A associação de uma série de influências ambientais como dietas com excesso de gordura ou falta de atividade física regular (sedentarismo) são importantes fatores que podem levar ao desenvolvimento da obesidade e dislipidemias. Portanto, a prática de atividade física regular, caracterizada pelo treinamento, mostra-se atualmente como parte de estratégias para combater problemas como dislipidemias. Sabe-se que o aumento do gasto calórico e a melhora no desempenho podem ser atingidos com treinamentos de endurance ou intermitentes, uma vez que ambos levam à alterações fisiológicas e metabólicas semelhantes. O treinamento intermitente é caracterizado pela execução de repetidas sessões de curtos ou longos períodos, preferencialmente de alta intensidade (aproximadamente 100% do VO2máx.), intercaladas por pausas ou períodos de menor intensidade, visando a recuperação do indivíduo. Dada a importância do fígado no metabolismo lipídico em repouso e no exercício foi nosso interesse avaliarmos o comportamento do fígado frente a oito semanas de treinamento intermitente de alta intensidade e comparar tais alterações às promovidas pelo treinamento de endurance em ratos, com especial atenção a síntese e secreção de VLDL. Os animais foram divididos em três grupos: sedentário (SD), treinamento contínuo (TC) e treinamento intermitente de alta intensidade (TI). Os dois protocolos de treinamento resultaram em menor ganho de peso em comparação com o grupo SD. Ainda o grupo TI apresentou maior secreção de VLDL em comparação aos grupos TC e SD. Além disso, a expressão gênica da MTP, proteína chave na montagem da VLDL e da LPL muscular responsável por catalisar a liberação de TAG da VLDL para captação pelo músculo esquelético demonstrou aumento no grupo TI em comparação ao SD. Tais resultados inferem que o treinamento intermitente modulou o transporte de TAG para a periferia e contribui para um efeito hipotrigliceridêmico após o exercício de alta intensidade / The combination of environmental influence, including high fat diets and lack of regular physical activity (sedentary lifestyle) are important factors leading to the development of obesity and dyslipidemias. Regular practice of physical activity, characterized by training, appears as important strategy to reduce such problems. Increased caloric expenditure and improvement of physical performance can be reached with endurance or intermittent training since both lead to similar physiological and metabolic adaptations. Intermittent training is characterized by the execution of repeated bouts of physical effort (high intensity, approximately 100% of VO2max.) Due to the importance of the liver in lipid metabolism during rest and exercise, we examined the adaptations of organ to 8 weeks of high intensity intermittent training, compared with the effect of endurance exercise with special attention to VLDL synthesis and secretion. The animals were randomized into three groups: sedentary (SD), continuous training (TC) and intermittent training (TI). Both training protocols resulted in reduced weight body gain compared with SD, although IT presents higher VLDL secretion, compared with TC and SD. In addition, gene expression of MTP, a key protein in the assembly of VLDL and LPL muscle responsible for catalyzing the release of TAG for VLDL uptake by skeletal muscle, showed an increase in TI compared to SD. These results suggest that intermittent training modulated the transport of TAG to the periphery and contributed to an hypotriglyceridemic effect caused by high intensity exercise

Les anomalies du métabolisme des lipoprotéines riches en triglycérides (LRTG), les chylomicrons et les VLDL exposent à des hypocholestérolémies lors d'un défaut de sécrétion et à des hypertriglycéridémies (HTG) majeures entraînant un risque athéromateux et de pancréatites aigües lors de l'altération de leur clairance. Nous avons diagnostiqué des patients présentant un défaut génétique de sécrétion des LRTG au décours de maladie de rétention des chylomicrons, d'abetalipoprotéinémie et d'hypobetalipoprotéinémie homozygote, causées respectivement par des mutations sur les gènes SAR1B, MTTP et APOB. Nous avons étudié le phénotype des 158 patients publiés avec mutation délétère et mis en évidence des différences portant principalement sur la stéatose hépatique, l'insulinorésistance et l'obésité. Nous avons également mis au point une méthode d'évaluation de l'activité post héparinique de la lipoprotéine lipase (LPL) par mesure de la lipolyse des triglycérides des VLDL in vitro, permettant l'exploration phénotypique des patients présentant une HTG sévère. Nous avons mis en évidence des activités LPL augmentées chez des patients présentant pourtant des antécédents d'HTG sévère et des déficits chez des patients ne présentant pas de mutation identifiable du gène LPL, laissant supposer l'existence de facteurs additionnels modulant l'expression ou l'activité de la LPL. Enfin des interrelations des multiples gènes impliqués dans le métabolisme des triglycérides modulent le phénotype. Elles soulèvent l'intérêt de l'exploration simultanée des principaux gènes impliqués dans les dyslipidémies, telle qu'elle sera effectuée par NGS, pour une meilleure compréhension de leur physiopathologie / Abnormal metabolism of triglyceride-rich lipoproteins (LRTG), chylomicrons and VLDL, can result in hypocholesterolemia in case of impaired secretion, or severe hypertriglyceridemia (HTG) and increased risk of atheroma and acute pancreatitis if clearance is affected. We explored patients suffering from genetic defect in the LRTG secretion (chylomicron retention disease, abetalipoproteinemia and homozygous hypobetalipoproteinemia) and identified mutations on respectively SAR1B, and MTTP and APOB gene. Then, we analysed the phenotype of 158 previously published patients with deleterious mutation (i.e. reported cases added to our cohort) and were able to highlight some specific differences like hepatic steatosis, insulin resistance and obesity. Furthermore we developed an assay to evaluate the lipoprotein lipase (LPL) functionality by measuring the triglyceride-VLDL lipolysis in vitro, and provide a reliable phenotypic exploration for patients with past history of severe hypertriglyceridemia. We found an increased LPL activity in some patients with severe hypertriglyceridemia but conversely showed deficits in other patients free from mutation on LPL gene. These results lead to hypothesize that additional factors might contribute to modulate the expression or the activity of LPL. Finally multiple genes of triglycerides metabolism interact together to additionally modulate phenotype. Of high interest is therefore the simultaneous exploration of the key genes involved in dyslipidemia, as provided by the new generation sequencing (NGS), for better understanding of all pathophysiological mechanisms

A associação de uma série de influências ambientais como dietas com excesso de gordura ou falta de atividade física regular (sedentarismo) são importantes fatores que podem levar ao desenvolvimento da obesidade e dislipidemias. Portanto, a prática de atividade física regular, caracterizada pelo treinamento, mostra-se atualmente como parte de estratégias para combater problemas como dislipidemias. Sabe-se que o aumento do gasto calórico e a melhora no desempenho podem ser atingidos com treinamentos de endurance ou intermitentes, uma vez que ambos levam à alterações fisiológicas e metabólicas semelhantes. O treinamento intermitente é caracterizado pela execução de repetidas sessões de curtos ou longos períodos, preferencialmente de alta intensidade (aproximadamente 100% do VO2máx.), intercaladas por pausas ou períodos de menor intensidade, visando a recuperação do indivíduo. Dada a importância do fígado no metabolismo lipídico em repouso e no exercício foi nosso interesse avaliarmos o comportamento do fígado frente a oito semanas de treinamento intermitente de alta intensidade e comparar tais alterações às promovidas pelo treinamento de endurance em ratos, com especial atenção a síntese e secreção de VLDL. Os animais foram divididos em três grupos: sedentário (SD), treinamento contínuo (TC) e treinamento intermitente de alta intensidade (TI). Os dois protocolos de treinamento resultaram em menor ganho de peso em comparação com o grupo SD. Ainda o grupo TI apresentou maior secreção de VLDL em comparação aos grupos TC e SD. Além disso, a expressão gênica da MTP, proteína chave na montagem da VLDL e da LPL muscular responsável por catalisar a liberação de TAG da VLDL para captação pelo músculo esquelético demonstrou aumento no grupo TI em comparação ao SD. Tais resultados inferem que o treinamento intermitente modulou o transporte de TAG para a periferia e contribui para um efeito hipotrigliceridêmico após o exercício de alta intensidade / The combination of environmental influence, including high fat diets and lack of regular physical activity (sedentary lifestyle) are important factors leading to the development of obesity and dyslipidemias. Regular practice of physical activity, characterized by training, appears as important strategy to reduce such problems. Increased caloric expenditure and improvement of physical performance can be reached with endurance or intermittent training since both lead to similar physiological and metabolic adaptations. Intermittent training is characterized by the execution of repeated bouts of physical effort (high intensity, approximately 100% of VO2max.) Due to the importance of the liver in lipid metabolism during rest and exercise, we examined the adaptations of organ to 8 weeks of high intensity intermittent training, compared with the effect of endurance exercise with special attention to VLDL synthesis and secretion. The animals were randomized into three groups: sedentary (SD), continuous training (TC) and intermittent training (TI). Both training protocols resulted in reduced weight body gain compared with SD, although IT presents higher VLDL secretion, compared with TC and SD. In addition, gene expression of MTP, a key protein in the assembly of VLDL and LPL muscle responsible for catalyzing the release of TAG for VLDL uptake by skeletal muscle, showed an increase in TI compared to SD. These results suggest that intermittent training modulated the transport of TAG to the periphery and contributed to an hypotriglyceridemic effect caused by high intensity exercise

Introduction/objectif : La dyslipidémie des sujets obèses insulinorésistants est principalement caractérisée par une augmentation plasmatique des LRT hépatiques (LRT-apo-B100) et intestinales (LRT-apo-B48). La chirurgie bariatrique, largement pratiquée dans le traitement de l'obésité, est associée à l'amélioration de nombreuses anomalies métaboliques. Nous avons étudié l'effet de la chirurgie bariatrique sur le métabolisme des LRT intestinales et hépatiques.Méthodes/résultats : Le métabolisme des LRT de 22 patients obèses non diabétiques bénéficiant d'une chirurgie bariatrique : sleeve gastrectomie (SG ; n=12) ou bypass gastrique (BP ; n=10) a été étudié par une méthode d'enrichissement isotopique stable (D3-L-Leucine) en alimentation continue. Chaque sujet a réalisé deux études cinétiques : une 1 mois avant et une 6 mois après la chirurgie. Le résultat principal est une diminution de la taille du pool de LRT-apo-B100 après une SG et un BP (p<0,01) expliquée par une augmentation du taux de clairance des LRT-apo-B100 (SG : p<0,05) sans diminution du taux de production. Le pool de LRT-apo-B48 est significativement réduit après une SG (p<0,05), sans explication claire à part une tendance à la diminution du taux de production. La diminution du pool de LRT-apo-B100 est significativement corrélée à la diminution de la concentration en apo-CIII dans le groupe entier.Conclusion : Cette étude est la première étude cinétique réalisée chez l'Homme explorant les mécanismes d'amélioration du métabolisme des LRT après une chirurgie bariatrique. Cette amélioration du métabolisme peut contribuer à la diminution de la mortalité cardiovasculaire observée après une chirurgie bariatrique. / Introduction and objective: The dyslipidemia of insulin-resistant obese patients is widely characterised by the elevation of plasma triglyceride-rich lipoproteins (TRL) of both hepatic (TRL-apoB-100) and intestinal (TRL-apoB-48) origin. Bariatric surgery is a well-established and effective modality for the treatment of obesity, and is associated with improvements in a number of metabolic abnormalities that are associated with obesity. Here, we have investigated the effect of bariatric surgery on intestinal and hepatic TRL metabolism. Approach and Results: Twenty two non-diabetic, obese subjects undergoing bariatric surgery: sleeve gastrectomy (SG; n=12) and gastric bypass (BP; n=10) were studied using a stable isotope (D3-L-Leucine) enrichment methodology, in the constant fed state. Each subject underwent two lipoprotein turnover studies: 1 month before and 6 months after surgery. The main finding was a reduction in TRL-apo-B100 concentration following both SG and BP procedures (P<0.01 for both), explained by an increase in TRL-apo-B100 fractional catabolic rate (P<0.05 for SG) without a reduction in production rate. TRL-apo-B48 concentration was significantly reduced following SG, with no clear explanation other than a trend towards reduction in production rate. The reduction of TRL-apo-B100 concentration was significantly associated with a reduction of plasma apo-CIII in the pooled group of patients undergoing bariatric surgery. Conclusions: This is the first human kinetic study to explore the mechanism of improvement of TRL metabolism following bariatric surgery. These effects may contribute to the decrease of cardiovascular mortality after surgery.

Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a negative regulator of the low-density lipoprotein receptor, and PCSK9 inhibition has become an important cholesterol-lowering therapeutic strategy. PCSK9 also associates with LDL particles, and evidence suggests that the activity of PCSK9 may be regulated by LDL binding. We have investigated the biochemistry of the interaction between PCSK9 and lipoproteins. Through mutagenesis and in-vitro binding assays, we found conserved motifs in the PCSK9 N-terminus that play a role in LDL binding. Through secondary structure studies using circular dichroism and computational modelling, we determined that the N-terminal region of the PCSK9 prodomain undergoes an environment-dependent structural shift that affects the ability of PCSK9 to bind LDL. We also found that the commonly found loss-of-function polymorphism R46L is capable of modulating this structural shift. Importantly, we found a surface-exposed region of the PCSK9 prodomain that maps a cluster of gain-of-function mutations (L108R, S127R, and D129G) that severely disrupt LDL binding. Through gel shift assays and density gradient centrifugation, we observed that PCSK9 shows remodeling-dependent ability to bind different classes of lipoprotein particles in vitro, binding strongly to LDL and IDL but showing barely detectable association to VLDL. Further, in human plasma, we found that lipoprotein-bound populations of PCSK9 shifted in response to differences in lipoprotein profiles between normolipidemic and hypercholesterolemic or hypertriglyceridemic subjects. Overall, elucidation of how lipoproteins regulate PCSK9 activity will reveal new targets for designing cholesterol-lowering therapeutics.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 123-140). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Young male rats fed a choline-deficient diet for three days accumulated triacylglycerol (TG) in the liver, and had reduced very low density lipoprotein (VLDL), but not high density lipoprotein (HDL), levels in the plasma. Cultured hepatocytes obtained from these rats were used as a model system to investigate how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis and lipoprotein secretion. When the cells were cultured in a medium free of choline and methionine, the secretion of TG and phosphatidylcholine (PC) was impaired. Supplementation of choline, methionine or lysophosphatidylcholine (lysoPC) to the culture medium increased the secretion of these lipids to normal levels, and stimulated PC biosynthesis. Fractionation of the secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of TG and PC from choline-deficient cells was mainly due to impaired secretion of VLDL. The secretion of HDL and lipid-free proteins (for example albumin), however, was not affected by choline and methionine deficiency. Supplementation of betaine and homocysteine also stimulated PC biosynthesis and enhanced hepatic VLDL secretion. However, supplementation of ethanolamine, N-monomethylethanol-amine or N, N-dimethylethanolamine did not correct the impaired VLDL secretion from the hepatocytes, although an active synthesis of phosphatidylmonomethyl-ethanolamine (PMME) and phosphatidyldimethylethanolamine was observed. Choline deficiency had no effect on the rate of incorporation of [³H]leucine into cellular apolipoprotein B, E and C or on the rate of disappearance of radioactivity from the labeled apolipoproteins. These results suggest that biosynthesis of PC is specifically required for hepatic VLDL (but not HDL) secretion, and any one of the three synthetic pathways, the CDP-choline pathway, methylation of phospha-tidylethanolamine (PE) or reacylation of lysoPC, is sufficient to provide the required PC. The total activity of cytidylyltransferase in liver was unchanged in choline deficiency. However, choline deficiency caused an abnormal distribution of cytidylyltransferase activity between rat liver cytosol and microsomes (mainly endoplasmic reticulum), a decrease in the cytosolic enzyme activity and an increase in the microsomal enzyme activity. In cultured hepatocytes from the choline-deficient rat, the abnormally distributed cytidylyltransferase activity could be rapidly reversed by the addition of choline, but not lysoPC, to the culture medium. The stimulated microsomal activity of cytidylyltransferase during choline deficiency might be a mechanism whereby the cells could more effectively utilize phosphocholine to maintain a normal CDP-choline level in the choline-deficient liver. Rat liver PE N-methyltransferase catalyzes all three transmethylation reactions in the conversion of PE to PC. The in vitro activity of PE N-methyltransferase was increased in choline-deficient livers using endogenous PE as the methyl group acceptor. However, no significant changes were observed in the enzyme activity when exogenous PMME was used as the methyl group acceptor. Addition of methionine to the cultured hepatocytes obtained from choline-deficient rats resulted in a concomitant reduction in cellular PE levels and the specific activity of PE-dependent methyltransferase. However, the specific activity of PMME-dependent methyltransferase was not significantly altered upon the addition of methionine. No change in PE N-methyltransferase activity was observed in the cultured hepatocytes supplemented with choline. Immunoblotting of PE N-methyltransferase, in crude liver microsomes and in membrane fractions of cultured hepatocytes, revealed that the enzyme mass was not altered by choline and methionine deficiency. Thus, hepatic PE N-methyltransferase is preserved in choline deficiency, and its activity is probably dependent on the availability of metabolic substrates (i.e. methionine and PE). / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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).

Hyperlipidemia caused by a diet high in saturated fat can lead to visceral fat weight gain, obesity, and metabolic syndrome. Being over-weight from visceral fat has been linked to increased risk of developing most age-related diseases and disability, along with a lower income potential and quality of life. However, researchers are just beginning to understand the biological mechanisms that regulate the conversion of excess calories into visceral fat storage rather than glycogen or muscle. Epidemiological studies have repeatedly shown a comorbid association between age-related diseases involving hyperlipemia and circulating levels of uric acid, but not a direct association. This study utilized archival data from 31 healthy, middle-aged adults, who participated in a randomized, double-blind, crossover clinical trial on blood markers of lipidemia and inflammation following a high saturated fat (HSF) verses a "healthy" polyunsaturated fat (PUFA) meal. This primary study was conducted and funded by the National Institute on Aging. A secondary analysis of this data using Pearson's correlation with least squares (2-tailed) regression modeling found that when stratified by gender, baseline uric acid level was an independent and significant predictor of the lipemic response from the HSF, but not the PUFA meal. The linear regression plots indicated that males with uric acid levels above 4.5, and females above 3.0 mg/dL, had a progressively increased lipemic response to the HSF meal. The public health utility of this finding may include the clinical use of the gender-specific linear regression plots of uric acid values to identify and advise individuals at risk for hyperlipidemia from a diet high in saturated fats.

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