observamos três fases distintas (a): o diagnóstico de CAD (T0) em que prevalecem hiperglicemia, insulinopenia e elevação de hormônios contrarreguladores; nesse momento, as concentrações de leptina foram menores que no grupo controle, provavelmente relacionadas à insuficiência de energia, estado hipercatabólico e elevação dos hormônios contrarreguladores; as concentrações de ghrelina foram menores que no grupo controle, apesar do hipercatabolismo, da hipoinsulinemia e da hiperglucagonemia, todas situações que fisiologicamente elevariam seus níveis, possivelmente devido à hiperglicemia marcante do momento; (b) durante o tratamento da CAD (T2 a T72): com redução gradual da glicemia até T24, elevação gradual da insulina, redução de glucagon, GH, cortisol e norepinefrina; nesse período, ocorreu elevação gradual da leptina após o início do tratamento com insulina, que atingiu níveis comparáveis ao GC no T72; redução da ghrelina (T4 menor que T72), provavelmente inibida pela hiperglicemia e por doses suprafisiológicas de insulina; e (c) após a resolução da CAD (TE): com hiperinsulinização; GH, cortisol e norepinefrina comparáveis ao GC, glucagon reduzido em relação ao GC, possivelmente supresso pelos altos níveis de insulina; as concentrações de leptina foram maiores que em T0 e comparáveis ao GC; os níveis de ghrelina, comparáveis ao diagnóstico e durante o tratamento da CAD, ainda significativamente menores que no GC, provavelmente influenciados pela hiperglicemia, hiperinsulinemia e baixos níveis de glucagon. CONCLUSÕES: as concentrações de leptina diminuídas ao diagnóstico de CAD tornam-se semelhantes em pacientes com DM1 estáveis em relação a indivíduos saudáveis, podendo ser um marcador de hipercatabolismo. As concentrações de ghrelina permaneceram baixas durante todo o estudo em pacientes diabéticos, independentemente da descompensação / INTRODUCTION: The role of glucoregulatory hormones in diabetic ketoacidosis have been investigated since 1970-80s and the elevation of growth hormone, cortisol and norepinephrine reduce the sensitivity to insulin. Leptin and Ghrelin have more recently been shown to regulate glucose and insulin metabolism; however, their functions in DKA are still controversial. The aims of this study were to analyze leptin, ghrelin and their relationships with other glucoregulatory hormones on diagnosis of diabetic ketoacidosis, during the first 72 hours of treatment and after recovery compared with healthy subjects. METHODS: We examined 25 DKA episodes in 22 patients who were admitted to the pediatric emergency department of a tertiary hospital in São Paulo, Brazil, from March 2010 to July 2013. These episodes were managed with fluids and electrolytes replacement and intermittent subcutaneous fast-acting insulin analogues. Samples for blood glucose, insulin, leptin, ghrelin, GH, cortisol, and catecholamines were obtained on admission (T0), during treatment (after 2, 4, 6, 12, 24 and 72 hours) and after discharge (TS). The control group (CG) was comprised by 21 healthy subjects, who submitted a single blood sample. Data were analyzed by ANOVA or Kruskal-Wallis to compare continuous variables during treatment, student t-test or Mann Whitney for comparisons between patients and controls, and Pearson or Spearman correlations between variables; p < 0.05 was considered to be significant. RESULTS: we observed three distinct phases: (a) on diagnosis of DKA (T0) where hyperglycemia, insulinopenia, and elevated glucoregulatory hormones prevail; leptin concentrations were lower than CG at this moment, probably related to energy insufficiency, hypercatabolic state, and elevated glucoregulatory hormones; ghrelin concentrations were lower than CG at this moment, despite hypercatabolism, hypoinsulinemia and hyperglucagonemia, situations that physiologically would increase them, possibly related to marked hyperglycemia at T0; (b) during DKA treatment (T2 to T72): with gradual reduction of blood glucose until T24, gradual rise of insulin; reduction of glucagon, GH, cortisol and norepinephrine. Leptin levels rises gradually after the start of insulin treatment and is comparable to control group at T72; reduction of ghrelin (T4 lower than T72), possibly inhibited by hyperglycemia and supraphysiological doses of insulin, all lower than CG; and (c) After DKA (TS), in an outpatient setting: with marked hyperinsulinization, GH, cortisol and norepinephrine were comparable to CG. Glucagon was lower than CG, possibly suppressed by high insulin levels; leptin was higher than T0 and comparable to CG; ghrelin levels were comparable to all samples during DKA treatment, and still significantly lower than CG, probably influenced by hyperglycemia, hyperinsulinemia and low glucagon levels. CONCLUSIONS: Low leptin levels were a marker of hypercatabolic state, with normalization of its concentrations with DKA resolution. Ghrelin was low in diabetic patients independent of metabolic derangements