This study was designed to investigate the mechanism(s) underlying the hypercatabolism of high density lipoprotein in Tangier disease (TD). Initially, the metabolism of normal HDL incubated in Tangier plasma in vitro was examined. Sufficient normal human HDL was added to TD plasma to raise the concentration of HDL-cholesterol to within normal levels. During incubation the concentration of HDL-cholesterol in the TD plasma fell by up to 50% in a time dependent manner. This was not seen in control samples treated in a similar manner. The loss of HDL-cholesterol in the TD could be completely accounted for by the loss of HDL-cholesteryl ester and was accompanied by a 2.3-fold increase in the concentration of HDL-triglyceride. These observations could not be accounted for by lecithin: cholesterol acytransferase activity, cholesteryl ester hydrolysis, or the triglyceride level in the TD plasma. However, preliminary evidence suggested that the activity of cholesteryl ester transfer protein in TD plasma is responsible for the changes in HDL-lipid composition. The resulting triglyceride-rich, cholesteryl-poor HDL was shown to have a normal affinity for the human skin fibroblast HDL receptor. However, this finding does not exclude other pathways of HDL catabolism that may contribute to the rapid turnover of modified HDL in TD plasma. The metabolism of normal HDL by TD fibroblasts and monocytes in vitro was also studied in an attempt to identify a cellular defect of HDL metabolism in TD. However, both TD fibroblasts and monocytes were normal with respect to their ability to bind/internalize and degrade normal HDL invitro. It is concluded that the hypercatabolism of normal HDL in TD involves alterations of HDL-lipid and protein composition prior to removal from the plasma component. Thus, these studies support the hypothesis that the defect in TD resides in the plasma and not in the cells of these patients. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21820号 / 農博第2333号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5192(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 三芳 秀人, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17986号 / 農博第2033号 / 新制||農||1019(附属図書館) / 学位論文||H26||N4811(農学部図書室) / 80830 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 植田 充美, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Lipoproteins are macromolecular assemblies of lipids and proteins that are essential for the transportation of the water insoluble lipids in the circulation. The major lipoproteins, are low density lipoproteins (LDL) and high-density lipoproteins (HDL). LDL, also called the “bad cholesterol”, delivers cholesterol to peripheral tissues and is directly correlated with increased risk of cardiovascular disease, stroke and type II diabetes. HDL, on the other hand, is called the “good cholesterol” as it removes excess cholesterol from peripheral tissues for elimination by the liver in a process called reverse cholesterol transport (RCT). HDL is therefore, cardioprotective. The first and rate-limiting step in HDL biogenesis involves interaction of apolipoprotein A-I (apoA-I), the major protein of HDL, with the membrane transporter ABCA1 to promote cholesterol efflux to form nascent HDL. Food additives and sugar substitutes have been widely consumed in recent years. While the FDA deems sweeteners such as stevia, sucralose, acesulfame and erythritol and food dyes such as Sunset Yellow and Allura Red safe for human consumption, their direct effects on HDL biogenesis are unknown. In this study, we examined effects of the above-mentioned additives and sugar substitutes on HDL biogenesis using macrophage-derived cells, J774. We used a fluorescent analog, BODPIY-cholesterol, to label the cholesterol pool and to measured its efflux by fluorescence. HDL biogenesis was determined by analyzing efflux media using native PAGE followed by immunoblotting using antibodies to apoA-I. Lipids were determined using cholera toxin subunit B to detect the ganglioside GM1. We found that all sweeteners and food dyes exerted inhibitory effects on cholesterol efflux and HDL biogenesis to varying degrees under physiologically-relevant concentrations. Steviol, the metabolite of stevia, appeared to exert the largest inhibition. It reduced cholesterol efflux by > 50% and HDL biogenesis was reduced the most, as compared to the other sugar substitutes. Under control conditions nascent HDL particles varying from less than 7 to about 12 nm diameter produced. However, under most experimental conditions, additives appeared to have an impact on the larger particles (8-12 nm diameter) as they were either reduced substantially or absent). Interestingly, the sweetener Erythritol at low concentration (5mg/ml) was inhibitory but at higher concentration (15 mg/ml) appears to reverse this effect. Since ABCA1 is essential for optimal cholesterol efflux and HDL biogenesis, we hypothesized that their inhibition by food additives and sugar substitutes was due to a decreased level of ABCA1. Surprisingly, we found that ABCA1 level under all conditions were either similar or higher than control levels. Therefore, we suggest that the additives used in this study presumably led to mislocalization of ABCA1 and/or caused a conformational change of the transporter. This could result in decreased binding of apoA-I thus leading to reduced cholesterol efflux resulting in decreased HDL biogenesis. On the basis of this study we suggest that the FDA guidelines should be modified and sugar substitutes and foods containing artificial dyes should be consumed in moderation.

No description available.

No description available.

Die chronische Herzinsuffizienz gehört zu den häufigsten internistischen Krankheitsbildern in Europa. Eine wichtige Rolle in der Therapie der chronischen Herzinsuffizienz spielt das moderate körperliche Ausdauertraining. HDL ist als Vasoprotektor bekannt und ist in der Lage, über die Regulation der endothelialen Stickstoffmonoxidsynthase (eNOS) die Dilatationsfähigkeit von Gefäßen zu regulieren. Da eine gestörte Endothelfunktion verbunden mit einer geringeren eNOS-Expression einen wichtigen Aspekt in der Pathophysiologie der Herzinsuffizienz darstellt, war das Ziel dieser Arbeit zunächst, die HDL-induzierte eNOS-Aktivierung und NO-Produktion in Endothelzellen bei chronisch Herzinsuffizienten mit der von Gesunden zu vergleichen. Des Weiteren wurde der Einfluss körperlichen Ausdauertrainings auf die HDL-Funktion bei chronischer Herzinsuffizienz untersucht. Dafür wurde HDL jeweils aus Blutserum von herzgesunden Probanden und Herzinsuffizienten vor und nach körperlichem Ausdauertraining isoliert. Damit wurden humane aortale Endothelzellen inkubiert und anschließend mittels Western Blot die HDL-induzierte Phosphorylierung der endothelialen Stickstoffmonoxidsynthase (Regulation der eNOS-Aktivierung), der Proteinkinase C-βII sowie der p70S6K ermittelt. Des Weiteren wurde ESR-spektroskopisch die HDL-induzierte NO-Produktion in Endothelzellen gemessen. Letztendlich bestand die Frage, worin der Unterschied zwischen HDL von Gesunden und HDL von Herzinsuffizienten besteht, der die funktionalen Differenzen erklären kann. Dazu wurde die Menge des HDL-gebundenen Malondialdehyds ermittelt. Die Endothelfunktion wurde sonographisch als Fluss-vermittelte Vasodilatation bestimmt. Die Ergebnisse der Untersuchungen belegen, dass die HDL-induzierte eNOS-Aktivierung bei Patienten mit chronischer Herzinsuffizienz im Vergleich zu Gesunden vermindert ist. Des Weiteren kann der Einfluss von HDL auf die eNOS-Aktivierung durch körperliches Ausdauertraining bei Patienten mit chronischer Herzinsuffizienz verbessert werden. Die Verbesserung der HDL-induzierten NO-Produktion korreliert dabei mit der verbesserten Fluss-vermittelten Vasodilatation. Als Unterschied zwischen HDL von Gesunden und dem von chronisch Herzinsuffizienten konnte bei den Letztgenannten eine höhere Menge von gebundenem Malondialdehyd nachgewiesen werden.

Introdução: A doença cardiovascular é a principal causa de morbimortalidade precoce em todo o mundo, e responde por grande parte dos gastos dos recursos destinados aos programas de políticas públicas. Neste contexto, a dieta representa uma importante ferramenta na redução dos fatores de risco cardiovasculares. Tendo em vista que inúmeros estudos mostram que o consumo de ômega 9 ou alimento fonte modifica positivamente diversos fatores de risco cardiovascular clássicos, se torna importante avaliar seu efeito sobre propriedades físico-químicas da LDL e da HDL, marcadores cardiometabólicos e oxidativos em indivíduos brasileiros com diferentes níveis de risco cardiovascular. Objetivo: Avaliar o efeito do consumo de azeite de oliva sobre parâmetros cardiometabólicos clássicos e novos em indivíduos com diferentes níveis de risco cardiovascular. Métodos: O estudo foi do tipo clínico prospectivo, aleatorizado, placebo controlado, duplo cego baseado em intervenção nutricional. Indivíduos de ambos os sexos, distribuídos em grupos azeite de oliva (AO) e placebo (PL) receberam durante 8 semanas 3 g/d de azeite de oliva ou placebo. Todos os indivíduos foram classificados quanto ao risco cardiovascular, seguindo os critérios estabelecidos pelo Escore de Risco de Framingham (ERF). Nos momentos basal, T=4S e T=8S foram determinados o perfil clínico, antecedentes familiares de doenças, pressão arterial, consumo alimentar e nível de atividade física. A partir do plasma ou soro, obtidos após 12 h de jejum, foram determinados o perfil lipídico, as apolipoproteínas, o tamanho da HDL e da LDL, o conteúdo de LDL(-) e de NEFAS e atividade da paraoxonase. A aderência à intervenção foi monitorada por meios diretos (marcadores bioquímicos) e indiretos (registro de intercorrências). Resultados: O azeite de oliva foi efetivo em reduzir concentração de triacilglicerois dos indivíduos em alto risco cardiovascular (p=0,023 no T=4S e p=0,049 no T=8S) e a de LDL-C dos indivíduos com risco cardiovascular intermediário (p=0,045 no T=8S) no atual estudo. Observou-se também redução significativa na LDL(-), quando a amostra foi estratificada pelo ERF. Demais parâmetros permaneceram inalterados em função do tempo da intervenção e do ERF. Conclusão: Baixa suplementação (3 g/d) de azeite de oliva promoveu redução dos triacilglicerois, LDL-C e da LDL(-). Portanto, recomenda-se a incorporação de azeite de oliva na dieta brasileira ainda que em baixas doses. Sugere-se também que estudos adicionais usando doses maiores sejam realizados no sentido de identificar potenciais benefícios cardioprotetores adicionais associados ao consumo de azeite de oliva. / Introduction: Cardiovascular disease is the leading cause of premature morbidity and mortality worldwide, and accounts for a large part of the costs of resources devoted to public policy programs. In this context, the diet is an important tool in managing and reducing the risk of cardiovascular disease. Given that numerous studies show that consumption of omega 9 or food source changes positively several classical cardiovascular risk factors, it becomes important to evaluate its effect on physicochemical properties of LDL and HDL, cardiometabolic and oxidative markers in Brazilian individuals with different levels of cardiovascular risk. Objective: This study aimed to evaluate the effect of consuming olive oil on classical and new cardiometabolic properties in individuals with different levels of cardiovascular risk. Methods: It was a clinical, prospective, randomized, placebo controlled, double blind study based on nutritional intervention. Individuals of both sexes, divided into groups olive oil (AO) and placebo (PL) for 8 weeks received 3 g/d of olive oil or placebo. All subjects were classified for cardiovascular risk following the criteria established by the Framingham Risk Score (FRS). At baseline period, T = 4W and T = 8W the clinical profile, the family history of diseases, blood pressure, food consumption and physical activity level were determined. From plasma or serum obtained after 12 h of fasting lipid profile, apolipoproteins, the size of LDL and HDL, LDL (-) and NEFAS content, and activity of paraoxonase were determined. Adherence to the intervention was monitored by direct means (biochemical markers) and indirect (register of complications). Results: The olive oil was effective in reducing the concentration of triacylglycerol of individuals at high cardiovascular risk (p = 0.023 at T=4W and p=0.049 at T=8W) and LDL-C in individuals with intermediate cardiovascular risk (p=0.045 at T=8W) in the current study. It was also observed a significant reduction in LDL (-) when the sample was divided by the FRS. However, changes in other parameters were not detected when comparing the intervention group and the placebo group. Conclusion: Even at low dosage, olive oil has proved to be beneficial in reducing triglycerides, LDL-C and LDL (-).It is therefore recommended the incorporation of olive oil in the Brazilian diet even in low doses. It is suggested that future studies to use higher doses in order to check additional benefits associated with olive oil consumption.

Introdução: Os efeitos do treinamento físico sobre o metabolismo de lípides têm sido bastante estudados, mas a situação diametralmente oposta, qual seja, a de pacientes acamados sob cuidados prolongados, tem sido pouco investigada. A avaliação de possíveis impactos derivados da imobilização é importante, pois o período de restrição ao leito pode gerar fatores de risco aterogênicos. Outro ponto relevante é a concentração e o aspecto funcional da HDL, que é fator de proteção anti-aterogênico e estudos têm mostrado a concentração diminuída em indivíduos sedentários. Objetivo: Investigar os efeitos da imobilização prolongada sobre o perfil de lípides, apolipoproteínas e a transferência de lípides para a HDL em pacientes acamados. Métodos: Foram estudados 23 pacientes acamados por um período maior que 90 dias de internação no Hospital Auxiliar de Suzano do Hospital das Clínicas da Faculdade de Medicina da USP. Foram avaliados o perfil lipídico, a concentração das apolipoproteínas, CETP, LCAT e LDL oxidada. No ensaio de transferência de lípides, as amostras de plasma foram incubadas com a nanopartícula artificial marcada com 3H-éster de colesterol, 14C-fosfolípides, 3H-triglicérides e 14C-colesterol livre. A quantificação da transferência de lípides da nanopartícula foi feita após a precipitação da fração não HDL. Os dados dos pacientes acamados foram comparados com os obtidos de 26 voluntários sedentários saudáveis, pareados por idade e sexo. Resultados: A média de internação dos acamados foi de 817 dias. As concentrações de colesterol não-HDL (148 ± 36 vs 125±40 mg/dL, p<0,05), LDL-C (124±31 vs 96±36 mg/dL, p<0,01), HDL-C (45±10 vs 36±13 mg/dL, p<0,01) foram menores no grupo acamado, enquanto que os triglicérides foram iguais entre os grupos. A apo A-I (134±20 vs 111±24 mg/dL) foi menor nos acamados (p<0,01), e a apo B não apresentou diferença entre os grupos. A LDL oxidada (53±13 vs 43±12 mg/dL) foi menor no grupo acamado (p<0,05), enquanto que a CETP e LCAT não diferiu entre os grupos. As transferências para HDL de éster de colesterol (6,24±1,1% vs 4,80±1,2%), colesterol livre (4,04±1,1% vs 3,05±1,1%), fosfolípides (19,06±1,3% vs 17,32±2,0%) e triglicérides (3,65±0,7% vs 3,06±0,6%) estavam diminuídas nos acamados comparado ao grupo sedentário (p<0,01). Conclusões: O sedentarismo extremo dos pacientes acamados afetou a concentração do HDL-C, apo A-I e as transferências lipídicas da nanopartícula para HDL. Mesmo a menor atividade física exercida no dia-a-dia dos sedentários pode ser determinante na concentração e no metabolismo da HDL. Apesar da menor concentração do LDL-C e os triglicérides não serem diferentes dos sedentários, o status de HDL mostrou-se alterado nos acamados. Devido à importante função anti-aterogênica da HDL, essas alterações metabólicas devem ser um motivo de atenção adicional na assistência a esses pacientes para a prevenção de eventos cardiovasculares. / Introduction: The effects of physical training on lipid metabolism have been deeply studied, but the diametrically opposite situation such as the long-term care bedridden patients, has been little investigated. The evaluation of immobilization impacts is important, because the bedridden period may take to atherogenic risk factors. Another relevant point is the concentration and functional aspects of HDL, that is an anti-atherogenic protection factor and studies have shown lower concentration in sedentary subjects. Objective: To investigate the effects of long-term immobilization on lipid profile, apolipoproteins and lipid transfer to HDL in bedridden patients. Methods: Twenty-tree bedridden patients under a period over than 90 days from the Auxiliary Hospital of the University of São Paulo Medical School in the city of Suzano, state of São Paulo were selected for the study. The lipid profile, apolipoproteins, CETP, LCAT and oxidized LDL concentration were evaluated. In the lipid transfer assay, the plasma samples were incubated with an artificial nanoparticle labeled with 3H-cholesteryl-esters, 14C-phospholipids, 3H-triglycerides and 14C-unesterified cholesterol. The lipids transferred from nanoparticle to HDL were quantified in the supernatant after chemical precipitation of non-HDL fractions. Data from bedridden patients were compared with those obtained from 26 healthy sedentary volunteers, paired for age and sex. Results: The average of hospitalization period of the bedridden was 817 days. The concentration of non-HDL cholesterol (148±36 vs 125±40 mg/dL, p<0.05), LDL-C (124±31 vs 96±36 mg/dL, p<0.01), HDL-C (45±10 vs 36±13 mg/dL, p<0.01), were lower in bedridden group, whereas the triglycerides were equal between the groups. The apo A-I (134±20 vs 111±24 mg/dL) was lower in bedridden (p<0.01), and the apo B was not different between the groups. The oxidized LDL (53±13 vs 43±12 mg/dL) was lower in bedridden group (p<0.05), whereas the CETP and LCAT was not different between the groups. The lipid transfer to HDL of cholesteryl-esters (6.24±1.1% vs 4.80±1.2%), unesterified cholesterol (4.04±1.1% vs 3.05±1.1%), phospholipids (19.06±1.3% vs 17.32±2.0%) and triglycerides (3.65±0.7% vs 3.06±0.6%) were decreased in bedridden patients compared to sedentary group (p<0.01). Conclusions: The extreme sedentary of bedridden patients affected the HDL-C and apo A-I concentration and the lipid transfer from nanoparticle to HDL. Even the low levels of physical activity exerted in the day-to-day life of sedentary subjects can be determinants of HDL concentration and metabolism. Despite their lower LDL-C and the triglycerides not different from sedentary subjects, the HDL status was clearly further altered in the bedridden. Due to the important anti-atherogenic functions of HDL, those metabolic alterations should be an additional matter of concern in the management of those patients to prevent cardiovascular events.

A ATP-binding cassette transporter A1 (ABCA1) é uma proteína transmembrana responsável pelo efluxo celular de colesterol e fosfolipídeos, que é um passo essencial para o transporte reverso do colesterol e para a biogênese da HDL. Polimorfismos do gene ABCA1 foram associados com risco de doença arterial coronariana, variações no perfil lipídico e diferenças na resposta a fármacos hipolipemiantes. Com a finalidade de avaliar os efeitos de polimorfismos do ABCA1 sobre a expressão gênica e a resposta a vastatinas, foram selecionados indivíduos normolipidemicos (NL, n=143) e hipercolesterolêmicos (HC, n=224). A resposta a atorvastatina (10 mg/dia/4 semanas) foi avaliada pelo perfil lipídico sérico em 141 indivíduos do grupo HC (ATORVA). DNA e RNA total foram extraídos de amostras de sangue periférico. Os polimorfismos de nucleotídeo único (SNP) G70943A (R219K), C-14T e C-105T, uma variante nova do ABCA1, foram detectados por PCR-RFLP e confirmados por seqüenciamento de DNA. A expressão de RNAm do ABCA1 em células mononucleares do sangue periférico (CMSP) foi analisada por PCR-duplex e PCR em tempo real, utilizando o gene GAPD como referência endógena. A freqüência do alelo -105T foi 1,4% em NL e 2,0% em HC. O alelo 70943A (genótipos GA+AA) foi associado com maior concentração sérica basal de apoAI (NL), de HDL-c (ATORVA) e com menores concentrações basais de triglicerídeos e VLDL-c e menor índice TG/HDL-c (HC e ATORVA) em comparação com o genótipo 70943GG (p<0,05). O polimorfismo C-105T está em desequilíbrio de ligação com o SNP C-14T (p=0,006). Portadores do alelo -105T (genótipos CT+TT), quando comparados aos portadores do genótipo -105CC, tiveram menores valores basais de triglicerídeos e VLDL-c, maior concentração de HDL-c e menor índice TG/HDL-c nos grupos HC e ATORVA e também maiores concentrações de apoAI e menor índice apoB/apoAI no grupo ATORVA (p<0,05). Nos grupos HC e ATORVA, os portadores do haplótipo -14CT+TT/-105CT+TT tiveram menores valores de triglicerídeos e VLDL-c basais, maiores concentrações de HDL-c e menor índice TG/HDL-c quando comparados aos portadores dos outros haplótipos (p<0,05). A expressão basal do ABCA1 foi menor nos HC que nos NL independentemente da taxa de expressão alta (GM1) ou baixa (GM2). Este efeito foi associado com os SNPs C-14T e G70943A SNPs. Após o tratamento com atorvastatina, a expressão de RNAm foi reduzida nos HC portadores do alelo - 14T em comparação com os portadores de alelo -14C. Esses resultados são sugestivos de que ABCA1 SNPs estão envolvidos na variação do perfil lipídico sérico e na expressão de RNAm em resposta a atorvastatina. / The ATP-binding cassette transporter A1 (ABCA1) is a transmembrane protein involved on cholesterol and phospholipid cellular efflux, which is an essential step for the reverse cholesterol transport and HDL biogenesis. Single nucleotide polymorphisms (SNPs) in the ABCA1 gene have been associated with increased risk of coronary heart disease, differences on serum lipid profile and response to lowering-cholesterol drugs. We have evaluated the influence of ABCA1 SNPs on mRNA expression and lipid-lowering response to atorvastatin. Normolipidemic (NL, n=143) hypercholesterolemic (HC, n=224) individuals were enrolled in this study and the response to atorvastatin (10 mg/day/4 weeks) was evaluated in HC individuals (ATORVA, n=141). Blood samples were collected for biochemical analyses, genomic DNA and total RNA extraction. SNPs G70943A (R219K), C-14T and C-105T, a novel variant of ABCA1, were detected by PCR-RFLP and confirmed for DNA sequencing. ABCA1 mRNA expression in peripheral blood mononuclear cells (PBMC) was analysed by PCR-duplex and Real Time PCR, using the GAPD as the endogenous reference. In HC and NL, the frequency of -105T allele was 2.0% and 1.4%, respectively. The 70943A allele (genotypes GA+AA) was associated with higher basal concentrations of apoAI (NL) and HDL-c (ATORVA) and lower triglyceride and VLDL-c and TG/HDL-c ratio (HC and ATORVA) than the 70943GG genotype (p<0.05). We found a linkage disequilibrium between C-14T and C-105T SNPs in HC group (p=0.006). Individuals carrying -105T allele (CT/TT genotypes), when compared with -105CC carriers, had lower basal concentrations of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio in HC and ATORVA groups and also higher concentration of apoAI and lower apoB/apoAI ratio in ATORVA group (p<0.05). In HC and ATORVA, individuals with -14CT+TT/-105CT+TT haplotype had lower basal values of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio than carries of others haplotypes (p<0,05). ABCA1 mRNA basal expression was lower in HC when compared to NL independently of high (GM1) or low (GM2) basal expression rate. This effect was associated with C-14T and G70943A SNPs. After atorvastatin treatment, mRNA expression was reduced in HC individuals carrying -14T allele in comparison with the -14C allele carriers. These results are sugestive that ABCA1 SNPs are involved on variation of serum lipid profile and mRNA expression in response to atorvastatin.