A suplementação dietética com AGCM induz resistência à insulina, redução de peso ponderal e aumento da adiposidade em ratos Wistar. Adipócitos isolados apresentam reduzidas captação de glicose estimulada por insulina e atividade/fosforilação da proteína AMPK. A expressão protéica do IR no tecido hepático está aumentada em animais tratados com AGCM com redução do grau de fosforilação, enquanto que o grau de fosforilação da proteína AKT permaneceu semelhante entre os grupos. Ilhotas pancreáticas isoladas apresentam redução na secreção de insulina quando incubadas com altas concentrações de glicose, diminuição do conteúdo total de insulina, hipersensibilidade a leucina e/ou arginina e aumento do percentual de morte celular com diminuída expressão da proteína AKT_1 . Desta forma, utilização em longo prazo dessa estratégia nutricional pode interferir no crescimento normal do indivíduo, na sensibilidade à insulina e possívelmente, desenvolvimento e instalação do diabetes. / The introduction of MCFA into diet induces insulin resistance, reduced body weight gain, and increased adiposity in Wistar rats. Isolated adipocytes have reduced insulin induced glucose uptake and phosphorylation/activation of AMPK protein. The insulin receptor protein expression is increased in liver of MCFA fed rats accompanied by reduced tyrosine phosphorylation, with similar AKT serine phosphorylation. Isolated pancreatic islets had reduced glucose stimulated insulin secretion due to high glucose exposure and reduced insulin content; higher insulin secretion induced by leucine and arginine, and increased apoptosis with reduced AKT protein level. In these regard, the chronic ingestion of MCFA may interfere with normal body growth, with the insulin sensitivity and may participate with the development of diabetes.

INTRODUÇÃO O câncer é uma das principais causas de morte no mundo, sendo responsável por cerca de 8 milhões de mortes por ano, segundo dados da OMS. As mortes por câncer são provocadas por tumores que se originam em órgãos como pulmão, fígado, estômago, intestino, mama e esôfago. As células-tronco mesenquimais (CTM) foram identificadas em vários órgãos e estudos da sua interação com células tumorais têm apresentado resultados indicando ação inibitória sobre alguns tipos de tumores. Para explorar essa questão foram analisados os efeitos sobre células tumorais de carcinoma hepatocelular humano (HepG-2) do meio condicionado (MC) obtido do cultivo de CTM isoladas de tecido adiposo (TA), líquido amniótico (LA) e geleia de Wharton (GW). MÉTODOS Os MCs foram coletados após 24 horas de incubação das CTM sub-confluentes com ?-MEM contendo 20% de soro fetal bovino (SFB). Os MCs foram centrifugados e passados através de filtros de 0,22 ?M e armazenadas a -20 °C. O MC da própria célula HepG-2 foi utilizado como controle. Os efeitos dos MCs sobre a proliferação de células HepG-2 foram testados por ensaio MTT, em várias concentrações após 24 h de incubação. O ciclo celular de células HepG-2 tratadas com MC a 25%, 50% ou 75% foi analisado por citometria de fluxo (coloração IP) utilizando o software Modfit LT. A expressão dos genes bcl-2, bcl-6, CCND1 foi analisada por RT-PCR. A proliferação celular foi avaliada pela expressão das proteínas survivina, Bcl-2, PCNA e Ki-67 e pela quantificação de mitocôndrias com corante MitoTracker, assim como pelo potencial de membrana mitocondrial por corante JC-1 Mitoscreen utilizando equipamento de high content analysis. RESULTADOS Os meios condicionados de células-tronco de tecido adiposo (MC-TA) não alteraram a proliferação de células tumorais HepG-2 e os meios condicionados de células-tronco de líquido amniótico (MC-LA) e de geleia de Wharton (MC-GW) provocam aumento da proliferação, confirmada pela contagem de células com núcleos corados com Hoescht 33342. A análise de alterações do ciclo celular demonstrou que a exposição de células HepG-2 aos meios MC-LA diminuem as células na fase G0/G1 e aumentam na fase G2/M do ciclo celular. A expressão dos genes CCND1, Bcl2 e Bcl6 que estão relacionados à proliferação e morte celular não apresentaram alteração. A quantificação das proteínas PCNA e survivina não apresentou alteração sob efeito dos MC, porém a comparação direta entre células tratadas com MC-LA e MC-TA indicou a tendência das células tratadas com MC-LA proliferarem. O percentual de células HepG-2 expressando a proteína Ki-67 foi significativamente menor em relação ao controle quando tratadas com MC-TA, não apresentando diferenças quando tratadas com MC-LA e MC-GW. A contagem de mitocôndrias evidenciou aumento de mitocôndrias nas células HepG-2 tratadas com MC-LA e efeito não significativo dos tratamentos com MC-TA e MC-GW. A diferença do potencial de membrana mitocondrial por JC-1 apresentou aumento da polarização nas células HepG-2 cultivadas com MC-TA. CONCLUSÃO Os resultados confirmam que as células-tronco mesenquimais diferem de acordo com a sua origem tecidual em sua ação na proliferação das células HepG-2. Mais estudos são necessários para estabelecer a causa destas ações, que não parecem ser devido a mediadores mais comuns na proliferação e morte celular / INTRODUCTION Cancer is a leading cause of death worldwide, accounting for about 8 million deaths per year, according to WHO data. Cancer deaths are caused by tumors that originate in organs such as lung, liver, stomach, bowel, breast and esophagus. The mesenchymal stem cells (MSCs) have been identified in many organs and studies of their interaction with tumor cells have shown results indicating an inhibitory effect on some types of tumors. To explore this question the effects of the conditioned medium (CM) obtained from mesenchymal stem cell isolated from adipose tissue (AT), amniotic fluid (AF) and Wharton jelly (WJ) on tumor cells of human hepatocellular carcinoma (HepG-2) were analyzed. METHODS The MSC CM was collected after 24 hours incubation of sub confluent MSC with ?-MEM containing 20% fetal bovine serum (FBS). The MSC CM were centrifuged and passed through 0.22 ?M filter and stored at -20° C. The CM of HepG-2 cell itself was used as control. The effects of contrast media on proliferation of HepG-2 cells were tested by MTT assay at various concentrations after 24 h of incubation. The cell cycle HepG-2 cells treated with CM at 25%, 50% or 75% was analyzed by flow cytometry (PI staining) using Modfit software LT. The expression of the genes Bcl-2, Bcl-6, CCND1 was analyzed by RT-PCR. Cell proliferation was assessed by the expression of survivin, Bcl-2, Ki-67 and PCNA proteins, and the quantization of mitochondrial by MitoTracker dye, as well as the mitochondrial membrane potential by JC-1 Mitoscreen dye using high content analysis equipment. RESULTS The conditioned media of mesenchymal stem cells (MSC) from adipose tissue (AT-CM) did not alter the proliferation of tumor HepG-2 cells and conditioned media of MSC cells from amniotic fluid (AF-CM) and Wharton jelly (WJ-CM) caused increased proliferation, confirmed by counting cells with nuclei stained with Hoechst 33342. The cell cycle analysis showed that exposure of HepG-2 cells to AF-CM means decrease the cells in G0 / G1 cell cycle phase and increase in phase G2 / M. The expression of Bcl2, Bcl6 and CCND1 genes that are related to proliferation and cell death did not change. The quantization of PCNA and survivin protein did not change under the effect of conditioned media, but a direct comparison between cells treated with AF-CM and AT-CM indicated the tendency of cells treated with AF-CM proliferate. The percentage of HepG-2 cells expressing the protein Ki-67 was significantly lower than the control when treated with AT-CM and no differences when treated with AF-CM and WJ-CM. The counting of mitochondria showed increased mitochondria in HepG-2 cells treated with AF-CM and no significant effect of treatment with WJ-CM and AT-CM. The difference in mitochondrial membrane potential by JC-1 showed an increase in polarization in HepG-2 cells cultured with AT-CM. CONCLUSION The results confirm that mesenchymal stem cells differ according to their tissue origin in its action on the proliferation of HepG-2 cells. More studies are needed to establish the cause of these actions, which seem to be not related to the most common mediators in cell proliferation and death

A cicatrização de feridas crônicas e de defeitos complexos representam desafios para a cirurgia plástica reconstrutiva. Novos tratamentos emergiram com a utilização de células tronco mesenquimais, com especial interesse para as derivadas de tecido adiposo (CTMs-TA), por possuir algumas vantagens em relação às derivadas da medula óssea. Algumas doenças poderiam se beneficiar com o uso das CTMs-TA, em particular, as feridas de pacientes portadores de anemia falciforme que ainda representam um desafio terapêutico. Neste estudo, investigou-se a existência de possíveis áreas doadoras preferenciais de CTMs-TA no tecido adiposo (TA) por meio da comparação de aspectos qualitativos e quantitativos das CTMs-TA derivadas de cinco diferentes áreas corporais. Posteriormente, analisou-se a influência do sobrenadante dessas células, rico em citocinas e fatores de crescimento, sobre a migração de fibroblastos de indivíduos normais e de portadores de anemia falciforme in vitro. Não foram observadas diferenças qualitativas entre as CTMs-TA das cinco áreas analisadas. A região do dorso apresentou número maior de CTMs, com diferença significativa em relação à região das coxas. A adição de sobrenadante produzido por CTMs-TA demonstrou aumento da velocidade de migração dos fibroblastos de forma similar para os normais e os falciformes. O microambiente desfavorável presente nas feridas falciformes parece exercer importante influência sobre esses fibroblastos, pois uma vez corrigido o microambiente com os meios de cultivo apropriados, as células apresentaram taxa de duplicação e velocidade de migração semelhante à dos fibroblastos normais in vitro. / The healing of chronic wounds and complex defects represents challenges for reconstructive plastic surgery. New treatments have emerged with the use of multipotent mesenchymal cells, with special interest for the adipose-derived stem cells (ADSCs) as it has some advantages over the bone marrow-derived. Some diseases could benefit from the use of ADSCs, in particular, the wounds of patients with sickle cell anemia that still represent a therapeutic challenge. In this study, we investigated the existence of possible preferential donor areas of ADSCs in adipose tissue by comparing qualitative and quantitative aspects of ADSCs derived from five different body areas. Later, we analyzed the influence of the supernatant of these cells, rich in cytokines and growth factors, on the migration of fibroblasts from healthy individuals and from patients with sickle cell anemia in vitro. No qualitative differences were observed among the ADSCs of the five areas analyzed. The dorsum presented a higher number of ADSCs, with a significant difference in relation to the thigh. Addition of supernatant produced by ADSCs has been shown to increase the rate of migration of fibroblasts in a similar way to healthy and sickle cells. The unfavorable microenvironment present in sickle wounds seems to exert a significant influence on these fibroblasts because once the microenvironment was corrected with the appropriate culture media, the cells had a doubling rate and migration rate similar to normal fibroblasts in vitro.

Numerous studies have investigated how bulk lipid metabolism is influenced in obesity and in particular how the composition of triglycerides found in the cytosol change with increased adipocyte expansion. However, in part reflecting the analytical challenge the composition of cell membranes, and in particular glycerophospholipids, an important membrane component, have been seldom investigated. Cell membrane components contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for numerous proteins and providing important pools for metabolites, such as choline for one-carbon metabolism and S-adenosylmethionine for DNA methylation. Here, I have conducted a comprehensive lipidomic and transcriptomic study of white adipose tissue in mice that become obese either through genetic modification (ob/ob genotype), diet (high-fat diet) or a combination of the two across the life course. Specifically, I demonstrated that the changes in triglyceride metabolism that dominate the overall lipid composition of white adipose tissue were distinct from the compositional changes of glycerophospholipids. These latter lipids became more unsaturated to maintain the fluidity and normal function of the membrane in the initiation of obesity but then turned saturated after long-term administration of HFD and aging. This suggests that while triglycerides within the adipose tissue may be a relatively inert store of lipids, the compositional changes occur in cell membranes with more far-reaching functional consequences in both obesity and aging. The two-phase change of phospholipids can be correlated well with transcriptional and one-carbon metabolic changes within the adipocytes. The transcriptomic study demonstrated that the lipid metabolic pathways regulated by the peroxisome, AMPK, insulin and PPARγ signaling were activated in the initiation of obesity but inhibited in the adipose tissue of old ob/ob mice along with up-regulated inflammation pathways. The brown and white adipose tissue of PPARα-knock-out mice were also studied by lipidomic tools to get a deeper understanding of the effect of the peroxisome and PPAR system on adipose tissue and lipid metabolism during obesity. Most of the lipids were increased and became more saturated and shorter in adipose tissues of PPARα null mice, which is in good accordance with the results of the former animal study. In conclusion, my work using different rodent models and multi-omics techniques demonstrated a protective metabolic mechanism activated in the initiation but impaired at the end of the processes of obesity and aging, which could be an explanation of the similarity of obesity and aging in terms of high incidence of the metabolic syndrome and related diseases.

Le tissu adipeux est traditionnellement décrit comme étant constitué de lobules : des entités de formes ovoïdales composées de cellules et de vaisseaux et faiblement connectées entre elles.Récemment, il a été montré qu’un potentiel métabolique spécifique (le browning) co-localise avec cette organisation en lobules au sein d’un même tissu. Dans ce travail de thèse, nous nous intéressons à décrire plus précisément l’organisation structurelle et fonctionnelle du tissu adipeux selon plusieurs aspects. Dans un premier temps, on s’attache à segmenter les lobules du tissu adipeux en utilisant une méthode de traitement d’image originale. Nous mettons en évidence une organisation 3D complexe et suivant plusieurs échelles. En particulier, il semble que le potentiel de browning soit également lié à une organisation structurelle particulière en clusters de lobules. Dans un second temps, à partir d’imagerie 3D, nous reconstruisons le réseau vasculaire entier du tissu adipeux et réalisons une simulation d’écoulements sanguins micro-vasculaires. Plusieurs hétérogénéités structurelles et fonctionnelles sont alors mises en valeurs à l’aide d’une analyse en communautés qui composent le tissu adipeux (par algorithme de clustering). Ces résultats confirment l’existence d’une zone centrale fortement vascularisée et qui se démarque également comme étant le lieu d’une perfusion sanguine d’intensité différente. Dans une dernière partie, nous abordons la question de transferts thermiques entre vaisseaux sanguins suivant des géométries simples mais pertinentes. Nous réalisons une étude systématique des paramètres adimensionnels clés du problème et mettons en évidence un invariant des échanges de chaleur : un optimum à faible nombre de Péclet (convection de même ordre que la diffusion). Nous introduisons également une méthode de calibration de paramètres effectifs dans le contexte des modèles homogénéisés de température à travers des tissus vascularisés. / Adipose tissue is traditionally described as consisting of lobules: ovoid-shaped entities composed of cells and vessels and weakly connected to each other. Recently, it has been shown that a specific metabolic potential (browning) colocalize with this organization in lobules within the same tissue. In this thesis work, we are interested in describing more precisely the structural and functional organization of adipose tissue from several aspects. We first perform a segmentation of adipose tissue lobules using an original image processing method. We highlight a complex 3D organization and relevant on several scales. In particular, it seems that browning potential is also linked to a particular structural organisation in clusters of lobules. In a second step, using 3D imaging, we reconstruct the entire vascular network of adipose tissue and simulate micro-vascular blood flow. Several structural and functional heterogeneities are then highlighted using an analysis in communities among adipose tissue (by clustering algorithm). These results confirm the existence of a highly vascularized central area that also stands out as the site of a more marked blood perfusion. In a last part, we approach the question of heat transfers between blood vessels following simple but relevant geometries. We carry out a systematic study of the key dimensionless parameters of the problem and highlight an invariant of heat exchanges: an optimum at low Péclet number (convection of the same order as diffusion). We also introduce a method of calibrating effective parameters in the context of homogenized temperature models across vascularized tissues.

Evidências na literatura demonstraram que o aumento da adiposidade confere maior suscetibilidade ao desenvolvimento de diabetes tipo 2, pois o tecido adiposo branco (TAB) atua na regulação da homeostasia energética e da sensibilidade à insulina através da sua atividade endócrina e de interações com reguladores neuroendócrinos. O treinamento físico aeróbio tem sido fortemente recomendado para a prevenção e tratamento do diabetes tipo 2, pois promove adaptações no metabolismo energético que contribuem diretamente para a melhora da resposta glicêmica e para o controle de peso corporal. Embora esteja claro na literatura o papel do treinamento físico contra o desenvolvimento de distúrbios no metabolismo da glicose e obesidade, uma lacuna de conhecimento ainda existe quando buscamos informações a respeito da participação metabólica do TAB na prevenção do diabetes tipo 2 através do treinamento físico aeróbio. Dessa forma, o presente estudo teve como objetivo testar a hipótese de que o efeito protetor do treinamento físico contra o desenvolvimento de diabetes tipo 2 é mediado por adaptações funcionais do TAB. Para isso, foram utilizados camundongos alimentados com dieta normocalórica e de cafeteria submetidos ou não ao treinamento físico aeróbio. O treinamento físico aeróbio foi eficaz para a prevenção do diabetes tipo 2, e essa resposta foi associada à menor adiposidade corporal resultante do aumento da lipólise e da capacidade oxidativa do TAB induzido pela maior ativação via da AMPK/ACC / Evidence in the literature have shown that increased adiposity confers greater susceptibility to developing type 2 diabetes and white adipose tissue (WAT) acts in the regulation of energy homeostasis and insulin sensibility through its endocrine activity and interaction with neuroendocrine regulators. Aerobic physical training has been strongly recommended for the prevention and treatment of type 2 diabetes because it promotes adaptations in the energy metabolism that contribute directly to the improvement of glycemic metabolism and body weight control. Although it is clear in the literature the role of physical training against the development of disturbances in the glucose metabolism and obesity, the role of WAT to prevent type 2 diabetes through physical training was poorly investigated. Thus, the present study aimed to test the hypothesis that the protective effect of physical training against the development of type 2 diabetes is mediated by functional adaptations of WAT. For this, we used mice fed with control or cafeteria diet and submitted or not to aerobic physical training. The physical training was effective for the prevention of type 2 diabetes, and this response was associated with lower body fat due to increased lipolysis and oxidative capacity of WAT induced by the activation of AMPK/ACC

A manutenção do estado progenitor durante o cultivo de células mesenquimais progenitoras derivadas do tecido adiposo (MSCs-TA), caracterizado pelo potencial de diferenciação e da capacidade de autorrenovação, é atualmente um dos maiores desafios da terapia celular. Sabendo da influência da angiogênese no desenvolvimento de tecidos de origem mesenquimal, avaliamos se um ambiente pro-angiogênico mimetizado em cultura forneceria condições para manutenção de um estado progenitor durante o processo de expansão celular. Utilizando como modelo de um ambiente pró-angiogênico o cultivo no meio EGM-2, o qual é suplementado pelos fatores de crescimento EGF, FGF-2, IGF e VEGF, nós demonstramos que a presença de tais fatores pró-angiogênicos é fundamental para a manutenção do estado progenitor de MSCs-TA em cultura. Verificamos que a presença de tais fatores de crescimento possibilitaram às MSCs-TA apresentarem um alto potencial de diferenciação adipogênico e osteogênico em comparação ao meio convencional DMEM/F12 e ao meio EBM, ausente de fatores. Além disso, o cultivo na presença de fatores pró-angiogênicos aumentou o potencial clonogênico das MSCs-TA, ao mesmo tempo em que aumentou a capacidade proliferativa destas células. Dentre os fatores de crescimento, EGF e FGF-2 foram responsáveis pelos efeitos mais robustos. Ao mesmo tempo, células cultivadas nas presença destas citocinas foram capazes de manter a morfologia fibroblastóide e apresentaram alta expressão do fator de pluripotência Klf-4. Em concordância com estes achados, o transplante subcutâneo de MSCs-TA cultivadas nestas condições mostrou que aquelas mantidas em EGM-2 geram um tecido semelhante ao tecido formado pela fração estromal vascular não cultivada. Estes resultados reforçam o papel do ambiente pró-angiogênico na manutenção do estado progenitor de MSCs-TA, e que tal estado foi proporcionado pela ação dos fatores de crescimento pró-angiogênicos EGF, FGF-2, IGF e VEGF nas células em cultivo, com destaque para as citocinas EGF e FGF-2. Em conclusão, o uso do ambiente pró-angiogênico no cultivo de MSCs-TA mostrou-se como uma abordagem promissora para a manutenção do estado progenitor destas células in vitro. / The maintenance of the progenitor state in the culture of adipose tissue derived- mesenchymal progenitor cell (TA-MSCs), characterized by the differentiation potential and self-renewal capability, is currently one of the major challenges of cell therapy. The information that the angiogenesis influences the development of mesenchymal tissues, has led us to evaluate how a pro-angiogenic environment mimicked in culture would provide conditions for maintaining a progenitor state during the cell expansion process. We designe a model for a pro-angiogenic environment in which cells grown in EGM-2 supplemented with the following growth factors: EGF, FGF-2, IGF and VEGF, and demonstrated that the presence of such pro-angiogenic growth factors was crucial for maintenance of the progenitor of AT-MSCs in culture. We observed that the presence of such growth factors allowed to AT-MSCs a high potential of adipogenic and osteogenic differentiation compared to conventional DMEM/F12 medium and the EBM medium, in the absence of the factors. Furthermore, the culture in presence of pro-angiogenic growth factors increased the clonogenic potential of AT-MSCs and increased the proliferative capability of these cells. Among the growth factors, EGF and FGF-2 were responsible for most robust effects. At the same time, cells cultured in the presence of these cytokines were able to maintaining the fibroblastoid morphology and presented high expression levels of Klf-4 pluripotency factor. In agreement with these observations, the subcutaneous transplantation of AT-MSCs cultured under these conditions showed that those cells kept in EGM-2 generated a tissue-like to tissue formed by the stromal vascular fraction uncultivated. These results reinforce the role of the pro-angiogenic environment in the maintenance of the progenitor state of AT-MSCs, and that such a state was provided by the action of the pro-angiogenic growth factors EGF, FGF-2, IGF and VEGF in cultured cells, highlighting EGF and FGF-2 cytokines. In conclusion, we showed that the use of a pro-angiogenic environment in AT-MSCs culture is a promising approach to the maintain the progenitor state of these cells in vitro.

Longtemps décrit comme un simple tissu de réserve énergétique, le tissu adipeux blanc est, depuis l’identification de la leptine en 1994, considéré comme un véritable organe endocrine. En effet, ce tissu secrète de nombreuses hormones et cytokines agissant de manière paracrine et endocrine pour contrôler le métabolisme énergétique. Par ailleurs, en plus des préadipocytes et des adipocytes, le tissu adipeux blanc contient également des cellules immunes innées et adaptatives ; lui conférant ainsi un rôle important dans le développement et le contrôle de l’immunité. Cependant, le rôle joué par le tissu adipeux blanc dans les infections - notamment pulmonaires - reste encore peu étudié. C’est dans ce cadre général que s’est inscrit ce travail de Thèse. La susceptibilité accrue des individus obèses (expansion du tissu adipeux blanc) à l’infection par le virus de la grippe (influenza) est largement étayée dans la littérature. Nous avons évalué l’impact de l’infection par le virus influenza sur le tissu adipeux blanc, chez des souris minces et des souris obèses. Nos résultats montrent que, de manière inattendue, le virus est détecté dans les tissus adipeux, sous-cutané (inguinal) et viscéral (périgonadique), de souris infectées par voie intra-nasale (détection du génome viral par RT-qPCR). La présence de virus dans le tissu adipeux est associée à l’augmentation de la sécrétion de cytokines pro- et anti-inflammatoires, à la diminution de l’expression de gènes impliqués dans la lipolyse et la lipogénèse, et à l’augmentation de l’expression des gènes impliqués dans l’induction d’une réponse immune anti-virale. De manière intéressante, l’infection par le virus influenza est associée au brunissement du tissu adipeux sous-cutané chez les souris minces. Chez les souris obèses, l’infection par le virus de la grippe n’induit pas l’effet dépôt spécifique observé chez la souris mince et ne montre pas de brûnissement au niveau du tissu adipeux sous-cutané 7 jours p.i. In vitro, nous montrons que le virus influenza peut infecter les préadipocytes et les adipocytes (lignée murine et cellules primaires humaines). Cependant, alors que le virus effectue la totalité de son cycle dans l’adipocyte, le préadipocyte libère très peu, voire pas, de nouveaux virions infectieux (PCR, transcriptomique, technique de plages de lyse, microscopie confocale et électronique). Ainsi nos résultats, très originaux, identifient le tissu adipeux blanc comme un nouveau tissu cible de l’infection par le virus de la grippe, in vivo. Au sein de ce tissu, les préadipocytes et les adipocytes sont potentiellement infectés par le virus, comme le montrent nos données in vitro, les adipocytes seuls permettant la production de nouvelles particules infectieuses.Contrairement à l’infection grippale, les données épidémiologiques et/ou expérimentales concernant la susceptibilité des obèses à l’infection par la bactérie Streptococcus pneumoniae sont contradictoires, du fait de l’utilisation de différents modèles d’obésité d’origine génétique et de sérotypes de pneumocoques. Dans ce projet, nous avons utilisé un modèle d’obésité d’origine nutritionnelle ; le modèle de souris nourries par un régime enrichi en lipides. Nous montrons que les souris obèses infectées (sérotype Sp1) développent un syndrome de type méningite, mortel, tandis que les souris minces contrôlent l’infection. Si les réponses pulmonaires à l’infection sont comparables entre les souris minces et obèses (dénombrement des colonies bactériennes, histologie, PCR, ELISA, cytométrie en flux), le nombre de bactéries dans le cerveau est significativement plus élevé chez les souris obèses, associé à une altération de la perméabilité de la barrière hématoencéphalique [...] / Since the identification of leptin in 1994, the white adipose tissue (WAT) is no longer considered to solely be an inert tissue storing fat. As an endocrine organ, the adipose tissue synthesizes and secretes several hormones and cytokines involved in the control of whole-body metabolism. In addition, besides preadipocytes and adipocytes, WAT also contains innate and adaptive immune cells; thereby contributing to the development and control of immunity. However, the role played by the adipose tissue during infections - notably during pulmonary infections, such as those resulting from influenza virus or Streptococcus pneumoniae infections - has barely been investigated. This defines the general scope of this work. Epidemiological and experimental data convincingly report that obese individuals are more susceptible to influenza infection. During this project, we therefore questioned whether influenza infection may impact on adipose tissues, both subcutaneous (inguinal, SCAT) and visceral (perigonadal, EWAT) depots, in lean and high-fat diet-induced obese mice. We unexpectedly showed that influenza virus is detected in SCAT and EWAT (quantification of the viral genome by RtqPCR) and that this was associated with drastic changes in the tissue such as: increased secretion of pro- and anti-inflammatory cytokines, decreased expression of genes involved in lipogenesis and lipolysis, as well as increased expression of genes related to the induction of anti-viral immunity. Interestingly enough, influenza infection was associated with the development of brown-like adipocytes in the SCAT, only in lean animals. Moreover, we demonstrated in vitro that preadipocytes and adipocytes (murine cell-line and human primary cells) are permissive to infection, yet with different outcomes. Indeed, only adipocytes allowed the release of new infectious particles (RtqPCR, transcriptomics, quantification of infectious particules on MDCK cells, confocal and transmission electron microscopy). Altogether, our findings revealed, for the first time, that the white adipose tissue, an organ at the crossroads of metabolism and immunity, is deeply affected by influenza infection and might thus be undervalued in influenza pathophysiology.In opposite to influenza infection, the impact of obesity on the outcome of Streptococcus pneumoniae (S.p.) infection remains uncertain, due to the different models (genetically-based obesity, and bacterial strains) used. During this work, we investigated S. pneumoniae (Sp1 strain, sublethal dose) infection in lean and high-fat diet-induced obese mice. We showed that obese mice died from sublethal S. pneumoniae infection, compared to lean animals. The increased mortality induced by infection did not result from impaired pulmonary response but rather from the development of a meningitis-like syndrom likely resulting from an increased bacterial dissemination through the bloodbrain barrier into the brain. We propose that the model of dietary obesity induced by consumption of fat-enriched diet, may be envisaged as a novel and valuable experimental model of memingitis to study Streptococcus pneumoniae travel through the blood-brain barrier and the subsequent immune consequences.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A obesidade está associada com a inflamação crônica atribuída à liberação de citocinas e adipocinas, à homeostase desregulda da glicemia e à dislipidemia. Intervenções nutricionais são frequentemente acompanhadas por episódios repetidos de perda e recuperação do peso, fenômeno conhecido como efeito sanfona ou ciclagem da massa corporal. Foram avaliados os efeitos da ciclagem da massa corporal sobre os parâmetros: eficiência alimentar, massa corporal (MC), perfil lipídico, metabolismo de carboidratos, indíce de adiposidade corporal, marcadores inflamatórios, esteatose hepática e triglicerídeo (TG) hepático em camundongos C57BL/6 machos que ciclaram a massa corporal duas ou três vezes consecutivas pela alternância de dieta hiperlipídica (high-fat, HF) e dieta padrão (standard-chow,SC). Após cada ciclo de dieta HF, os animais ficavam cada vez mais pesados e, após cada ciclo de dieta SC, os animais perdiam cada vez menos peso. A ciclagem da massa corporal provocou flutuação nas reservas de gordura e nos lipídeos sanguíneos. O colesterol total dos animais, após mudança da dieta HF para dieta SC, apresentou redução dos seus valores, assim como os TG plasmáticos. No teste oral de tolerância à glicose, após o perído de ingestão da dieta HF, os animais apresentaram intolerância à glicose e, após a troca para dieta SC, os animais continuaram com intolerância à glicose. Em relação as adipocinas e citocinas, a leptina, resistina e o fator de necrose tumoral (TNF) alfa séricos aumentaram após o ciclo da dieta HF e diminuíram após a troca por dieta SC. Ao contrário, a adiponectina sérica diminuiu após dieta HF e aumentou após troca por dieta SC. A IL-6 aumentou após ingestão da dieta HF, porém após a troca para dieta SC, a IL-6 permaneceu elevada. Enquanto o MCP-1 não variou durante as trocas de dietas. A expressão da adiponectina no tecido adiposo diminuiu após a dieta HF e os valores permaneceram reduzidos mesmo após a troca para dieta SC. As expressões da leptina e IL-6 no tecido adiposo aumentaram após dieta HF e continuaram aumentados mesmo após a troca para dieta SC. Da mesma forma, a esteatose hepática e os TG hepáticos não reduziram após a mudança da dieta HF para dieta SC. Tanto a dieta HF, como a ciclagem da massa corporal são relevantes para o remodelamento do tecido adiposo e provoca repercussões nos lipídios séricos, na homeostase da glicose, na secreção de adipocinas e provoca acúmulo de gordura no fígado. A troca para dieta SC e redução da MC não são capazes de normalizar a secreção de adipocinas no tecido adiposo e nem das citocinas pró-inflamatórias que permaneceram aumentadas. A esteatose hepática e os TG hepáticos também não são recuperados com a troca para dieta SC e redução da massa corporal. Estes resultados indicam que a ciclagem da MC cria um ambiente inflamatório, que é agraado com adipocinas alteradas, intolerância à glicose e acúmulo de gordura no fígado / Obesity is associated with low-grade chronic inflammation attributed to release of cytokines and adipokines and to dysregulated glucose-insulin homeostasis and dyslipidemia. Nutritional interventions such as dieting are often accompanied by repeated bouts of weight loss and regain, a phenomenon known as weight cycling (WC). In this work we studied the effects of WC on the parameters: feed efficiency, body mass (BM), blood lipids, carbohydrate metabolism, adiposity, inflammatory markers, hepatic steatosis and hepatic triglyceride (TG) in C57BL/6 male mice that WC two or three consecutive times by alternation of a high-fat (HF) diet with standard chow (SC). The body mass (BM) grew up in each cycle of HF feeding, and decreased after each cycle of SC feeding. After three consecutive WC, less marked was the BM reduction during SC feeding, while more severe was the BM increase during HF feeding. After each cycle of the HF diet body mass grew up in each cycle of HF feeding, became increasingly heavier and after each cycle of SC feeding, the animals lost less weight. The WC mass caused fluctuations in fat reserves and blood lipids total cholesterol, after shift the HF diet by SC diet showed a reduction of their values and plasma TG. The oral glucose tolerance test after the regular intake of HF diet, the animals showed glucose intolerance and, after switching to SC diet, the animals continued with glucose intolerance. Regarding the adipokines and cytokines, leptin, resistin and tumor necrosis factor alpha (TNF) serum increased after the cycle of HF feeding and decreased after the switch to SC feeding. In contrast, serum adiponectin decreased after HF feeding and increased after dietary exchange for SC. The IL-6 increased after intake of HF diet, but after switching to SC feeding, which remained elevated, while the MCP-1 was not changed during the shift of diets. The expression of adiponectin in adipose tissue decreased after the HF feeding and the values remained low even after switching to diet SC. The expression of leptin, IL-6 in the adipose tissue increased after HF feeding and remained elevated after the change to SC diet. Similarly, hepatic steatosis, and hepatic TG not reduced after the change to the diet HF diet SC. The results of the present study showed that both the HF diet and WC are relevant to BM evolution and fat pad remodeling in mice, with repercussion in blood lipids, homeostasis of glucose-insulin, adipokine levels and causes fat accumulation in the liver. The simple reduction of the BM during a WC is not able to recover the high levels of adipokines in the adipose tissue and serum as well as the pro-inflammatory cytokines. Hepatic steatosis and hepatic TG are not recovered with the change to SC feeding and body mass reduction. These findings indicate that the WC creates an inflammatory environment; with altered adipokines in association with WC which potentially aggravates the chronic inflammation attributed to dysregulated production and release of adipokines in mice

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O aumento da longevidade evidenciou nas mulheres os efeitos deletérios da deficiência ovariana como, a obesidade central e as doenças neurodegenerativas. Os hábitos alimentares modernos favorecem o consumo excessivo de gorduras, que contribuem para efeitos adversos sobre a saúde, incluindo alteração da composição corporal e da função nervosa. Assim, objetivamos estudar como a dieta hiperlipídica (DH), contendo óleo de soja, associada à perda da função gonadal influenciam o comportamento e o desenvolvimento corporal de ratas adultas. Ratas Wistar adultas foram ovariectomizadas (OVX) ou pseudo-operadas (C) e após 7d passaram a receber dieta contendo 4%(normo) ou 19%(hiperlípidica) de óleo de soja: C4 (n=29), OVX4 (n=30), C19 (n=30), OVX19 (n=30). Foram avaliados a massa e o comprimento corporais e a ingestão alimentar, e aspectos comportamentais de ansiedade, através de teste no Labirinto em Cruz Elevado (LCE); de busca pela novidade, por testes no Campo Vazado (CV); e aprendizagem/memória avaliada no labirinto aquático radial de 8 braços (LAROB). Ao final dos testes, os animais foram anestesiados, exsanguinados e o tecido adiposo intra-abdominal foi coletado e pesado. O soro foi utilizado para dosagens de triglicerídeo (TG), VLDL-c, colesterol (COL), HDL-c e estradiol. A ingestão alimentar média e o comprimento corporal não diferiram entre os grupos, mas o grupo OVX19 mostrou ganho significativo (13,6%) de massa corporal mais precocemente que os demais. O mesmo padrão foi observado quanto à massa de tecido adiposo, que foi 86% maior no OVX19. Os TG, VLDL e COL aumentaram cerca de 28% no OVX19, porém sem alteração de HDL-c (33,762,2 mg/dl). A DH e a ovariectomia se mostraram ansiogênicos e quando associados, a DH parece reverter o efeito ansiogênico da castração. Padrão similar de resposta foi observado na resposta ao teste de busca por novidade. No teste de aprendizagem/memória o OVX4 apresentou maior tempo de latência no primeiro dia, e todos responderam de forma semelhante nos demais dias. Conclui-se que a deficiência de estradiol acompanhada da ingestão de dieta hiperlipídica mantém o desempenho cognitivo, o comportamento emocional e a motivação, embora prejudique a adiposidade e o metabolismo lipídico. / The increase in longevity in women showed the deleterious effects of ovarian failure such as central obesity and neurodegenerative diseases. The modern feeding habits are associated with increased consumption of fats that contribute to adverse health effects, including changes in body composition and nervous function. The aim of present study was to assess how a hyperlipidic diet (HD) containing soybean oil, coupled with the loss of the gonadal function influence behavior and body development of adult rats. Adult female rats were ovariectomized (OVX) or pseudo-op (C) and after 7d began to receive a diet containing 4% (normo) or 19% (hyperlipidic) of soybean oil: C4 (n = 29), OVX4 (n = 30), C19 (n = 30), OVX19 (n = 30). We evaluated the length and body mass and food intake, and behavioral aspects of anxiety through the test Plus Maze (EPM), the search for novelty, for testing in the field not (CV) and learning / memory evaluated in the maze Water radial eight arms (LAROB). At the end of testing, animals were anesthetized, exsanguinated and intra-abdominal adipose tissue was collected and weighed. The serum was used for total serum triglyceride (TG), VLDL-C, cholesterol (COL), HDL-C and estradiol. The average dietary intake and body length did not differ between groups, but the group showed significant gain OVX19 (13.6%) body mass earlier than the others. The same pattern was observed for adipose tissue mass, which was 86% higher in OVX19. TG, VLDL and COL increased by about 28% in OVX19, but no change in HDL-C (33.76 2.2 mg / dl). The DH and ovariectomy proved anxieties and when combined, the DH appears to reverse the anxiogenic effect of castration. Similar pattern of response was observed in response to the test to search for novelty. In the test of learning/memory the OVX4 showed higher latency on the first day, and all responded similarly in the other days. It is concluded that deficiency of estradiol followed by the intake of fat diet keeps the cognitive, emotional behavior and motivation, although detrimental to adiposity and lipid metabolism.