Overproduction of intestinal chylomicrons (CM) has been proposed to contribute to fasting and post-prandial (PP) dyslipidemia and may accelerate the development of cardiovascular disease (CVD) during obesity, insulin resistance (IR) and diabetes. However, the impact of morphological changes in intestinal mucosa structure have not been investigated during IR and intestinal dyslipidemia. The first objective of this thesis was to characterize intestinal villi morphology and to determine whether a morphological relationship exists with enterocytic apoB48 (a marker of CM), and intestinal lymph secretion of apoB48 in the obese and IR JCR:LA-cp rat. The second objective was to assess the impact of n-3 PUFA supplementation on PP dyslipidemia in the JCR:LA-cp rat. Intestinal hypertrophy was observed in IR rats, corresponding to an increase in intestinal and lymphatic apoB48 expression. Further, a dietary intervention of n-3 PUFA showed lower PP plasma concentrations of apoB48 and PP plasma inflammatory markers. We conclude that intestinal hypertrophy may contribute to intestinal CM overproduction during obesity and IR. Additionally, dietary n-3 PUFA improves PP lipemia and the associated PP inflammatory response in the JCR:LA-cp rat model. / Nutrition and Metabolism

Emulsões artificiais sem proteína simulando quilomicrons e remanescentes de quilomícron foram preparadas por sonicalcação de trioleína, lecitina, colesteril oleato e colesterol em solução aquosa. A seguir foram ultracentrifugadas em gradiente descontínuo de densidade. As emulsões, marcadas com 3H-trioleína e 14C-colesteril oleato foram injetadas via intra-arterial em ratos controle e em ratos tratados com Triton WR1339, sulfato de protamina e heparina, medindo-se a seguir a remoção plasmática do colesteril-ester e dos triglicérides, durante dez minutos. O Triton WR 1339 e a protamina inibiram a lipólise dos quilomícrons artificiais, diminuindo a remoção destas partículas do plasma. O Triton WR 1339 mostrou ser mais efetivo que a protamina nestes efeitos. Por outro lado, a heparina promoveu uma lipólise rápida e brusca nos quilomícrons artificiais, assim como uma aceleração na remoção destas partículas do plasma. Em contraste flagrante com esses resultados, o metabolismo dos remanescentes de quilomícron não foi consideravelmente afetado pelo tratamento com Triton e heparina. Estas experiências indicam que as emulsões artificiais reproduzem o comportamento metabólico dos quilomícrons e remanescentes de quilomícron naturais, em condições em que a atividade da lipase lipoproteica esteja alterada. / Protein-free emulsions models of chylomicrons and chylomicron remnants were prepared by sonicating triolein, lecithin., cholesteryl oleate and cholesterol in aqueous saline media, followed by ultracentrifugation in density gradient solution. The 3H-triolein and 14C-cholesteryl oleate labeled emulsions were injected into the carotid artery of control rats and rats treated with Triton WR 1339, protamine sulphate and heparin. Plasma removal of both labels was measured during ten minutes in two minutes intervals. Triton WR 1339 and protamine sulphate strongly inhibited lipolysis of chylomicron-like emulsions leading to delayed removal of the particles from blood. Triton WR 1339 de appeared to be more effective than protamine to elicit these effects. On the other hand, heparin produced instantaneous lipolysis of the chylomicron-like particles markedly enhancing its removal from plasma. Contrarily, chylomicron remnant-like emulsions were not considerably affected either by Triton WR 1339 or by heparin treatment. The above described results obtained with artificial emulsions support current concepts on the metabolic behavior of natural chylomicron and remnant submitted to changes in lipoprotein lipase action.

Dietas vegetarianas oferecem baixo conteúdo calórico, baixos níveis de gordura saturada, colesterol, proteína animal e mais altos de gordura polinsaturada, carboidratos, fibras, magnésio, boro, folato e antioxidantes. Todos esses nutrientes influenciam nos fatores de risco de doenças cardiovasculares como hipertensão arterial, diabetes mellitus, obesidade e dislipidemias. Níveis plasmáticos de colesterol total, colesterol de lipoproteína de baixa densidade (LDL), de lipoproteína de densidade muito baixa (VLDL) e triglicérides em indivíduos vegetarianos são menores, em vários estudos, quando comparados a indivíduos onívoros. O metabolismo de quilomícrons (Qm) e dos seus produtos de degradação pela lipase lipoprotéica, os remanescentes, lipoproteínas que transportam os lípides da dieta na circulação sanguínea, não foi avaliado até o momento e está relacionado à aterosclerose. O objetivo deste estudo foi avaliar a cinética plasmática de quilomícrons artificiais marcados com triglicérides (TG-3H) e éster de colesterol (EC-14C) e aspectos qualitativos da HDL, em 18 indivíduos ovolacto-vegetarianos (excluem da alimentação carne, frango e peixe), 21 indivíduos veganos (não consomem nenhum alimento de origem animal), há pelo menos 5 anos e 29 indivíduos onívoros (consomem alimentos de origem animal), todos normolipidêmicos, não diabéticos e sem uso de medicamentos hipolipemiantes. Após a injeção endovenosa dos Qm artificias, foram colhidas amostras de sangue em tempos pré-estabelecidos durante 60 minutos. A radioatividade em cada uma das amostras foi medida para o cálculo da taxa fracional de remoção (TFR) dos lípides radioativos, através de análise compartimental. Foram realizadas as determinações bioquímicas nesses indivíduos e calculada a atividade da lipase lipoprotéica pós-heparina, em ensaio in vitro. Verificou-se também a transferência de lípides de uma nanoemulsão lipídica para a lipoproteína de alta densidade (HDL) e o diâmetro dessa lipoproteína. A remoção plasmática dos remanescentes de quilomícrons avaliada pela taxa fracional de remoção do éster de colesterol da emulsão, foi maior nos veganos (p<0,05) e ovolacto-vegetarianos (p<0,05) comparando-se aos onívoros, enquanto que o processo de lipólise dos quilomícrons, avaliado pela taxa fracional de remoção dos triglicérides da emulsão e pela medida da atividade da lipase lipoprotéica in vitro foi similar nos três grupos avaliados. Não foram encontradas diferenças significativas nas concentrações plasmáticas de HDLC, VLDL e triglicérides entre os grupos estudados .No grupo vegano, as concentrações plasmáticas de LDL-C e glicose foram menores quando comparadas apenas ao grupo dos onívoros (p<0,05, p<0,01; respectivamente). Já em relação ao colesterol total, os indivíduos veganos apresentaram menores níveis quando comparados tanto aos ovolacto-vegetarianos (p<0,05) como aos onívoros (p<0,01). Não observou-se diferença na transferência de lípides da nanoemulsão para a HDL e no tamanho da HDL entre os três grupos. Os resultados do presente estudo sugerem que a dieta vegana e ovolacto-vegetariana aceleram a remoção dos remanescentes de quilomícrons, lipoproteínas aterogênicas, o que pode estar relacionado, aos efeitos benéficos dessa dieta em relação a doenças em relação ao processo aterosclerótico / Vegetarian diets provide less caloric content, low levels of saturated fats, cholesterol and animal protein while providing at the same time providing high levels of polyunsaturated fats, carbohydrates, fibers, magnesium, borium, folate and antioxidants. All these nutrients have an influence upon the cardiovascular diseases such as hypertension, diabetes mellitus, obesity and dyslipidemias. Studies have shown that total cholesterol plasmatic levels, low density lipoprotein (LDL), very low density lipoprotein (VLDL) and triglycerides in vegetarian individuals are lower when compared to omnivores individuals. Chylomicron metabolism (Qm) and its lipoprotein lipase degradation products, the remnants, lipoproteins that transport dietary lipids in the blood has not yet been evaluated, although it is related to atherosclerosis. This study evaluated the plasmatic kinetics of artificial chylomicrons marked with triglycerides (3HTG) and cholesterol esters (14C-CE), as well as the qualitative aspects of HDL in 13 ovolacto-vegetarians (no ingestion of meat, chicken or fish), 10 vegan individuals (no ingestion of any food of animal origin) for more than 5 years and 14 omnivores individuals (ingestion of animal origin foods). All participants were normolipidemic, non diabetic and using no hypolipemiants medication. After an intravenous injection containing artificial Qms, blood samples were collected during 60 minutes at predetermined times. The radioactivity of each sample was measured and the fractional clearance rate (FCR) calculated for the radioactive lipids using compartmental analysis. Biochemical determinations were undertaken in order to calculate the post-heparin lipoprotein lipase activity in vitro. Also evaluated were the lipid transfer of the lipid nanoemulsion to the HDL and the diameter of the HDL particles. The plasma removal of the chylomicron remnants evaluated by the fractional clearance rate of the cholesteryl ester of the emulsion was greater in the vegan (p<0,05) and ovolacto-vegetarian (p<0,05) groups when compared to the omnivore group. The lipolysis of the chylomicron evaluated by the fractional clearance rate of the emulsion triglycerides and by the in vitro lipoprotein lipase activity assay was similar in the three studied groups. There were no differences in the plasma concentrations of HDL-C, VLDL-C and triglycerides between the three groups. The plasma concentrations of LDL-C and glucose of the vegan group were lower than omnivore group (p<0.05 and p<0.01, respectively). In regards to total cholesterol, vegans present lower levels when compared to both ovolactovegetarians (p<0.05) and omnivores (p<0.01). No difference was found in the transfer of nanoemulsion lipids to HDL or in the size of the HDL of all three groups. These results suggest that the vegetarian diet can improve the metabolism of chylomicron remnants, atherogenic lipoproteins, which can be related to the beneficial effects of this diet upon the atherosclerosis process.

Orally administered drugs are generally absorbed by the small intestine and transported either to the lymphatic system or to the hepatic portal system. In general, lipid soluble drugs and vitamins are transported by the small intestine to the lymphatics, and water-soluble drugs are transported to the hepatic portal system. By avoiding the early hepatic first pass effect, the lymphatic transport system may increase drug bioavailability. In addition to its transport systems, the small intestine may affect drug bioavailability through drug uptake, intestinal first pass effect, recruitment of drugs by chylomicrons, formation and secretion of chylomicrons, and enterohepatic circulation. All of these factors should be considered when formulating orally administered lipophilic drugs. Our data also suggest that Caco-2 cells may serve as a valuable in vitro model to study the intestinal transport of orally administered drugs.

Orally administered drugs are generally absorbed by the small intestine and transported either to the lymphatic system or to the hepatic portal system. In general, lipid soluble drugs and vitamins are transported by the small intestine to the lymphatics, and water-soluble drugs are transported to the hepatic portal system. By avoiding the early hepatic first pass effect, the lymphatic transport system may increase drug bioavailability. In addition to its transport systems, the small intestine may affect drug bioavailability through drug uptake, intestinal first pass effect, recruitment of drugs by chylomicrons, formation and secretion of chylomicrons, and enterohepatic circulation. All of these factors should be considered when formulating orally administered lipophilic drugs. Our data also suggest that Caco-2 cells may serve as a valuable in vitro model to study the intestinal transport of orally administered drugs.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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L’altération du métabolisme postprandial des lipides et l’inflammation chronique associée apparaissent comme des éléments majeurs de la physiopathologie de l’obésité. L’implication de l’absorption intestinale d’endotoxines bactériennes du microbiote au cours de la digestion des lipides a été mise en évidence. Cependant la modulation de ces phénomènes par différentes quantités de lipides différemment structurés reste mal caractérisée, notamment chez les obèses. Nous avons mis en place un protocole clinique, en cross-over randomisé, visant à étudier chez des sujets normo-pondérés et obèses les conséquences de la digestion de matière grasse consommée, soit sous forme tartinée en différentes quantités (10 g ou 40 g), soit sous forme finement émulsionnée (40 g) sur le métabolisme postprandial des lipides et des endotoxines. Nous avons ainsi mis en évidence que l’augmentation plasmatique des chylomicrons, suite à une augmentation de la quantité de matière grasse ingérée, était plus précoce et plus importante chez les normo-pondérés que chez les sujets obèses, avec la sécrétion de plus gros chylomicrons suite à 40 g. Lorsque 40 g de matière grasse est émulsionnée, nous montrons qu’elle aboutit à un pic de triglycérides des chylomicrons plus précoce et plus élevé, reflétant une absorption facilitée des lipides, et de manière plus marquée chez l’obèse. Nous montrons aussi que cet état émulsionné aboutit à une β-oxydation plus élevée des lipides exogènes sur la journée, sans différence de perte fécale. Une endotoxémie postprandiale est également observée suite aux différents repas. L’accumulation postprandiale d’endotoxines, notamment présentes dans les chylomicrons, augmente avec la quantité de matière grasse tartinée en corrélation avec l’aire sous courbe des chylomicrons chez les obèses. En complément, l’absorption lipidique in vitro par des cellules Caco-2 était plus importante suite à l’incubation de milieux de lipolyse d’émulsions stabilisées par du caséinate que de la lécithine. Enfin, un test de digestion a été réalisé avant et après une chirurgie de by-pass gastrique pour identifier si une diminution drastique de l’absorption lipidique modifiait l’endotoxémie. Suite à l’opération, les patients sont davantage exposés aux endotoxines après la prise d’une émulsion au petit-déjeuner. En revanche, la LBP, protéine de transport des endotoxines proinflammatoire, diminue significativement à jeun et en postprandial suite à l’opération. L’ensemble de ces travaux démontrent qu’en plus des effets de la quantité de lipides ingérée sur la lipémie et l’absorption d’endotoxines associée, la structure de la matière grasse joue un rôle important dans la modulation du devenir métabolique des acides gras. La structuration des lipides alimentaires pourrait donc être spécifiquement adaptée afin d’optimiser le métabolisme lipidique postprandial, notamment chez des personnes obèses. / The alteration of postprandial lipid metabolism and associated chronic inflammation emerge as major elements in the obesity pathophysiology. The involvement of the intestinal absorption of endotoxin from microbiota during lipid digestion was recently highlighted. However, the modulation of these phenomena by different amounts of differently structured lipids remains poorly characterized, especially in obese people. We developed a cross-over randomized clinical study to explore in normal weight and obese subjects the consequences of fat digestion, consumed either spread on bread in different amounts (10 g or 40 g) or finely emulsified (40 g), on postprandial metabolism of lipids and endotoxins. We have demonstrated that the increase in plasma chylomicrons, after increase in the amount of fat ingested, was earlier and greater in normal-weight than in obese subjects, with the secretion of larger chylomicrons after consumption of 40 g of spread fat. When 40 g of fat is emulsified, we show that it leads to an earlier and higher peak of chylomicron triglycerides, reflecting facilitated absorption of fat, and more significantly in obese subjects. We also show that emulsified fat results in higher β-oxidation of exogenous lipids over the day, with no difference in fecal excretion. Postprandial endotoxemia was also observed in response to different meals. The postprandial accumulation of endotoxins, present in chylomicrons, increases with the amount of spread fat ingested and it is correlated with the area under the curve of chylomicrons in obese subjects. In addition, the in vitro lipid absorption by Caco-2 cells was greater following incubation with lipolysis media of emulsions stabilized by caseinate than lecithin. Finally, a digestion test was conducted before and after gastric bypass surgery to identify whether a drastic reduction in lipid absorption altered metabolic endotoxemia. After surgery, patients are more exposed to endotoxins in the morning after emulsion consumption at breakfast. However, LBP, a proinflammatory protein transporting endotoxins, significantly decreases after surgery. Altogether, these studies demonstrate that in addition to the metabolic effects of dietary fat intake on lipemia and associated endotoxemia, the fat structure also plays an important role in the modulation of further fatty acid handling. Structuring of dietary lipids could thus be specifically adapted to optimize postprandial lipid metabolism, especially in obese people.

La protéine CD36 est un senseur aux AGLC au niveau des papilles gustatives et des entérocytes, impliqué dans la détection gustative des lipides et dans l’optimisation de la synthèse des CM. Notre 1er objectif a été de déterminer si le syndrome métabolique (MS) était associé à une altération de la détection oro-intestinale des AGLC. A l’aide de modèles murins de MS (régimes riches en AGS), nous avons montré que le MS est associé à une moindre sensibilité gustative aux sucres et aux lipides et à une altération de la synthèse des CM à l’origine d’une hypertriglycéridémie postprandiale. Cette modification est associée à une absence de dégradation de CD36 classiquement médiée par les AGLC. Cette altération conduit à un retard d’induction de l’expression des gènes impliqués dans la synthèse de CM moins bien dégradés au niveau sanguin. Selon nos résultats l’hyperinsulinémie en MS pourrait être à l’origine de l’absence de dégradation de CD36. Au niveau oral il a aussi été montré que l’absence de dégradation de CD36 est associée à une altération de la signalisation calcique probablement à l’origine d’un seuil de détection plus élevé des AGLC. Le MS se caractérise donc par un problème de détection des lipides au niveau de la sphère oro-digestive qui favoriserait hyperphagie et hypertriglycéridémie postprandiale. Le 2e objectif a été de déterminer si le XXS (polyphénols) pouvait prévenir le MS. La supplémentation en XXS diminue la prévalence du MS, du fait d’une action anti-obésité associée à un maintien du seuil de détection des lipides et une augmentation de l’activité. Ainsi, la détection oro-intestinale des lipides semble être une cible pertinente pour lutter contre la mise en place du MS / CD36 is a LCFA sensor in gustatory papillae and enterocytes. CD36 is implicated in the gustatory detection of lipids and in optimized CM synthesis. Our first goal was to determine if the metabolic syndrome (MS) was associated with alteration of the oro-intestinal detection of LCFA. Using a murine model of diet induced MS (saturated high fat diets) we shown that the MS is associated with a decrease in lipids and sugar gustatory sensitivity and an alteration in CM synthesis which contributes to the postprandial hypertriglyceridemia. This modification is associated with an absence of CD36 protein degradation classically triggered by LCFA. This alteration leads to a delay in the stimulation of the gene expression involved in the synthesis of CM less well cleared into the blood. Our data shows that the hyperinsulinemia on MS could cause the abolition of CD36 protein degradation. Other data obtained on gustatory papillae demonstrates that the absence of CD36 degradation is associated with an alteration of the LCFA induced calcium-signaling and that probably causes the increase in LCFA detection threshold. Thus, the MS is characterized by an alterated dietary lipids detection by the oro-intestinal tract which could promote overeating and postprandial hypertriglyceridemia. The second goal was to determine if the XXS (polyphenols) could prevent the appearance of the MS. The XXS supplementation decreases the syndrome prevalence, by exerting an anti-obesity activity associated with a LCFA detection threshold preservation and an increased activity. Thus, dietary lipids detection by gustatory papillae and intestine could represent a relevant target in order to prevent MS appearance