L’obésité et le surpoids sont aujourd’hui des problèmes de santé majeurs au niveau mondial touchant près de 1,9 milliard d’adultes. Cet état physiologique se définit comme une accumulation excessive de masse grasse dans l’organisme avec des conséquences néfastes sur la santé. L’obésité tient son origine d’un déséquilibre de la balance énergétique résultant d’une consommation excessive de calories par rapport aux dépenses énergétiques. En accord avec cette idée, les lipides alimentaires représentent les macronutriments les plus caloriques dont la biodisponibilité est assurée par l’intestin. L’accumulation excessive de ces lipides dans les tissus tel que le foie, le tissu adipeux et les vaisseaux sanguins déclenche une inflammation bas-bruit. Cette inflammation bas-bruit est souvent associée à une augmentation modérée de l’endotoxémie métabolique, c’est-à-dire du taux sanguin des lipopolysaccharides bactériens provenant du microbiote intestinal.Dans un premier temps, les travaux de cette thèse avaient pour objectif de définir la contribution de l’intestin dans la mise en place de cette endotoxémie métabolique à travers le rôle du récepteur CD36. En effet, il est montré que ce récepteur participe à la biodisponibilité des lipides en favorisant leur absorption et la formation des chylomicrons qui véhiculent les lipopolysaccharides. Les données obtenues ont permis de montrer que CD36 participe aux situations d’endotoxémie métabolique en régulant l’écologie microbienne du site d’absorption des lipides alimentaires.Dans un second temps, ces travaux se sont intéressés à la contribution de la réponse inflammatoire dans les mécanismes d’absorption des lipides alimentaires au travers du rôle de l’inflammasome NLRP3. Ils ont permis de montrer que cet inflammasome participe à la digestion et à l’absorption des lipides alimentaires lors d’un régime hyperlipidique obésogène.L’ensemble de ces données permet d’établir le rôle majeur de l’intestin dans la mise en place de l’endotoxémie et des processus inflammatoires conduisant à une situation d’obésité. De plus, le récepteur CD36 et l’inflammasome NLRP3 pourrait constituer des cibles thérapeutiques de choix pour limiter la mise en place de cet état physiologique. / Nowadays, obesity and overweight are major health problems affecting nearly 1.9 billion adults. This physiological state is defined as an excessive accumulation of fat mass in the body with harmful consequences on health. Obesity is caused by a disequilibrium in the energy balance resulting from an excessive calorie consumption in relation to the energy expenditure. According to this concept, dietary fats represent the most caloric macronutrients whose bioavailability is ensured by the intestine. Excessive accumulation of these lipids in tissues such as liver, adipose tissue and blood vessels triggers low-grade inflammation. This low-grade inflammation is often associated with a moderate increase in metabolic endotoxemia, i.e. an increase in the blood level of bacterial lipopolysaccharides coming from the intestinal microbiota.First, the aim of this thesis was to elucidate the contribution of the intestine to the development of the metabolic endotoxemia through the role of the CD36 receptor. Indeed, this receptor participates in the bioavailability of dietary lipids by promoting their absorption and the formation of chylomicrons able to carry the lipopolysaccharides. The data obtained showed that CD36 could participates in metabolic endotoxemia situations by regulating the microbial ecology of the food lipid absorption site.In a second step, this work focused on the contribution of the inflammatory response to the absorption mechanism of dietary lipids through the role of the NLRP3 inflammasome. It showed that this inflammasome is involved in the digestion and absorption of dietary lipids in high-fat diet-induced obesity.The data obtained allow to establish the major role of the intestine in the development of endotoxemia and inflammation during the development of obesity. In addition, the CD36 receptor and the NLRP3 inflammasome may be therapeutic targets to limit the development of this physiological condition.

The clearance of circulating glucose and long-chain fatty acids (FA) into skeletal muscle involves the translocation of glucose transporter GLUT4, fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm) and fatty acid transport protein (FATP) 1 and 4 to the plasma membrane (PM). FAT/CD36 also appears to participate in the regulation of mitochondrial FA oxidation. Metabolic challenges are known to increase FA transport and/or oxidation, but whether this is solely attributable to the translocation of FAT/CD36 to the sarcolemma and/or mitochondria is unknown. Moreover, the signaling and trafficking pathways involved in the translocation of FA transporters are largely unexplored. In this thesis it was found that FA transport was markedly increased following insulin (+2.9-fold) or contraction (+1.7-fold) stimulation of skeletal muscle, along with the PM contents of FAT/CD36 (+78%, +55%,), FABPpm (+61%, +62%), FATP1 (+84%, +61%) and FATP4 (+60%, +66%) (p<0.05). Upon combining the two stimuli, only the translocation of FAT/CD36 (+179%) and FATP1 (+125%) to the PM was additive, suggesting that these transporters may reside in distinct insulin-sensitive and contraction-sensitive intracellular compartments. The translocation of FA transporters may involve the insulin-signaling protein Akt2. It was found that insulin-stimulated FA transport and PM translocation of FA transporters was essentially prevented in Akt2 knockout mice. Following contraction, FA transport was also markedly blunted, along with an impaired translocation of both FAT/CD36 and FATP1, but not FABPpm or FATP4. FA oxidation and mitochondrial FAT/CD36 appearance were also inhibited following muscle contraction in knockout mice (p<0.05). Whether the GLUT4 trafficking protein Munc18c is important for the vesicular re-distribution of FA transporters to the PM or mitochondria was also investigated. FA uptake was comparably increased 1.4 fold with insulin and contraction in both wildtype and heterozygous Munc18c-/+ mice, as were PM FA transporters FAT/CD36 (+82%, +84%), FABPpm (+39%, +43%), FATP1 (+40%, +38%) and FATP4 (+33%, +32%) (p<0.05). Contraction-stimulated mitochondrial FA oxidation was also increased similarly in wildtype (+39%) and Munc18c-/+ mice (+33%). These studies demonstrate that a number of FA transporters are involved in upregulating skeletal muscle FA transport, although their signaling and trafficking pathways may differ from that of GLUT4.

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Estudos mostram que indivíduos portadores de modificações ateroscleróticas precoces e mais graves possuem um ou mais fatores e marcadores equivalentes de risco cardiovascular na vida adulta como, dislipidemia, hipertensão, obesidade e hiperglicemia. Objetivos. Identificar marcadores plasmáticos, histológicos e imunohistoquímicos de gênese e progressão da aterosclerose humana precoce e crônica em amostras de plasma e fragmentos vasculares de pacientes submetidos à revascularização miocárdica. Casuística e Métodos. Foram avaliadas amostras de plasma e tecido vascular de 23 pacientes de ambos os gêneros, 44 a 73 anos, em dois grupos > 56 e < 56 anos, da Unidade de Cirurgia de Revascularização Miocárdica (RM) /Hospital Ana Nery/UFBA, com indicação de RM, entre Dez/2009 e Jun/2012. Foi determinado perfil lipídico, calculados indicadores de risco cardiovasculares e marcações para os receptores LDL-r, CD36 e CD68 em fragmentos de aorta e torácica interna. Resultados. Os dados do perfil lipídico entre os grupos dito crônico (> 56 anos) e dito precoce (< 56 anos) foram diferentes (p < 0,05). Os índices calculados no grupo < 56 anos estavam acima dos valores de referência. Em ambos os grupos, o índice de Gazziano (TG/HDL-C) indicou presença de LDL pequenas e densas. O grupo < 56 anos, mostra valores de tamanho de LDL inferiores (7,1 ± 1,4 vs 4,15 ± 2,9) quando comparados ao grupo > 56 anos, respectivamente. O não-HDL-C mostrou-se mais graves no grupo < 56 anos em função da hipertrigliceridemia (234±58mg/dL; p=0,0006). Nos estudos histológicos e imunohistoquímicos, observou-se aterosclerose discreta e marcações com diferentes intensidades entre os dois grupos para LDL-r, CD36 e CD68. Nos < 56 anos, observou-se estrias lipídicas, macrófagos degenerados, cristais de colesterol, basofilia de fibras elásticas e pontos hemorrágicos. A marcação para LDL-r nos >56 anos, mostrou-se difusa. Na torácica interna, as marcações foram focais e de baixa intensidade, porém, menos intensas do que nos < 56 anos. A marcação CD36 na torácica interna foi intensa nos pacientes < 56 anos, quando comparada com os cortes dos > 56 anos. A marcação para CD68 nos cortes de aorta foi intensa em ambos os grupos, porém, nos cortes de torácica interna a marcação foi tênue independente do grupo avaliado. Para o CD36, os dados apontam para risco em < 56 anos, em função da marcação intensa observada, esse receptor está implicado na gênese do processo aterosclerótico. Conclusões. Pode-se conceber que existe interação importante entre os marcadores plasmáticos e teciduais atuando no evento aterogênico, sendo mais graves em pacientes com menos de 56 anos. / Introduction. Although cardiovascular disease (CVD) often manifest in adulthood, the atherosclerotic process begins in childhood. Studies show that individuals with early atherosclerotic changes possess one or more equivalent factors and markers of cardiovascular risk in adult life as dyslipidemia, hypertension, obesity and hyperglycemia. Objective. Identify plasma markers, histological and immunohistochemical features of the genesis and progression of atherosclerosis in early human plasma samples and fragments of vascular patients undergoing myocardial revascularization. Casuistic and Methods. Samples of plasma and vascular tissue from 23 patients of both genders, 44 to 73 years, in two groups >56 and <56 years, from Myocardial Revascularization (MR) Surgery Unit / Ana Nery’s Hospital / UFBA, indicated to MR, between Dec/2009 Jun/2012. Lipid profile, calculated indices of cardiovascular risk markers and receptors for LDL-r, CD36 and CD68 in fragments and internal thoracic aorta was determined. Results. Lipid profile data between groups said as chronic (> 56 years) and said as precocious (<56 years) were different (p <0.05). The indices in the group <56 years were above the reference values. In both groups, the rate of Gazziano (TG / HDL-C) indicates the presence of small, dense LDL. The group <56 years, shows lower LDL size values (7.1 ± 1.4 vs 4.15 ± 2.9) when compared to > 56 years group, respectively. The non-HDL-C was more severe in the group <56 years due to hypertriglyceridemia (234 ± 58mg/dL, p = 0.0006). In histological and immunohistochemical studies, were observed discrete atherosclerosis and marks with different intensities between the two groups for LDL-r, CD36 and CD68. In <56 years, were observed lipid streaks, degenerate macrophages, cholesterol crystals, basophilia of elastic fibers and bleeding points. The markup for LDL-r in > 56 years was diffused. In internal thoracic, the tissue marks were of low intensity and focally localized, but less intense when compared to <56 years group. The internal mammary CD36 mark was high in patients <56 years, when compared with > 56 years. The marks for CD68 in aorta was intense in both groups, however, in internal thoracic was tenuous independent of evaluated group. For CD36, the data point to risk from <56 years, due to the intense labeling observed, this receptor is really implicated in the pathogenesis of atherosclerosis. Conclusion. It is conceivable that there is significant interaction between the plasma and tissue markers acting in atherogenic event, being most severe in patients younger than 56 years.

No description available.

The multifunctional fatty acid (FA) binding protein, Cluster of Differentiation 36 (CD36), has been found to be expressed in a variety of tissues where it is involved in multiple fat-related biological processes, such as lipid metabolism in mammals and the detection of lipid-like pheromones in insects. As identified in the apical membranes of taste cells, along with functional evidence in behavioral and cellular level, its involvement in the gustatory FAs detection is suggested. Nonetheless, whether CD36 acts as a direct lipid sensor or as a chaperone protein that facilitates the function of FA-activated G protein coupled receptors (GPCRs), such as taste cell expressing GPR120, remains to be determined. To characterize the role of CD36 in FA signaling, either as a primary receptor or in concert with GPCRs, I utilized human embryonic kidney 293 (HEK293) cell lines that express the different combination of the LCFA receptor GPR120 and CD36. By using intracellular calcium imaging, the presence of CD36 increased the cell sensitivity to LA slightly in GPR120+ cells. Treating the CD36+GPR120+ cells with CD36 inhibitor, sulfo-N-succinimidyl oleate (SSO), resulted in a large reduction, but not abolishment of the LA activated response, which was absent in CD36+GPR120- cells. To investigate the role of CD36 in FA transduction specifically in taste, a mouse taste bud-derived (TBD) cell line, TBD-a1, was used. Knockdown of CD36 by RNA interference in these cells reduced but did not eliminate their intracellular calcium responses to LA. In vivo, isolated taste cells from CD36-KO mice and WT mice were compared for their FA sensitivity. CD36-KO cells were capable of responding to LA with the concentration-response curve not shifted significantly compared to WT cells. However, SSO significantly reduced the LA response in WT mice. At the behavioral level, responsiveness to LA in CD36-KO mice was not eliminated comparing to WT mice after formation of a conditioned taste aversion to LA. These data suggest that CD36 is a protein that facilitates the activation of GPR120 by FAs instead of a primary receptor for FAs itself. In the taste system, CD36 is not required but may facilitate activity in FAs responsive pathways.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21133号 / 農博第2259号 / 新制||農||1057(附属図書館) / 学位論文||H30||N5107(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 金本 龍平, 准教授 井上 和生 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The glycoprotein CD36 is expressed in the plasma membrane (PM) of many cell types that surround or contact arteries, including macrophages, myocytes, and endothelial cells. CD36 binds oxidized low density lipoprotein (oxLDL), which promotes atherosclerosis, and fatty acids (FA), which promotes their cellular uptake. To gain insights into the molecular mechanisms of uptake, HEK293 cells expressing CD36 were studied by cell biological and fluorescence methods. To test our hypothesis that the PM is not an impermeable barrier to FA and that FA move into cells by diffusion via their uncharged form, we first applied biophysical fluorescence spectroscopy to directly measure transmembrane FA movement and membrane fluidity. Expression of CD36 in HEK293 cells did not increase either transport across the PM or the fluidity of the PM compared to HEK293 cells without CD36; however, CD36 enhanced intracellular FA esterification. Furthermore, the widely used “inhibitors” of FA transport did not alter either the rapid FA transmembrane diffusion in HEK293 cells or diffusion in control experiments with protein-free phospholipid bilayers. To gain new insights into the physiological relevance of FA binding to CD36, we applied surface plasmon resonance (SPR) to quantify FA and oxLDL binding to the ectodomain of CD36. Structurally distinct FA [saturated, monounsaturated (cis and trans), polyunsaturated, ω-3, ω-6, and oxidized FA] were pulsed in a solubilized form (bound to methyl-β-cyclodextrin) across SPR channels, generating real-time association and dissociation binding curves. With the exception of the oxidized FA hydroxyoctadecadienoic acid (HODE), all FA tested bound to CD36 with rapid association and dissociation kinetics similar to human serum albumin. In addition, FA increased oxLDL binding to CD36. To investigate whether FA affect CD36-mediated oxLDL uptake in live cells, we monitored fluorescent oxLDL (Dii-oxLDL) uptake using confocal microscopy. Addition of exogenous FA to serum-free media enhanced dose-dependent oxLDL uptake. Exceptions were ω-3 FA, which bound to CD36, and HODE, which did not bind to CD36, demonstrating FA structure-specific effects on a major function of CD36 and a new mechanistic link between atherosclerosis and high levels of FA in obese and Type-II diabetic individuals.

No description available.

No description available.

No description available.