Made available in DSpace on 2014-06-11T19:23:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-29Bitstream added on 2014-06-13T19:29:46Z : No. of bitstreams: 1 fassini_pg_me_arafcf.pdf: 613112 bytes, checksum: f00daa1b71f5ee79d5673101f5d6e0e8 (MD5) / Universidade Estadual Paulista (UNESP) / A globulina 11S (glicinina) é a proteína predominante da soja e tem sido extensivamente caracterizada. Além das suas propriedades nutricionais, há evidências de sua participação na redução das concentrações lipídicas do soro. Por outro lado, o fármaco rosuvastatina tem suas ações demonstradas no metabolismo lipídico, por reduzir a fração LDL-colesterol, bloqueando a chave do processo de produção de colesterol hepático. Embora a eficiência do medicamento seja comprovada, é importante considerar a alimentação como uma possível ação somatória, tendo em vista os graves problemas ocasionados pelas alterações lipídicas no organismo. À vista do exposto, a presente pesquisa teve por objetivo estudar o efeito da glicinina isolada da soja em comparação ao fármaco rosuvastatina em animais submetidos à dieta hipercolesterolêmica. Foram elaboradas duas dietas, uma padrão, contendo caseína como proteína (AIN-93M), e outra hipercolesterolêmica, composta pela dieta padrão adicionada de 1% de colesterol e 0.5% de ácido cólico. A proteína glicinina de soja (300 mg/kg de peso corporal) e o fármaco rosuvastatina (10 mg/Kg de peso corporal), ambos solubilizados em solução salina, foram administrados por gavagem. Foram utilizados ratos machos Wistar mantidos em gaiolas individuais sob condições adequadas. Os animais foram separados em cinco grupos (n = 9): 1) grupo padrão (STD) recebeu a dieta padrão; 2) grupo hipercolesterolêmico (HC) recebeu apenas a dieta hipercolesterolêmica; 3) grupo (HC+11S) recebeu a dieta e a proteína glicinina da soja; 4) grupo (HC+ROS) recebeu a dieta e o fármaco; 5) grupo (HC+ROS+11S) recebeu a dieta, a proteína e o fármaco. Ao final de 28 dias os animais foram sacrificados e o sangue removido para análises bioquímicas de colesterol total (CT), HDL-colesterol (HDL-C) e triglicérides (TG) plasmático, CT e TG hepático... / The 11S globulin (glycinin) is the predominant protein of soybeans and has been extensively characterized. In addition to its nutritional properties there is evidence of its action in reducing serum lipid concentrations. Moreover, the drug rosuvastatin has demonstrated its effects on lipid metabolism, by reducing LDL cholesterol and blocking the key enzyme in the process of cholesterol production in the liver. Although the efficiency of the drug is proven, it is important to consider the sum of its action and that of food, in view of the serious problems caused by changes in body fat. In view of the foregoing, the aim of the present study was to investigate the effects of isolated soy glycinin, as compared to the drug rosuvastatin, in rats subjected to a hypercholesterolemic diet. Two diets were prepared, a standard diet, containing casein as protein (AIN-93M), and a hypercholesterolemic, consisting of the standard diet plus 1% cholesterol and 0.5% cholic acid. The glycinin (300 mg/kg body weight) and the rosuvastatin (10 mg/kg body weight), both dissolved in saline, were administered by gavage. Male Wistar rats were kept in individual cages under appropriate conditions. The animals were divided into five groups (n=9): 1) standard group (STD) received the standard diet; 2) hypercholesterolemic group (HC) received the hypercholesterolemic diet alone; 3) group HC+11S received the HC diet and the glycinin; 4) group HC+ROS received the HC diet and the drug; 5) group HC+ROS+11S received the HC diet, the protein and the drug. After 28 days, the animals were sacrificed and the blood removed for biochemical analysis of total plasma cholesterol (TC), HDL-cholesterol (HDL-C) and triglycerides (TG), and hepatic TC and TG. The results indicated that the experimental HC diet was able to induce hypercholesterolemia and that a single daily dose of the isolated protein was appropriate for a comparative... (Complete abstract click electronic access below)

Orientadores: Helena Coutinho Franco de Oliveira, Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T19:29:15Z (GMT). No. of bitstreams: 1 Bonfleur_MariaLucia_D.pdf: 1633140 bytes, checksum: 765548b2268a05188490bc90cf1939b8 (MD5) Previous issue date: 2007 / Resumo: Neste trabalho, investigamos se a hipercolesterolemia primária per se, independente de dieta rica em gordura, afeta a homeostase glicêmica e a secreção de insulina estimulada por vários secretagogos em animais knockout para o receptor de LDL (LDLR-/-). Além disso, investigamos os possíveis mecanismos envolvidos na liberação deste hormônio neste modelo animal. Podemos resumir nossos achados da seguinte maneira: camundongos LDLR-/- apresentam hiperglicemia pós-prandial, hipoinsulinemia, intolerância à glicose e sensibilidade periférica à insulina normal. Nós demonstramos que, as alterações na homeostase glicêmica ocorrem em parte, por uma diminuição da sensibilidade das ilhotas à glicose. A secreção de insulina é normal na presença de baixa concentração de glicose, entretanto na presença de 11,1 mmol/l, as ilhotas de animais LDLR-/- liberam menos insulina que as ilhotas controles. A secreção de insulina estimulada por outros secretagogos metabolizáveis (leucina e KIC) também está reduzida nas ilhotas dos animais knockout. O conteúdo total de insulina e DNA são similares entre os grupos, sugerindo que as alterações na secreção de insulina não ocorrem devido a diferenças no tamanho e/ou número de células b. Observamos uma redução na primeira e segunda fase de secreção de insulina estimulada por 11,1 mmol/l de glicose. A oxidação da glicose está reduzida, enquanto a metabolização da leucina está aumentada. Quando adicionamos agentes despolarizantes (KCl, Arginina e Tolbutamida), observamos uma redução da secreção de insulina tanto em concentrações basais quanto estimulatórias de glicose. Na presença de 11,1 mmol/l de glicose e carbacol (agonista colinérgico) ou PMA (ativador da proteína-quinase C), a secreção de insulina foi semelhante entre os grupos LDLR- /- e controles. Entretanto, quando estimulamos a secreção com forskolin ou IBMX, que aumentam os níveis de AMPc, observamos redução na liberação de insulina pelas ilhotas dos animais LDLR-/- em comparação com os controles. A expressão protéica da fosfolipase C (PLCb2) está aumentada enquanto que a expressão da proteína-quinase A (PKA) está reduzida nas ilhotas dos animais LDLR-/-. Assim, observamos que camundongos LDLR-/- apresentam alterações na homeostase glicêmica independente de dieta rica em gordura, provocada por redução na secreção de insulina devido, em parte à redução do metabolismo da glicose, bem como, redução na expressão da PKA / Abstract: In this work, we investigated whether primary hyperlipidemia per se, independently of a high-fat diet, affects glycemia and insulin secretion stimulated by several secretagogues in hypercholesterolemic low-density lipoprotein receptor knockout mice (LDLR-/-). In addition, we investigated the possible mechanisms involved in the release of this hormone. We found that, besides higher total cholesterol and triglyceride plasma concentrations, glucose plasma levels were increased and insulin decreased in LDLR-/- compared to the wild type (WT) mice. LDLR-/- mice presented impaired glucose tolerance, but normal whole body insulin sensitivity. In addition, we also demonstrate that the main cause of the impaired glucose homeostasis is a reduced pancreatic islet insulin secretion ability following fuel secretagogue stimuli. LDLR-/- mice have impaired insulin secretion in response to glucose without alterations in the pancreatic total insulin and DNA contents. These findings support the idea that the decreased response to glucose cannot be explained by differences islet size or number of beta cells, but it is probably caused by a defect in the secretory process. Glucose oxidation was 30% lower and L-leucine oxidation 60% higher in LDLR-/- islets than in WT islets. At basal (2.8 mmol/l) and stimulatory (11.1 mmol/l) glucose concentrations, the insulin secretion rates induced by depolarizing agents such as KCl, L-arginine and tolbutamide were significantly reduced in LDLR-/- when compared with WT islets. Insulin secretion induced by the PKA activators, forskolin and IBMX, in the presence of 11.1 mmol/l glucose, was lower in LDLR-/- islets, and it was normalized in the presence of the PKC pathway activators, carbachol and PMA. Western blotting analysis showed that phospholipase C-b2 expression was increased and PKA-a decreased in LDLR-/- compared with WT islets. In conclusion, we demonstrate that genetic hypercholesterolemia, due to complete deficiency of LDLR, impairs the beta cell insulin secretion, leading to hyperglycemia without affecting body insulin sensitivity. The lower insulin secretion in LDLR-/- mice islets may be explained by reduced glucose metabolism and expression of PKA / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Elevated plasma cholesterol is a risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) hinders the uptake of low-density lipoprotein cholesterol (LDL-c) by mediating degradation of LDL receptors (LDLRs) in the liver. Gain-of-function (GOF) mutations in PCSK9 cause familial hypercholesterolemia (FH). In normolipidemic human plasma, 30-40% of PCSK9 is bound to LDL particles, and this association with LDL inhibits PCSK9’s ability to mediate LDLR degradation in cultured cells. To further investigate the physiological relevance of this interaction, we analyzed natural GOF mutations in PCSK9 and assessed their effects in vitro on LDL binding, LDLR binding and LDLR degradation. Our results indicate that several GOF mutations severely inhibit LDL binding compared to wild type (WT) PCSK9, and only modestly affect LDLR affinity and LDLR degradation. These findings shed light on the potential physiological relevance of the PCSK9-LDL interaction, which may have an inhibitory effect on PCSK9 activity in vivo.

Hypercholesterolemia represents a major risk factor of cardiovascular disease. A number of experimental models is used for study of hypercholesterolemia pathogenesis and therapy. This thesis concentrates on characterization of one of these models. Prague hereditary hypercholesterolemic (PHHC) rat is such a suitable model for study of hypercholesterolemia. Although the majority of plasma cholesterol is transported by high density lipoprotein in PHHC rat fed standard diet, PHHC rat fed cholesterol diet develops hypercholesterolemia comparable to that of humans. The advantage of this model is that hypercholesterolemia develops without the need for addition of bile acids or other hepatotoxic substances to the diet. The hypercholesterolemia of PHHC rat is caused by slowed down catabolism of cholesterol-rich very low density lipoprotein (VLDL). These cholesterol-rich particles are synthesized in the liver. We found out that PHHC rat fed 1% cholesterol diet accumulates cholesteryl esters (CE) in the liver and also in the VLDL. Acyl-CoA:cholesterol acyltransferase (ACAT) and microsomal triglyceride transfer protein (MTP) may participate in the increased incorporation CE into VLDL. We found out no difference in ACAT and MTP activities in the liver between PHHC rats and control animals. Neither ACAT activity...

LDL-receptor mutations and familial defective apolipoprotein B-100 (codon 3500) (FOB), the known causes of monogenic hypercholesterolemia (MH), have similar clinical features. The nature of the mutations responsible for MH in South Africans of Indian origin was previously unknown. Similarly, the mutations in the LDL-receptor gene of a South African Black FH homozygote had also not been characterised. The aim of this thesis was to identify and analyse the LDL-receptor mutations in the Indian homozygotes NS, D, AV and AA and in the Black homozygote JL. In addition, the possible importance of FOB as a cause of MH in South Africans was also assessed. The patient NS was characterized as having two "Null" LDL-receptor alleles. His skin fibroblasts expressed no detectable LDL-receptor protein and very low levels of LDL-receptor mRNA of approximately normal size. Since NS' s LDLreceptor promoter sequence was normal, his alleles are likely to harbour exonic point mutations or minor rearrangements that cause premature stop codons. The patient D was found to be a heteroallelic homozygote. Two new point mutations in the LDL receptor, Asp₆₉ -Tyr and Glu₁₁₉-Lys, were identified. D's fibroblasts expressed about 30% of the normal surf ace complement of receptors that bound LDL poorly. This low number could at least be partially explained by their decreased stability. These mutations were not identified in any other Indian FH or hypercholesterolemic patients. Patients AV and AA were both shown to be homoallelic homozygotes for the Pro₆₆₄ -Leu mutation. This mutation was identified in 4 unrelated Muslim families of Gujerati origin suggesting that the mutation arose from this area in India. Contrary to previous reports (Knight et al. 1990, Soutar et al. 1989), neither LOL nor β-VLDL binding were shown to be affected by this mutation. These mutant receptors were rapidly degraded. Thus the disease FH in these subjects is presumably due to the low steady-state level of mature receptors that are functionally normal but exhibit accelerated turnover. The Pedi FH homozygote, JL, expressed very few LOL receptors due to decreased receptor synthesis associated with low mRNA levels and not due to enhanced degradation. One of JL's LOL receptor alleles has a 3 b.p. deletion in repeat 1 of the promoter (G. Zuilani, H. Hobbs and L.F. de Waal, personal communication). The nature of the defect in his other allele is unknown. The importance of FOB as a cause of monogenic hypercholesterolemia in the South African Indian, "Coloured" and Afrikaner populations was determined by screening hypercholesterolemic subjects with or without xanthomata. The absence of FOB in such patients, in whom the relevant common or founder South African mutations were excluded, suggested that this disorder was rarer in these groups than in North America and Europe. FOB was identified in two different families of mixed British and Afrikaner ancestry. One family contained individuals who were heterozygous for the FOB mutation, as well as the FH Afrikaner-1 and the FH Afrikaner-2 LOL-receptor mutations. In addition, 4 compound heterozygotes, who had both FOB and the FH Afrikaner-1 mutation and one individual whu inherited all 3 defects, were identified. This family allowed us to characterise the compound heterozygotes with one mutant LOLreceptor allele and FOB as having a condition that was probably intermediate in severity between the FH heterozygote and homozygote states.

No description available.

Abstract available in the Journal of Investigative Medicine.

Background: Hypercholesterolemia is a leading risk factor for cardiovascular diseases, with an average of 102.3 million American diagnosed with Hypercholesterolemia. Medical conditions, lifestyle habits, hereditary factors, and psychiatric illnesses have been associated with Hypercholesterolemia. A few studies postulate that Hypercholesterolemia leads to brain changes that underlie depressive illness. Depression is anticipated to overtake chronic diseases like hypertension in developed nations by 2030, and over 17 million US adults suffer from depression. Tennessee ranks 9th (22.36%) out of 50 states with the highest depression rates, with the incidence rising steadily. As a major public health concern needing urgent attention, it is imperative to establish the association between depression and Hypercholesterolemia among adults in Tennessee. Methods: We used cross-sectional data from the 2021 Behavioral Risk Factor Surveillance System a nationally representative U.S. telephone-based survey of adults aged 18 years, and extracted data for Tennessee (n=4,788). Logistic regression analyses were conducted to test the association between diet, physical activity, depression, no past month exercise, high body mass index, substance use, and high cholesterol (outcome). We controlled for income, race, educational status, health insurance status, and age. Results: Overall, 36.1% (n=1,726) of participants in our sample reported high cholesterol. Results of our logistic regression model revealed that depression (OR =1.37, 95% CI, 1.19, 1.58), High body mass index (OR=1.75, 95% CI, 1.52,1.99), no past month exercise (OR=1.45, 95% CI, 1.27,1.66), male gender (OR =1.16, 95% CI, 1.03, 1.32) and low income (OR =1.33, 95% CI, 1.15, 1.53) were associated with high cholesterol. Furthermore, participants with high cholesterol are 37% more likely to report depression. On the other hand, cigarette use, e-cigarette use, alcohol use, no insurance, and marijuana use were not significantly linked. Conclusions: There is a need for awareness and prevention of Hypercholesterolemia in patients managed for depression, as they might die from the complications of high cholesterol rather than the psychiatric illness itself. It is important to intensify the existing programs and interventions for the prevention of Hypercholesterolemia, which would favorably impact on the burden of depression among adults in Tennessee. Also, studies should be done on the outcomes of cholesterol-reducing medications to prevent Hypercholesterolemia in patients with chronic diseases and depressed patients.

L’hypercholestérolémie familiale à transmission autosomique dominante (ADH), caractérisée par une élévation des taux plasmatiques en cholestérol total et LDL-C, est due à des altérations de 4 gènes : LDLR, APOB, PCSK9 et APOE. L’objectif principal de cette thèse est d’identifier de nouveaux gènes impliqués dans l’ADH. L’identification de nouveaux gènes sera suivie de l’étude des mécanismes physiopathologiques liés à leurs mutations. Un deuxième objectif est de calculer le score génétique (GRS) chez tous les individus appartenant à 5 familles où une mutation FH a déjà été identifiée afin de déterminer si une forme polygénique expliquerait les cas de phénocopies observés. Parallèlement, nous avons mené une étude dans la population libanaise caractérisée par une fréquence élevée de dyslipidémie et qui représente un outil d’étude remarquable au plan génétique du fait de l’existence d’une forte homogénéité du fond génétique.Ce projet de recherche a permis de révéler un gène candidat pouvant être impliqué dans l’ADH : LRP6. De plus il a permis de remettre en question le rôle du récepteur LRP6 jusqu’à présent considéré comme un protagoniste important dans l’internalisation des LDL. Des études supplémentaires sont encore nécessaires afin de confirmer ou non l’implication de ce gène dans l’ADH et de déterminer son rôle exact dans le métabolisme du cholestérol. Concernant le score polygénique, nous avons montré que le GRS ne peut pas être considéré comme un outil de diagnostic pour différencier les sujets avec une hypercholestérolémie monogénique de ceux avec une hypercholestérolémie polygénique et ne peut pas être utilisé pour expliquer les cas de phénocopies / Atherosclerosis and its cardiovascular complications are the leading causes of morbidity and mortality in industrialized countries. Hypercholesterolemia is one of the major cardiovascular risk factors and it affects one in 20 subjects in the general population. Autosomal dominant hypercholesterolemia (ADH), characterized by elevated plasma total cholesterol and LDL-C levels, is due to alterations in 4 genes: LDLR, APOB, PCSK9 and APOE. The fundamental work of Brown and Goldstein revealed the important role of the mutations in the LDLR gene in ADH and contributed to the development of a major class of cholesterol-lowering drugs: statins. Similarly, the discovery by Abifadel et al. in 2003 of the first hypercholesterolemic mutations of PCSK9 was the starting point of an adventure which resulted, 12 years later, in the development of a new class of cholesterol-lowering drugs: anti-PCSK9 antibodies. The main objective of this thesis is to discover new genes, major genetic factors and modifiers involved in ADH. The identification of new genes will be followed by the study of the pathophysiological mechanisms linked to their mutations. A second objective of this work is to calculate the genetic risk score (GRS) in all individuals belonging to 5 families where a mutation responsible of the hypercholesterolemic phenotype has been already identified in order to determine whether a polygenic form would explain the phenocopies observed in these families. In parallel to these two projects, we conducted a study in the Lebanese population which is characterized by a high incidence of dyslipidemia. In this population, it is interesting to conduct genetic studies because of the existence of a limited number of sub-populations that constitute "genetic isolates" with a high homogeneity of their genetic background, making it easier to study many hereditary diseases such as familial hypercholesterolemia. The results obtained in this project revealed a candidate gene that could be involved in ADH: LRP6. Moreover, it allowed us to question about the exact role of the LRP6 receptor until now considered as an important protagonist in the internalization of LDL particles. Further studies are still needed to confirm whether or not this gene is involved in ADH and to determine its exact role in cholesterol metabolism. Concerning the genetic score, we have shown that the GRS does not seem to be a reliable diagnostic tool to identify polygenic hypercholesterolemia at the individual level. The 6-SNP score did not give us a clear answer and thus we cannot use the GRS to identify phenocopies within ADH families

A hipercolesterolemia familial (HF) é uma doença autossômica dominante considerada como uma das formas mais graves de hiperlipidemia, assim como, a principal causa de morbi-mortalidade por ser o principal fator desencadeante da aterosclerose. A alteração primária e mais freqüente da HF incide no gene do receptor da LDL (LDLr), sabe-se que mais de 1600 mutações são descritas na literatura e a principal consequência dessas alterações resultam no comprometimento da remoção da LDL, aumentando a concentração plasmática. Atualmente, o ultrasequenciamento genômico permite gerar muitos dados, que podem identificar novas mutações gênicas de forma eficiente, reprodutiva e rápida. No entanto, somente a validação da nova mutação por atividade funcional pode realmente estabelecer a associação com a doença. O presente estudo tem como objetivo realizar a análise da atividade do receptor da LDL, identificadas através do sequenciamento de alto rendimento, no gene LDLr realizado pelo nosso grupo de pesquisa e correlacionar com dados clínicos, in vitro, in silico e estrutural. Para cumprir esta meta, os linfócitos T dos portadores de HF foram isolados do sangue periférico, cultivados e submetidos a estímulo para a expressão de receptores da LDL, incubados com LDL marcada para avaliação de ligação e interiorização pelas células de cada paciente. Dos 30 pacientes selecionados para esse estudo, 63% apresentaram mutação no LDLR, sendo que quase todas as variantes (p.Gly373Asp, p.Asp601His, p.Ile488Thr, p.Gly549Asp, p.Gly592Glu e Gly681Asp) são localizadas no segundo domínio entre os éxons 7 ao 14. De acordo com o docking molecular a variante p.Gly592Glu (rs137929307), que já foi identificada na população polonesa, espanhola e brasileira, já relacionada com a HF, pode aumentar a interação do LDLr com a ApoB e consequentemente o modo de interação entre as proteínas, no estudo in vitro foi possível notar um aumento tanto na média de fluorescência da ligação e da ligação e interiorização em relação a quantidade de LDLr na superfície celular. / Familial hypercholesterolemia (HF) is an autosomal dominant disease considered as one of the most severe forms of hyperlipidemia, as well as the main cause of morbidity and mortality because it is the main triggering factor for atherosclerosis. The primary and more frequent alteration of the HF affects the LDL receptor gene (LDLr), it is known that more than 1600 mutations are described in the literature and the main consequence of these alterations results in the compromise of the LDL removal, increasing the plasma concentration. Nowadays, genomic ultrasequencing allows the generation of many data, which can identify new gene mutations efficiently, reproductively and rapidly. However, only the validation of the new functional activity mutation can actually establish association with the disease. The aim of the present study was to analyze LDL receptor activity, identified by high-throughput sequencing, in the LDLr gene performed by our research group and to correlate with clinical, in vitro, in silico and structural data. To meet this goal, the T lymphocytes from the HF carriers were isolated from the peripheral blood, cultured and challenged for the expression of LDL receptors, incubated with labeled LDL for binding assessment and internalization by the cells of each patient. Of the 30 patients selected for this study, 63% had a mutation in LDLR, and almost all variants (p.Gly373Asp, p.Asp601His, p.Ile488Thr, p.Gly549Asp, p.Gly592Glu and Gly681Asp) are located in the second domain between exons 7 to 14. According to the molecular docking the variant p.Gly592Glu (rs137929307), which has already been identified in the Polish, Spanish and Brazilian population, already related to HF, can increase the interaction of LDLr with ApoB and consequently the mode of interaction between proteins, in the in vitro study it was possible to note an increase in both the mean fluorescence of binding and binding and internalization in relation to the amount of LDLr on the cell surface.