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Made available in DSpace on 2015-07-22T20:50:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-11-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Objetivos: As lipoproteínas oxidadas e os anticorpos anti-LDL oxidada (anti-LDLox) têm sido detectados no plasma e em lesões ateroscleróticas em humanos. No entanto, o papel destes autoanticorpos na proteção vascular ou na patogênese das síndromes coronarianas agudas (SCA) permanece não elucidado. Nós examinamos a relação entre os títulos de IgG humana anti-LDLox com marcadores de risco para a doença cardiovascular. Métodos: Títulos de autoanticorpos anti-LDLox foram mensurados em indivíduos portadores de hipertensão arterial em estágio 1 (n=94), sem outros fatores de risco, e em indivíduos com síndrome metabólica após recente síndrome coronariana aguda (n=116). Os autoanticorpos contra a LDL oxidada pelo cobre foram avaliados por ELISA. Resultados: pacientes com hipertensão arterial apresentaram menor índice de massa corpórea e circunferência abdominal, maiores níveis de pressão arterial sistólica e diastólica quando comparados aos portadores de SCA (p<0,001). O HDL-C e a Apo A1 foram maiores, enquanto os triglicérides e a Apo B foram menores nos pacientes do grupo hipertensão em estágio 1 (p<0,0001). Os títulos de anticorpos anti-LDLox foram maiores no grupo hipertensão comparados aos do grupo SCA, e os hipertensos do primeiro grupo apresentaram níveis de PCR menores do que indivíduos com SCA (p<0,0001). A análise conjunta de ambos os grupos mostrou, em análise univariada, significante correlação inversa para a PCR (r=-0,284), IMC (r=-0,256), circumferência abdominal (r=-0,368), apo B (r=-0,191) e glicemia (r=-0,303) e correlações positivas entre pressão arterial sistólica e diastólica (r=0,319 e r=0,167, respectivamente), HDL-C e Apo A1 (r=0,224 e r=0,257, respectivamente), com os títulos de anticorpos anti-LDLox (p<0,02). Regressão linear múltipla mostrou que a PCRas, glicemia e circunferência abdominal permaneceram independente e negativamente associados com os títulos de anticorpos anti-LDLox. Conclusões: nossos resultados sugerem que os títulos baixos de anticorpos circulantes anti-LDLox possam estar associados com maior risco cardiovascular. / Objectives: Oxidized lipoproteins and antibodies anti-oxidized LDL (anti-oxLDL) have been detected in human plasma and in atherosclerotic lesions. However, the role of these autoantibodies in the maintenance of health or in the pathogenesis of acute coronary syndromes (ACS) remains unclear. We examined the relationship of human IgG antibodies anti- ox LDL with cardiovascular disease risk markers. Methods: Titers of human anti-oxLDL were measured in hypertensive subjects in stage 1 (n=94) without other risk factors, and in individuals with metabolic syndrome after recent acute coronary syndrome (n=116). Autoantibodies against copper ion oxidized LDL were measured by ELISA. Results: Hipertensive patients presented lower BMI, waist circunference, higher blood pressure levels than those with ACS (p<0.001). HDL-C and Apo A1 were higher, whereas triglycerides and Apo B were lower in those with hypertension stage 1 (p<0.0001). Anti-oxLDL titers were higher in hypertensive patients compared to those with acute coronary syndromes, and hypertensive patients presented lower hs-CRP than those with ACS (p<0.0001). Taken into account both populations, univariate analysis showed small, but significant inverse correlations between the hs-CRP (r=-0.284), BMI (r=-0.256), waist circunference (r=-0.368), apo B (r= -0.191), and blood glucose (r= - 0.303) and positive correlations between systolic and diastolic blood pressure (r=0.319 and r=0.167, respectively), HDL-C and Apo A1 (r=0.224 and r=0.257, respectively), with anti-ox LDL titers (p<0.02). After multiple linear regression, hs-CRP, fasting glycemia and waist circunference remained independently associated with anti-oxLDL. Conclusions: Our results suggest that low titers of circulating anti-oxLDL antibodies may be associated with increased cardiovascular risk. / TEDE / BV UNIFESP: Teses e dissertações

A cinética plasmática de uma emulsão lipídica, que se liga aos receptores de LDL, foi estudada em 23 pacientes com Doença Arterial Coronariana (DAC) e em 16 indivíduos sadios (não apresentaram lesões obstrutivas, nem irregularidades, após serem submetidos à cineangiocoronariografia). Foram selecionados 39 participantes, sendo 30 do sexo masculino e 9 do sexo feminino, na faixa etária de 30 a 84 anos, com índice de Massa Corpórea (IMC) entre 21 e 41 ( kg/m2 ) , divididos em 2 grupos: DAC e Sadio. Após jejum de 12 horas, foram coletadas amostras de sangue total para determinação dos níveis séricos de lipídios e apolipoproteínas. A seguir foram injetadas, endovenosamente, emulsões com marcação radioativa, constituídas por lípides puros (40 mg), trioleína (TG) 1 mg, oleato de colesterol (CE) 20 mg, colesterol (CL) 0,5 mg e os isótopos radioativos 14C-oleato de colesterol e 3H-colesterol. 3H-colesterol. A cada participante foi administrado um volume da emulsão, correspondente à atividade de 37 kBq para o isótopo 14C e de 74 kBq para o isótopo 3H. Amostras de sangue foram colhidas nos intervalos 0,08 , 1, 4, 10 e 24 h , após a aplicação da emulsão. O colesterol-éster e o colesterol livre foram isolados e tiveram suas radioatividades medidas em cintilador. As taxas fracionais de remoção do 14C-oleato de colesterol e do 3H-colesterol livre, foram estimadas por análise compartimental, com auxílio de um programa computacional. Observou-se que: - não há diferença entre os dois grupos, em relação à idade, porcentagem de indivíduos do sexo masculino e feminino e o IMC. Pacientes do grupo DAC apresentam níveis de HDL-colesterol (p=0,0005) e de apolipoproteína A 1 ( p=0,009), reduzidos em relação aos indivíduos do grupo Sadio. As taxas fracionais de remoção do 14C-oleato de colesterol não diferiram entre os grupos DAC e Sadio (0,107&#177; 0,026 horas -1VS 0,064&#177; 0,009 horas -1, p=ns ). A taxa fracional de remoção do 3H-colesterol livre foi maior no grupo DAC do que no Sadio (0,152&#177;0,014 horas -10,096&#177; 0,019 horas-1, p=0,032). A taxa fracional de remoção do 3H-colesterol livre correlacionou-se diretamente com os níveis plasmáticos de triglicérides (r=0,37, p=0,002) e inversamente com os de HDL-C(r=-0,47, p=0,002) e LDL-C (r= -0,35, p = -0,03). Não houve diferença significativas entre os perfis lipídicos e de apolipoproteínas dos pacientes do grupo DAC e os dos indivíduos do grupo Sadio / The plasma kinetics of a lipidic emulsion that binds to LDL receptors was studied in patients with coronary artery disease (CAD) and in control subjects without CAD as documented by coronary angiography. CAD group consistent in 23 subjects (20 male and 3 of female sex , 37-70 year 21-37 body mass index), whereas the healthy control group had 16 subjects (10 male,30-84 aged, 21-41 body mass index). The emulsion was prepared by ultrassonic irradiation of a lipid mixture in aqueous buffer and purified by a two-step ultracentrifugation procedure. The emulsion composition was lipids (40 mg), triglycerides (1 mg), cholestero! (0,5 mg), cholesterol oleate( 20 mg) and was labeled with C14 and 3H . The emulsion vvas injected intravenously into the subjects after a 12 h fast and plasma samples were colected at 0.08, 1, 4, 10 and 24 hour time periods after the injection for determination of the plasma decaying curves of the radioactive lipids. \\/\\lith this purpose, the plasma samp!es were lipid extracted and the lipids were separated by thin-Iayer chromatography. Radioactivity was counted in a liquid scintillation solution. The plasma Fractional Clearance Rate (FC R, in h -1 ) was estimated from the decaying curves by the method of minimum squares, with the aid of a computational software. The two study groups did not differ regarding age, sex, and BMI. The CAD group had smaller plasma HDL cholesterol and apolipoprotein (apo) A1 values, but LDL and VLDL cholesterol as well as apo B, were similar between the two groups. 14C-cholesteryl ester FCR-of the CAD group was not different from the controls (CAD: and Healthy:,p). However, 3H-cholesteroi FCR was greater in the CAD group than in the controls (0,152 &#177;0,014 h -1 vs 0,096&#177; 0,019 h -1 ,p=0,032 ). 3H cholesterol FCR was positively correlated with triglyceride plasma concentration ( r=0,37, p=0,002) and negatively correlated with HDL (r= -0,47, p=0,002) and LDL (r= -0,35, p=0,03). Therefore, the emulsion particles were similarly removed from the circulation in both CAD and healthy groups. Disturbances of cholesterol esterification and removaI from the plasma of free-cholesterol are related with presence od CAD.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: The regulation of LRP in the CNS. Includes bibliographical references. Also available online through Digital Dissertations.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2016. / Made available in DSpace on 2017-05-23T04:21:08Z (GMT). No. of bitstreams: 1 345667.pdf: 5164258 bytes, checksum: a6d6e4f807243c1d227b1ae23222193a (MD5) Previous issue date: 2016 / A hipercolesterolemia familiar é uma doença do metabolismo das lipoproteínas causada principalmente por mutações no gene do receptor de lipoproteína de baixa densidade (LDL). A resultante perda da função do receptor de LDL (LDLr) tem como principal consequência o aumento das concentrações plasmáticas de colesterol. Estudos clínicos reportam, com certa frequência, a comorbidade entre a hipercolesterolemia e transtornos de humor, como a depressão. Além disso, aumento na permeabilidade da barreira hematoencefálica, redução na proliferação celular hipocampal adulta e prejuízos comportamentais de aprendizado e memória foram reportadas em camundongos LDLr-/- (nocautes para o LDLr, modelo animal de hipercolesterolemia familiar). O primeiro objetivo deste estudo foi verificar o comportamento de camundongos LDLr-/- em testes preditivos para a depressão. Sabendo que a neurogênese hipocampal adulta é importante para a manutenção do humor e da cognição, o segundo objetivo foi verificar se a comorbidade entre a hipercolesterolemia e a depressão envolve alterações na neurogênese hipocampal adulta. Nossos resultados demonstraram que os camundongos LDLr-/- apresentaram comportamento tipo-depressivo (anedonia, diminuição do auto-cuidado e da motivação) em testes preditivos para depressão. Este comportamento tipo-depressivo foi revertido pelo tratamento antidepressivo repetido (fluoxetina, 7 dias). Os camundongos LDLr-/- também apresentaram aumento na atividade da monoamina oxidase (MAO) A no córtex cerebral e no hipocampo. Neste estudo as análises comportamentais também revelaram comprometimento de função cognitiva dependente do giro denteado (GD) hipocampal, o que corroborou a redução na proliferação e neurogênese das células precursoras neuronais (CPNs) observada nesta estrutura. Em outra etapa experimental, em cultivo primário de CPNs isoladas do GD de camundongos C57BL/6 adultos, foi caracterizada uma maior expressão gênica de enzimas de síntese de colesterol e do LDLr no estágio proliferativo, enquanto os genes do LRP1 e de enzimas de degradação têm sua expressão aumentada durante a diferenciação celular. Corroborando os resultados obtidos em camundongos LDLr-/-, tanto a exposição destas células à LDL humana isolada, quanto o silenciamento gênico do LDLr (LDLr siRNA), reduziram a proliferação de CPNs. O tratamento com LDL também reduziu a diferenciação neuronal de CPNs, induziu acúmulo de gotículas lipídicas intracelulares e inibiu a expressão gênica de enzimas de síntese do colesterol e do LDLr. Por sua vez, o LDLr siRNA reduziu também os níveis de RNAm do LRP1. A análise de microarray revelou que a LDL inibiu fortemente processos celulares relacionados ao metabolismo do colesterol. O tratamento com siRNA alterou a expressão relativa de muitos genes, no entanto a análise de ontologia não revelou associação significativa destes genes com processos celulares específicos. Ainda, ambos os tratamentos aumentaram a capacidade de reserva das CPNs no teste de função mitocondrial. Em conjunto, estes resultados caracterizam o fenótipo tipo-depressivo e a redução da neurogênese hipocampal adulta em um modelo experimental de hipercolesterolemia familiar, sendo que a neurogênese parece ser influenciada tanto pela presença da LDL no nicho neurogênico quanto pela função do LDLr. Conjuntamente, estes dados destacam o papel do metabolismo do colesterol na neurogênese adulta e reforçam as observações clínicas que associam a hipercolesterolemia à depressão.<br> / Abstract : Familial hypercholesterolemia is a lipoprotein metabolism disorder caused primarily by mutations in the low-density lipoprotein receptor (LDLr) gene, causing loss of function of the LDLr and increased plasma cholesterol. Clinical studies frequently report comorbidity between hypercholesterolemia and depression. Supporting those observations, pre-clinical studies from our group have shown impairments in the CNS of LDLr-/- mice (knockout mice for the LDLr, a murine model of familial hypercholesterolemia). Increased blood brain barrier permeability, reduced hippocampal cell proliferation, and learning and memory impairments in behavioral tasks are some of the CNS abnormalities observed in these mice. The first aim in this study was to verify the behavior of LDLr-/- mice in predictive tasks for depression. Considering that adult neurogenesis is thought to play a role in both mood regulation and cognition, the following objective in this study aimed to avaluate if an impairment in proliferation and differentiation of hippocampal adult neural stem cells (aNSC) may be underlying the co-morbidity between hypercholesterolemia and depression. Endorsing this hypothesis, in the present study we show that LDLr-/- mice present a depressive-like behavior (anhedonia, reduction in self-care and motivational behavior). This depressive-like behavior was abolished by repeated antidepressant treatment (fluoxetine, 7 days). In addition, the LDLr-/- mice presented increased MAO-A activity in the cerebral cortex and hippocampus. Moreover, a deficit in dentate gyrus (DG)-dependent cognitive task was observed in these mice using a DG-dependent behavioral test, corroborating the reduction in DG cell proliferation and neurogenesis. Primary culture of aNSCs isolated from the DG of adult C57BL/6 mice demonstrated that the expression of enzymes involved in cholesterol synthesis (HMG-CoA reductase and squalene synthase) and of LDLr peaks during the proliferation stage of the neurogenic process. On the other hand, the expression of LRP1, cholesterol 24-hydroxylase, and sterol 27-hydroxylase is up-regulated during the differentiation stage. In agreement with the observations from the LDLr-/- mice, exposure to both human LDL as well as silencing of the LDLr (using an LDLr siRNA) reduced cell proliferation and/or neuronal differentiation of aNSCs monolayers. LDL treatment was also associated with an increase in the number of lipid droplets and a down-regulation of mRNA levels of the LDLr and enzymes involved in cholesterol synthesis, whereas LDLr siRNA also reduced LRP1 gene expression. Microarray analysis showed that LDL down-regulated cholesterol metabolism. On the other hand, LDLr siRNA altered the relative expression of hundreds of genes, however a gene ontology analysis failed to define a clear relationship with cellular functions. In addition, both treatments increased the reserve capacity of aNSCs to respond to mitochondrial stress. Altogether, this study describes the depressive-like phenotype and deficits in adult hippocampal neurogenesis in an experimental model of familial hypercholesterolemia. These deficits may result from LDL being present in the neurogenic niche and from compromised LDLr function. In conclusion, the present data implicate cholesterol metabolism as a modulator of adult hippocampal neurogenesis and support the observed co-morbidity between hypercholesterolemia and depression.

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

A cinética plasmática de uma emulsão lipídica, que se liga aos receptores de LDL, foi estudada em 23 pacientes com Doença Arterial Coronariana (DAC) e em 16 indivíduos sadios (não apresentaram lesões obstrutivas, nem irregularidades, após serem submetidos à cineangiocoronariografia). Foram selecionados 39 participantes, sendo 30 do sexo masculino e 9 do sexo feminino, na faixa etária de 30 a 84 anos, com índice de Massa Corpórea (IMC) entre 21 e 41 ( kg/m2 ) , divididos em 2 grupos: DAC e Sadio. Após jejum de 12 horas, foram coletadas amostras de sangue total para determinação dos níveis séricos de lipídios e apolipoproteínas. A seguir foram injetadas, endovenosamente, emulsões com marcação radioativa, constituídas por lípides puros (40 mg), trioleína (TG) 1 mg, oleato de colesterol (CE) 20 mg, colesterol (CL) 0,5 mg e os isótopos radioativos 14C-oleato de colesterol e 3H-colesterol. 3H-colesterol. A cada participante foi administrado um volume da emulsão, correspondente à atividade de 37 kBq para o isótopo 14C e de 74 kBq para o isótopo 3H. Amostras de sangue foram colhidas nos intervalos 0,08 , 1, 4, 10 e 24 h , após a aplicação da emulsão. O colesterol-éster e o colesterol livre foram isolados e tiveram suas radioatividades medidas em cintilador. As taxas fracionais de remoção do 14C-oleato de colesterol e do 3H-colesterol livre, foram estimadas por análise compartimental, com auxílio de um programa computacional. Observou-se que: - não há diferença entre os dois grupos, em relação à idade, porcentagem de indivíduos do sexo masculino e feminino e o IMC. Pacientes do grupo DAC apresentam níveis de HDL-colesterol (p=0,0005) e de apolipoproteína A 1 ( p=0,009), reduzidos em relação aos indivíduos do grupo Sadio. As taxas fracionais de remoção do 14C-oleato de colesterol não diferiram entre os grupos DAC e Sadio (0,107&#177; 0,026 horas -1VS 0,064&#177; 0,009 horas -1, p=ns ). A taxa fracional de remoção do 3H-colesterol livre foi maior no grupo DAC do que no Sadio (0,152&#177;0,014 horas -10,096&#177; 0,019 horas-1, p=0,032). A taxa fracional de remoção do 3H-colesterol livre correlacionou-se diretamente com os níveis plasmáticos de triglicérides (r=0,37, p=0,002) e inversamente com os de HDL-C(r=-0,47, p=0,002) e LDL-C (r= -0,35, p = -0,03). Não houve diferença significativas entre os perfis lipídicos e de apolipoproteínas dos pacientes do grupo DAC e os dos indivíduos do grupo Sadio / The plasma kinetics of a lipidic emulsion that binds to LDL receptors was studied in patients with coronary artery disease (CAD) and in control subjects without CAD as documented by coronary angiography. CAD group consistent in 23 subjects (20 male and 3 of female sex , 37-70 year 21-37 body mass index), whereas the healthy control group had 16 subjects (10 male,30-84 aged, 21-41 body mass index). The emulsion was prepared by ultrassonic irradiation of a lipid mixture in aqueous buffer and purified by a two-step ultracentrifugation procedure. The emulsion composition was lipids (40 mg), triglycerides (1 mg), cholestero! (0,5 mg), cholesterol oleate( 20 mg) and was labeled with C14 and 3H . The emulsion vvas injected intravenously into the subjects after a 12 h fast and plasma samples were colected at 0.08, 1, 4, 10 and 24 hour time periods after the injection for determination of the plasma decaying curves of the radioactive lipids. \\/\\lith this purpose, the plasma samp!es were lipid extracted and the lipids were separated by thin-Iayer chromatography. Radioactivity was counted in a liquid scintillation solution. The plasma Fractional Clearance Rate (FC R, in h -1 ) was estimated from the decaying curves by the method of minimum squares, with the aid of a computational software. The two study groups did not differ regarding age, sex, and BMI. The CAD group had smaller plasma HDL cholesterol and apolipoprotein (apo) A1 values, but LDL and VLDL cholesterol as well as apo B, were similar between the two groups. 14C-cholesteryl ester FCR-of the CAD group was not different from the controls (CAD: and Healthy:,p). However, 3H-cholesteroi FCR was greater in the CAD group than in the controls (0,152 &#177;0,014 h -1 vs 0,096&#177; 0,019 h -1 ,p=0,032 ). 3H cholesterol FCR was positively correlated with triglyceride plasma concentration ( r=0,37, p=0,002) and negatively correlated with HDL (r= -0,47, p=0,002) and LDL (r= -0,35, p=0,03). Therefore, the emulsion particles were similarly removed from the circulation in both CAD and healthy groups. Disturbances of cholesterol esterification and removaI from the plasma of free-cholesterol are related with presence od CAD.

The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein that mediates the uptake of plasma LDL and thereby provides cholesterol to cells. During its synthesis in the endoplasmic reticulum, the LDL receptor folds and forms disulfide bonds in multiple cysteine-rich repeats. N- and 0-linked oligosaccharide chains are added in the endoplasmic reticulum and processed during passage through the Golgi apparatus, en route to the cell surface. The aim of this thesis was to study the influence of post-translational events on the synthesis of the LDL receptor. Experiments addressed: 1) the necessity of the compartmental organisation of the secretory pathway for the glycosylation of the LDL receptor; 2) the requirements for the formation of disulfide bonds; 3) the role for the chaperone, calnexin, in the folding of the LDL receptor; and 4) the manner in which folding was disrupted by mutations. Experiments were performed in cultured cells that were incubated with [³⁵S]methionine. Biosynthetically-labelled LDL receptor was immunoprecipitated and was analysed by SOS polyacrylamide gel electrophoresis.