Lipoprotein lipase (LPL) is the main enzyme hydrolyzing triglyceride-rich lipoproteins in plasma. Proteoglycan-bound LPL on the vascular endothelium represent the functional pool of active enzyme. LPL is regulated in a tissue specific manner according to metabolic demands. Rapid regulation of LPL activity is necessary to provide free fatty acids for storage or energy production. This regulatory mechanism appears to be post-translational and requires synthesis of other protein/proteins. Recently it was demonstrated that angiopoietin-like protein 4 (ANGPTL4) is involved in the metabolism of plasma triglycerides and that it is able to inhibit LPL activity in vitro. These properties were linked to the N-terminal coiled-coil domain of ANGPTL4 (ccd-ANGPTL4), but the mechanism for the inhibition was not known. The aim of this thesis was to investigate the molecular mechanism for inhibition of LPL by ccd-ANGPTL4, to characterize regions in ccd-ANGPTL4 that are important for inactivation of LPL and to study the role of ANGPTL4 for regulation of LPL in vivo. Binding of ccd-ANGPTL4 to LPL was demonstrated by several methods, including surface plasmon resonance. The interaction was transient and resulted in conversion of the enzyme from catalytically active dimers to inactive monomers with decreased affinity for heparin. We have shown that ANGPTL4 mRNA in rat adipose tissue turns over rapidly and that changes in the ANGPTL4 mRNA abundance were inversely correlated to LPL activity, both during the fed to fasted and the fasted to fed transitions. We conclude that ANGPTL4 is a fasting-induced controller of LPL in adipose tissue, acting extracellularly on the native conformation of LPL in an unusual fashion, like an unfolding molecular chaperone. Site directed mutagenesis was used to explore regions in ccd-ANGPTL4 important for inactivation of LPL, and for binding of ANGPTL4 to heparin. Others had shown that ccd-ANGPTL4 forms higher oligomers. Structure prediction analyses demonstrated that the coiled-coil domain of ccd-ANGPTL4 probably forms three consecutive α-helices with strong hydrophobic faces, and that there are clusters of positively charged residues both on the helices and in intervening sequences. We made replacements of hydrophobic residues, positively charged residues, cysteine residues and negatively charged residues in ccd-ANGPTL4. In addition, helix-breaking proline residues were introduced in all three helices. We found that hydrophobic residues are important for oligomer formation. The higher oligomers appeared to be stabilized by disulfide bonds, but cysteines are not crucial for oligomerization. Introduction of Pro-residues in the first and second helix prevented formation of higher oligomers and reduced the ability of ccd-ANGPTL4 to inactivate LPL. We found that negatively charged residues in ccd-ANGPTL4 are important for inactivation of LPL. A heparin binding site was localized in the C-terminal end of ccd-ANGPTL4 (amino acid residues 114-140). To investigate whether LPL is differently processed in different depots of adipose tissue we measured the levels of LPL mRNA, protein and activity in omental and subcutaneous adipose tissue in human subjects undergoing elective surgery. Our results show that, although the expression level of LPL was higher in subcutaneous adipose tissue, the specific LPL activity (ratio of activity over the LPL protein mass) was higher in omental adipose tissue. Interestingly, the levels of ANGPTL4 mRNA were lower in omental compared to subcutaneous adipose tissue in most of the studied subjects. This difference can possibly explain the higher specific activity of LPL in omental adipose tissue and indicated that ANGPTL4 is involved in regulation of LPL activity also in humans. LPL produced by macrophages in the artery wall promotes local accumulation of lipids in these cells, and thereby plays an important role in development of atherosclerosis. The known association between type 2 diabetes and atherosclerosis forwarded us to study production of LPL by THP-1 macrophages under hyperglycemic conditions and under treatment with a peroxisome proliferator-activated receptor delta (PPARδ) agonist (GW501516). We found that LPL activity (but not LPL mass) produced by macrophages was decreased by GW501516. The loss of LPL activity coincided with increased level of ANGPTL4 mRNA, indicating that the agonist regulates LPL activity through expression of ANGPTL4. This effect was even more pronounced in cells grown under hyperglycemic conditions. Our data suggest that a suitable PPARδ agonist, like GW501516, may have protective effects against development of atherosclerosis in subjects with diabetes type 2.

L’hypertriglycéridémie (hyperTG) est une dyslipidémie fréquente, caractérisée par une augmentation de la concentration plasmatique en triglycérides (TG). L’hyperTG est considérée comme un facteur de risque indépendant de la maladie cardiovasculaire, particulièrement de la maladie coronarienne athérosclérotique. Plusieurs facteurs environnementaux et génétiques ont été associés avec l’hyperTG. Cependant, près de 90% des cas d’hyperTG primaire sont encore incomplètement caractérisés au niveau moléculaire. Dernièrement, la protéine GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1), qui a un rôle clef dans le métabolisme des TG, a été associée à l’expression d’hyperTG sévère et rare chez l’humain. Ce mémoire présente les résultats de nos travaux qui ont été effectués afin d’identifier de nouvelles bases moléculaires associées à l’expression de l’hyperTG dans le locus du gène GPIHBP1. Nous avons observé que le polymorphisme GPIHBP1 g.-469G>A (rs72691625), dont la fréquence de l’allèle mineure a été évaluée à 19,6% dans notre échantillon, serait associé à l’expression d’hyperTG (TG ≥ 2mmol/L) dans une population canadienne-française. Ce polymorphisme est associé à un risque 1,67 fois plus grand d’exprimer une triglycéridémie ≥ 2mmol/L chez les porteurs hétérozygotes et 5,7 fois plus grand chez les porteurs homozygotes, comparativement aux non-porteurs. Ce risque d’hyperTG serait exacerbé par la présence concomitante d’une mutation hypertriglycéridémiante dans le gène codant pour la lipoprotéine lipase. La présence de ce polymorphisme serait particulièrement associée à l’expression de la dysbêtalipoprotéinémie familiale et de l’hypertriglycéridémie familiale endogène. GPIHBP1 g.-469G>A est le premier polymorphisme fréquent identifié dans le promoteur du gène à être associé avec l’expression d’hyperTG. GPIHBP1 émerge de plus en plus comme un gène candidat intéressant pour la recherche de nouvelles bases moléculaires pouvant expliquer certaines formes d’hyperTG primaire fréquente. / Hypertriglyceridemia (hyperTG) is a frequent dyslipidemia referring to an increased fasting plasma triglyceride (TG) level ≥ 2 mmol/L. HyperTG is an independent risk factor for cardiovascular disease, such as coronary artery diseases. Several environmental and genetic factors have been associated with hyperTG. Although several gene factors were associated with hyperTG, nearly 90% of cases of primary hyperTG are still incompletely characterized at the molecular level. Recently, few cases of rare and severe hyperTG have been associated with some rare polymorphisms in the gene coding for GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1). This manuscript resumes our research regarding the identification of new molecular bases associated with the expression of frequent hyperTG subtypes in the gene locus GPIHBP1. Our results show that the GPIHBP1 g.-469G>A polymorphism (rs72691625), whose the minor allele frequency was estimated to 19.6% in our sample, was associated with the expression of hyperTG (TG ≥ 2 mmol/L) in a French-Canadian population. Subjects heterozygous and homozygous for this polymorphism respectively had a 1.67-fold and 5.70-fold increased risk to exhibit plasma TG levels ≥ 2mmol/L as compared to non-carriers. This increased risk of hyperTG observed in g.-469A carriers seems to be exacerbated by the concomitant presence of a frequent loss-of-function lipoprotein lipase gene variant. This polymorphism seems also particularly associated with dysbetalipoproteinemia and familial hypertriglyceridemia. The g.-469G>A polymorphism is the first common polymorphism in the GPIHBP1 gene promoter to be associated with the expression of hyperTG. GPIHBP1 emerges as a significant candidate for the molecular based of primary hyperTG.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T14:03:58Z No. of bitstreams: 1 Juliana Stolze Efeito...2015.pdf: 1337321 bytes, checksum: 6fdad9cde6d0cc06bd37fdbe677450ae (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T14:04:13Z (GMT) No. of bitstreams: 1 Juliana Stolze Efeito...2015.pdf: 1337321 bytes, checksum: 6fdad9cde6d0cc06bd37fdbe677450ae (MD5) / Made available in DSpace on 2016-02-15T14:04:13Z (GMT). No. of bitstreams: 1 Juliana Stolze Efeito...2015.pdf: 1337321 bytes, checksum: 6fdad9cde6d0cc06bd37fdbe677450ae (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Introdução: as doenças cardiovasculares acometem milhares de pessoas no mundo. Destas, a doença arterosclerótica está entre as de maior morbimortalidade. Para a avaliação da necessidade de intervenções hemodinâmicas e/ou revascularização miocárdica, há a necessidade da realização do cateterismo (CATE), procedimento de imagem indicado para evidenciar pontos de obstrução e determinar a melhor estratégia cirúrgica. Para a realização do CATE utiliza-se heparina sódica (5000 UI) in bolus. Atualmente, sabe-se que a heparina interfere no remodelamento de partículas lipoproteicas por liberação da lipoproteína lipase (LPL) e da lipase hepática (LH), essa ação pode alterar o transporte reverso do colesterol (TRC), em função de modificações no metabolismo das lipoproteínas. Métodos: foram selecionados por conveniência 20 pacientes, 10 do sexo masculino e 10 do sexo feminino, ambos os sexos, entre 45 e 73 anos, admitidos no Hospital Ana Neri, submetidos à cineangiocoronariografia (CATE). Todas as determinações laboratoriais foram realizadas antes e depois do CATE. Resultados: houve aumento significativo da atividade da lipase e diminuição da concentração dos triglicérides depois do CATE na análise geral e estratificada pelo sexo (p<0,05; Teste t pareado). A razão HDL-C/apoA aumentou significativamente depois do CATE, já a razão LDL-C/apoB não aumentou, nem diminuiu nas análises geral e estratificada por sexo. Enquanto a razão de risco cardiovascular TG/HDL-C diminuiu significativamente, a ApoB/apoA aumentou significativamente na análise geral e estratificada por sexo depois do CATE. As análises de correlações tiveram comportamentos diferentes, sendo a significância estatística encontrada dependente do grupo analisado (geral, masculino e feminino). A concentração do não-HDL-C, semelhante à determinação da haptoglobina, tiveram diminuição significativa na análise geral e no sexo masculino depois do CATE (p<0,05; Teste t pareado), o grupo feminino não mostrou significância. As taxas de incorporação de colesterol livre e fosfolípides não foram significativas depois do CATE. Conclusão: A administração in bolus de heparina sódica interfere no remodelamento de partículas lipoproteicas, sendo este fato evidenciado pelas variações das razões de risco, tais como, HDL-C/apoA, TG/HDL-C. O percentual de incorporação dos fosfolípides e colesterol livre na HDL mostrou-se influenciado em relação ao sexo, o que o torna relevante dado aos resultados encontrados. A utilização de razões de risco e ainda suas correlações mostraram-se melhores indicadores de desfecho sugestivo de doença cardiovascular nessa casuística do que quando avaliados apenas os marcadores séricos do perfil lipídico isoladamente. / Introduction: cardiovascular diseases affect thousands of people worldwide. Of these, the atherosclerotic disease is one of the most morbidity and mortality. To evaluate the need for hemodynamic interventions and / or CABG, the catheterization (CATE) is performed, an imaging procedure to evidence obstruction and to determine the best surgical strategy. To perform CATE, is necessary to use in bolus sodium heparin (5000 IU). Currently, it is known that heparin interferes with the remodeling of the lipoprotein particles by releasing lipoprotein lipase (LPL) and hepatic lipase (HL), this action may alter the reverse cholesterol transport (TRC), by changes in lipoprotein metabolism. Methods: were selected by convenience 20 patients, 10 male and 10 female, both gender, between 45 and 73 years old, admitted to the Hospital Ana Neri, who underwent coronary angiography (CATE). All laboratory measurements were performed before and after CATE. Results: were significant increase in lipase activity and decreased concentration of triglycerides after CATE, in the overall analysis and stratified by sex (p<0.05, paired t test). The HDL-C/apoA ratio increased significantly after CATE, since the LDL-C/apoB ratio has not increased or decreased in the general analysis, and stratified by gender. While TG/HDL-C cardiovascular risk ratio decreased significantly, ApoB/apoA increased significantly in the overall analysis, and stratified by sex after CATE. The correlation analysis had different behaviors, and the statistic significance found, were dependent of the group analyzed (generally male and female). The concentration of non-HDL-C, similar to the determination of haptoglobin, had a significant decrease in the overall analysis and in males after CATE (p<0.05, paired t-test), the female group do not show significance. The free cholesterol and phospholipids incorporation rates were not significant after the CATE. Conclusion: The administration of in bolus sodium heparin interferes in lipoprotein particles remodeling, by evidences from risk ratios variations, such as HDL-C/apoA, and TG/HDL-C. The percentage of phospholipids and free cholesterol incorporation in HDL shows sex influences, which makes it relevant to the obtained results. The use of hazard ratios and their correlations were better surrogate markers at these casuistic of cardiovascular disease than when serum markers of lipid profile were evaluated alone.

Le syndrome de chylomicronémie multifactorielle (MCS) est une maladie complexe au cours de laquelle les valeurs de triglycérides (TG) dépassent 10 mmol/L. Le MCS se manifeste à l'âge adulte et a une prévalence d’environ 1 adulte sur 600 au Québec. Deux conditions doivent être réunies pour développer cette maladie : une composante génétique (oligogénique ou polygénique) ainsi que la présence de facteurs de risque reliés au style de vie (une alimentation riche en gras et en sucres raffinés, une consommation excessive d'alcool, un diabète non contrôlé ou l'obésité). Le MCS est une condition de santé grave, puisqu’il augmente considérablement le risque de pancréatites aigües et peut doubler le risque de maladies cardiovasculaires. Actuellement, il n’y a pas d’étude d’intervention nutritionnelle, réalisée dans cette population, qui permette de connaitre l’approche nutritionnelle la plus bénéfique. Ce mémoire présente les résultats d’une étude croisée randomisée dont l’objectif était d’évaluer l’impact d’une diète faible en lipides et d’une diète faible en glucides sur le profil lipidique à jeun et postprandial chez des patients atteints de MCS en fonction de la présence d’un variant rare à l’état hétérozygote du gène de la lipoprotéine lipase (LPL). Les résultats de cette étude suggèrent qu’une diète faible en lipides permettrait une diminution plus importante des TG chez les sujets porteurs d’un variant rare à l’état hétérozygote de la LPL et pourrait ultimement contribuer à réduire le risque de pancréatite aigüe sur le long terme. / Multifactorial chylomicronemia syndrome (MCS) is a complex disease in which triglyceride (TG) values exceed 10 mmol/L. MCS occurs in adulthood and has a prevalence of approximately 1 in 600 adults in Quebec. Two conditions must be met to develop this disease: a genetic component (oligogenic or polygenic) as well as the presence of lifestyle risk factors (a diet high in fat and refined sugars, excessive alcohol consumption, uncontrolled diabetes or obesity). MCS is a serious health condition, as it significantly increases the risk of acute pancreatitis and can double the risk of cardiovascular disease. Currently, there are no nutritional intervention studies conducted in this population to determine the most beneficial nutritional approach. This thesis presents the results of a randomized crossover study whose objective was to evaluate the impact of a low-fat diet and a low-carbohydrate diet on the fasting and postprandial lipid profile in patients with SCD according to the presence of a rare heterozygous lipoprotein lipase (LPL) gene variant. The results of this study suggest that a low-fat diet would result in a greater reduction in TGs in subjects with a rare heterozygous variant of LPL and may ultimately help reduce the risk of acute pancreatitis in the long term.

Dislipidemia é implicada no processo aterosclerótico do LES. A descrição de aLPL no LES associado a hipertrigliceridemia levou-nos a analisar esse anticorpo no contexto da inflamação envolvida na aterogênese. aLPL foi encontrado em 38% dos pacientes com LES com altos níveis de triglicérides. Correlação positiva significante foi observada entre aLPL e PCR, VHS, SLEDAI, anti-DNA, anti-cardiolipina e CH100 baixo. Análise de regressão múltipla confirmou a forte associação entre aLPL e PCR. Esses dados dão suporte à associação entre inflamação, resposta imune e dislipidemia, introduzindo o aLPL como um novo componente nos complexos eventos da aterogênese do LES / Dyslipidemia is implicated in the atherosclerosis process of SLE. The description of aLPL in SLE associated with hypertrigliceridemia prompted us to analyze this antibody in the context of the inflammation involved in the atherogenesis. aLPL was found in 38 por cento of SLE patients with high levels of triglycerides. Significant positive correlation was observed between aLPL and CRP, ESR, SLEDAI, anti-DNA, anti-cardiolipin and low CH100. Multiple regression analysis confirmed the strong association between aLPL and CRP. These data support the link between inflammation, immune response and dyslipidemia, introducing anti-LPL as new player in the complex events of atherogenesis in SLE

Objetivos: Muitas questões permanecem sobre as causas da aterosclerose acelerada nos pacientes com doenças inflamatórias sistêmicas como a artrite reumatóide (AR). Estudos na população geral sugeriram que além da inflamação existe uma participação patogênica da auto-imunidade na aterosclerose e discutem a possível associação dos anticorpos contra fosfolípides e proteínas de choque térmico (Hsp). O objetivo deste estudo foi investigar a presença de anticorpos contra fosfolípides, beta2-glicoproteína 1 (beta2-gp1), lipoproteína lipase (LPL) e Hsp em pacientes com AR e avaliar a associação entre estes anticorpos com a presença de aterosclerose subclínica de carótidas. Métodos: Anticorpos contra cardiolipina (aCL) IgG e IgM, beta2-gp1 IgG, IgM e IgA , Hsp 60 e Hsp 65 foram testados por ELISA em um grupo de 71 pacientes com AR comparado com 53 indívíduos controles não portadores de AR, de idade e sexo similar. Foram excluídos os pacientes com HAS, diabetes melitos e os fumantes em ambos os grupos. Níveis de lipoproteínas, parâmetros clínicos da AR, questionário de avaliação de saúde (HAQ), escore de atividade da doença (DAS) 28, velocidade de hemossedimentação (VHS) e proteína C reativa (PCR) foram avaliadas. A associação entre a presença dos anticorpos aCL, beta2-gp1, Hsp 60 e Hsp 65 com os parâmetros clínicos de atividade da doença, com a presença das placas de aterosclerose e com a medida da espessura íntimomedial (IMT) da carótida comum, usando ultra-som (US) modo B de alta resolução foram pesquisadas. Resultados: A idade média no grupo com AR foi 48,93 ± 12,31 vs. 45,37 ± 9,37 no grupo controle saudável (p = 0,20); 90,1% no grupo com AR eram do sexo feminino vs. 86,8% no grupo controle (p = 0,56); índice de massa corporal (IMC) foi 25,72 ± 4,57kg/m² no grupo com AR vs. 26,40 ± 4,52kg/m² no grupo controle (p = 0, 69); Os níveis de colesterol, LDL, triglicerídeos e a relação CT/HDL não foram diferentes quando comparamos os 2 grupos (p > 0,05). O nível de HDL foi maior no grupo com AR vs. grupo controle com 60,56 ± 14,40mg/dl e 54,52 ± 11,55 respectivamente (p = 0,05). A média da medida da IMT foi 0,721 ± 0,16 mm na AR e 0,667 ± 0,14mm no grupo controle, e a IMT dos pacientes com AR foi maior naqueles com idade acima dos 50 anos (P < 0,001). No grupo com AR, 14,1% dos pacientes tinham placas nas carótidas vs. 1,9% dos indivíduos saudáveis (p = 0,02) e no grupo com AR, as placas foram mais frequentes nos pacientes acima dos 50 anos (p = 0,004). No grupo AR, 5,6% tinham anticorpos aCL IgG vs. 3,8% no grupo controle (p > 0,05); 14,1% apresentavam aCL IgM vs. 7,5% (p > 0,05); 43,7% tinham anti-beta2-gp1 IgA vs. 40,8% no grupo controle (p > 0,05). A prevalência de anti-beta2-gp1 IgG e IgM e anti-LPL não foi diferente entre os pacientes com AR e o grupo controle ( p > 0,05). A presença dos anticorpos anti-Hsp 60 e 65 na AR e no grupo controle não foram diferentes (p > 0,05), mas os títulos de anticorpos contra Hsp 65 e beta2-gp1 IgM foram maiores no grupo com AR ( p = 0,007 e p = 0,03 respectivamente). Nós não encontramos associação entre a presença e os títulos dos anticorpos aCL IgG e IgM, beta2-gp1 IgG, IgM e IgA, LPL e Hsp 60 e 65 com a presença de placas nas carótidas ou com a medida da IMT (p > 0,05). Discussão: Este estudo confirma achados anteriores da maior prevalência de aterosclerose carotídea nos pacientes com AR e sua correlação com idade, colesterol e LDL. Embora tenha se encontrado uma tendência a maior presença de anticorpos nos pacientes com AR, não houve relação entre a presença da aterosclerose mais prevalente nos pacientes com AR, com a auto-imunidade dirigida contra cardiolipina, beta2-gp1 ou Hsp. / Purpose: Many questions remain unanswered about the causes of accelerated atherosclerosis in patients with inflammatory systemic diseases such as rheumatoid arthritis (RA). Some studies have suggested the role of autoimmunity besides inflammation in the pathogenesis of atherosclerosis in general population and have also discussed the possible association with antibodies directed to phospholipids and heat shock proteins (Hsp). The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoprotein1 (beta2-gp1), lipoprotein lipase (LPL) and Hsp in RA subjects and evaluate the association between these antibodies with the presence of subclinical carotid atherosclerosis. Methods: Tests to antibodies against cardiolipin (aCL) IgG and IgM, beta2-gp1 IgG, IgM and IgA ,Hsp 60 and Hsp 65 were done by ELISA test in a group of 71 RA subjects compared with 53 age and sex-matched non-RA subjects. Smoking, diabetic and hypertensive patients were excluded in both groups. The lipoprotein levels, clinical parameters of RA, Health Assessment Questionnaire (HAQ), Disease Activity Score (DAS) 28, Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) were evaluated. The association between the presence of antibodies against cardiolipin, beta2-gp1 and Hsp 60 and 65 with the clinical parameters of disease activity in RA, and with the presence of plaques and mean intimo-medial thickness (IMT) of common carotid using high-resolution B-mode ultrasound were assessed. Results: Mean age in RA group was 48.93 ± 12.31 vs. 45.37 ± 9.37 in healthy control group (p = 0.20); 90.1% were women in RA group vs. 86.8% in healthy control (p = 0.56); body mass index (BMI) were 25.72 ± 4.57 in RA group vs. 26.40 ± 4.52 in healthy control (p = 0.69). The levels of cholesterol, LDL, triglycerides, CT/HDL didn t have difference between the two groups (p > 0.05). The HDL was higher in RA group vs. control group with 60.56 ± 14.40mg/dl and 54.52 ± 11.55 respectively (p = 0.05). The mean IMT was 0.721 ± 0.16mm in RA and 0.667 ± 0.14mm in control group, and the IMT was higher in patients older than 50 years among RA subjects (p < 0.001). In RA subjects, 14.1% had carotid plaques vs. 1.9% in healthy controls (p = 0.02). In RA group, the carotid plaques were more frequent in patients older than 50 years (p = 0.004). In RA group, 5.6% had antibodies against cardiolipin IgG vs. 3.8% in control group (p > 0.05); 14.1% in RA group had anti-cardiolipin IgM vs. 7.5% (p > 0.05); 43.7% had anti-beta2-gp1 IgA vs 40.8% in control group (p > 0.05). The presence of anti-beta2-gp1 IgG and IgM, and anti-LPL didn t have significant difference between the groups (p > 0.05). The prevalence of antibodies to Hsp 60 and Hsp 65 were similar in RA and in control group (p > 0.05), but the titers of antibodies against Hsp 65 and beta2-gp1 IgM were higher in RA group (p = 0.007 and p = 0.03 respectively). We didn t find relationship between antibodies against cardiolipin IgG and IgM, or beta2-gp1 IgG, IgM and IgA, LPL, Hsp 60 and 65 with mean IMT or plaque carotid (p > 0.05). Discussion: This study confirms the great prevalence of carotid atherosclerosis in RA subjects and its correlation with age, cholesterol and LDL. Although it was found a tendency to have more autoantibodies in RA subjects, there weren t any link between atherosclerosis in RA with autoimmunity against cardiolipin, beta2-gp 1, LPL or Hsp.

Objetivos: Muitas questões permanecem sobre as causas da aterosclerose acelerada nos pacientes com doenças inflamatórias sistêmicas como a artrite reumatóide (AR). Estudos na população geral sugeriram que além da inflamação existe uma participação patogênica da auto-imunidade na aterosclerose e discutem a possível associação dos anticorpos contra fosfolípides e proteínas de choque térmico (Hsp). O objetivo deste estudo foi investigar a presença de anticorpos contra fosfolípides, beta2-glicoproteína 1 (beta2-gp1), lipoproteína lipase (LPL) e Hsp em pacientes com AR e avaliar a associação entre estes anticorpos com a presença de aterosclerose subclínica de carótidas. Métodos: Anticorpos contra cardiolipina (aCL) IgG e IgM, beta2-gp1 IgG, IgM e IgA , Hsp 60 e Hsp 65 foram testados por ELISA em um grupo de 71 pacientes com AR comparado com 53 indívíduos controles não portadores de AR, de idade e sexo similar. Foram excluídos os pacientes com HAS, diabetes melitos e os fumantes em ambos os grupos. Níveis de lipoproteínas, parâmetros clínicos da AR, questionário de avaliação de saúde (HAQ), escore de atividade da doença (DAS) 28, velocidade de hemossedimentação (VHS) e proteína C reativa (PCR) foram avaliadas. A associação entre a presença dos anticorpos aCL, beta2-gp1, Hsp 60 e Hsp 65 com os parâmetros clínicos de atividade da doença, com a presença das placas de aterosclerose e com a medida da espessura íntimomedial (IMT) da carótida comum, usando ultra-som (US) modo B de alta resolução foram pesquisadas. Resultados: A idade média no grupo com AR foi 48,93 ± 12,31 vs. 45,37 ± 9,37 no grupo controle saudável (p = 0,20); 90,1% no grupo com AR eram do sexo feminino vs. 86,8% no grupo controle (p = 0,56); índice de massa corporal (IMC) foi 25,72 ± 4,57kg/m² no grupo com AR vs. 26,40 ± 4,52kg/m² no grupo controle (p = 0, 69); Os níveis de colesterol, LDL, triglicerídeos e a relação CT/HDL não foram diferentes quando comparamos os 2 grupos (p > 0,05). O nível de HDL foi maior no grupo com AR vs. grupo controle com 60,56 ± 14,40mg/dl e 54,52 ± 11,55 respectivamente (p = 0,05). A média da medida da IMT foi 0,721 ± 0,16 mm na AR e 0,667 ± 0,14mm no grupo controle, e a IMT dos pacientes com AR foi maior naqueles com idade acima dos 50 anos (P < 0,001). No grupo com AR, 14,1% dos pacientes tinham placas nas carótidas vs. 1,9% dos indivíduos saudáveis (p = 0,02) e no grupo com AR, as placas foram mais frequentes nos pacientes acima dos 50 anos (p = 0,004). No grupo AR, 5,6% tinham anticorpos aCL IgG vs. 3,8% no grupo controle (p > 0,05); 14,1% apresentavam aCL IgM vs. 7,5% (p > 0,05); 43,7% tinham anti-beta2-gp1 IgA vs. 40,8% no grupo controle (p > 0,05). A prevalência de anti-beta2-gp1 IgG e IgM e anti-LPL não foi diferente entre os pacientes com AR e o grupo controle ( p > 0,05). A presença dos anticorpos anti-Hsp 60 e 65 na AR e no grupo controle não foram diferentes (p > 0,05), mas os títulos de anticorpos contra Hsp 65 e beta2-gp1 IgM foram maiores no grupo com AR ( p = 0,007 e p = 0,03 respectivamente). Nós não encontramos associação entre a presença e os títulos dos anticorpos aCL IgG e IgM, beta2-gp1 IgG, IgM e IgA, LPL e Hsp 60 e 65 com a presença de placas nas carótidas ou com a medida da IMT (p > 0,05). Discussão: Este estudo confirma achados anteriores da maior prevalência de aterosclerose carotídea nos pacientes com AR e sua correlação com idade, colesterol e LDL. Embora tenha se encontrado uma tendência a maior presença de anticorpos nos pacientes com AR, não houve relação entre a presença da aterosclerose mais prevalente nos pacientes com AR, com a auto-imunidade dirigida contra cardiolipina, beta2-gp1 ou Hsp. / Purpose: Many questions remain unanswered about the causes of accelerated atherosclerosis in patients with inflammatory systemic diseases such as rheumatoid arthritis (RA). Some studies have suggested the role of autoimmunity besides inflammation in the pathogenesis of atherosclerosis in general population and have also discussed the possible association with antibodies directed to phospholipids and heat shock proteins (Hsp). The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoprotein1 (beta2-gp1), lipoprotein lipase (LPL) and Hsp in RA subjects and evaluate the association between these antibodies with the presence of subclinical carotid atherosclerosis. Methods: Tests to antibodies against cardiolipin (aCL) IgG and IgM, beta2-gp1 IgG, IgM and IgA ,Hsp 60 and Hsp 65 were done by ELISA test in a group of 71 RA subjects compared with 53 age and sex-matched non-RA subjects. Smoking, diabetic and hypertensive patients were excluded in both groups. The lipoprotein levels, clinical parameters of RA, Health Assessment Questionnaire (HAQ), Disease Activity Score (DAS) 28, Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) were evaluated. The association between the presence of antibodies against cardiolipin, beta2-gp1 and Hsp 60 and 65 with the clinical parameters of disease activity in RA, and with the presence of plaques and mean intimo-medial thickness (IMT) of common carotid using high-resolution B-mode ultrasound were assessed. Results: Mean age in RA group was 48.93 ± 12.31 vs. 45.37 ± 9.37 in healthy control group (p = 0.20); 90.1% were women in RA group vs. 86.8% in healthy control (p = 0.56); body mass index (BMI) were 25.72 ± 4.57 in RA group vs. 26.40 ± 4.52 in healthy control (p = 0.69). The levels of cholesterol, LDL, triglycerides, CT/HDL didn t have difference between the two groups (p > 0.05). The HDL was higher in RA group vs. control group with 60.56 ± 14.40mg/dl and 54.52 ± 11.55 respectively (p = 0.05). The mean IMT was 0.721 ± 0.16mm in RA and 0.667 ± 0.14mm in control group, and the IMT was higher in patients older than 50 years among RA subjects (p < 0.001). In RA subjects, 14.1% had carotid plaques vs. 1.9% in healthy controls (p = 0.02). In RA group, the carotid plaques were more frequent in patients older than 50 years (p = 0.004). In RA group, 5.6% had antibodies against cardiolipin IgG vs. 3.8% in control group (p > 0.05); 14.1% in RA group had anti-cardiolipin IgM vs. 7.5% (p > 0.05); 43.7% had anti-beta2-gp1 IgA vs 40.8% in control group (p > 0.05). The presence of anti-beta2-gp1 IgG and IgM, and anti-LPL didn t have significant difference between the groups (p > 0.05). The prevalence of antibodies to Hsp 60 and Hsp 65 were similar in RA and in control group (p > 0.05), but the titers of antibodies against Hsp 65 and beta2-gp1 IgM were higher in RA group (p = 0.007 and p = 0.03 respectively). We didn t find relationship between antibodies against cardiolipin IgG and IgM, or beta2-gp1 IgG, IgM and IgA, LPL, Hsp 60 and 65 with mean IMT or plaque carotid (p > 0.05). Discussion: This study confirms the great prevalence of carotid atherosclerosis in RA subjects and its correlation with age, cholesterol and LDL. Although it was found a tendency to have more autoantibodies in RA subjects, there weren t any link between atherosclerosis in RA with autoimmunity against cardiolipin, beta2-gp 1, LPL or Hsp.

Dislipidemia é implicada no processo aterosclerótico do LES. A descrição de aLPL no LES associado a hipertrigliceridemia levou-nos a analisar esse anticorpo no contexto da inflamação envolvida na aterogênese. aLPL foi encontrado em 38% dos pacientes com LES com altos níveis de triglicérides. Correlação positiva significante foi observada entre aLPL e PCR, VHS, SLEDAI, anti-DNA, anti-cardiolipina e CH100 baixo. Análise de regressão múltipla confirmou a forte associação entre aLPL e PCR. Esses dados dão suporte à associação entre inflamação, resposta imune e dislipidemia, introduzindo o aLPL como um novo componente nos complexos eventos da aterogênese do LES / Dyslipidemia is implicated in the atherosclerosis process of SLE. The description of aLPL in SLE associated with hypertrigliceridemia prompted us to analyze this antibody in the context of the inflammation involved in the atherogenesis. aLPL was found in 38 por cento of SLE patients with high levels of triglycerides. Significant positive correlation was observed between aLPL and CRP, ESR, SLEDAI, anti-DNA, anti-cardiolipin and low CH100. Multiple regression analysis confirmed the strong association between aLPL and CRP. These data support the link between inflammation, immune response and dyslipidemia, introducing anti-LPL as new player in the complex events of atherogenesis in SLE

Type 2 diabetes is usually caused by a combination of pancreatic β-cell failure and insulin resistance in target tissues like liver, muscle and fat. Insulin resistance is characterised by an impaired effect of insulin to reduce hepatic glucose production and to promote glucose uptake in peripheral tissues. The focus of this study was to further elucidate cellular mechanisms for insulin resistance that may be of relevance for type 2 diabetes in humans. We used rat and human adipocytes as an established model of insulin’s target cells. Glucocorticoids, e.g. cortisol, can induce insulin resistance in vivo. In the present study, pretreatment of rat adipocytes in vitro for 24 h with the cortisol analogue dexamethasone produced a downregulation of glucose uptake capacity as well as a marked depletion of cellular insulin receptor substrate 1 (IRS-1) and protein kinase B (PKB), two proteins suggested to play a critical role in the intracellular signal transduction pathway of insulin. The amount of phosphorylated PKB in response to acute insulin treatment was decreased in parallel to total PKB content. The basal rate of lipolysis was enhanced, but insulin’s antilipolytic effect was not consistently altered following dexamethasone pretreatment. Alterations in blood glucose as well as insulin levels may be of great importance for cellular as well as whole-body insulin resistance. High glucose (≥15 mM) for 24 h induced a decrease in glucose uptake capacity in rat adipocytes and IRS-1 content was reduced whereas IRS-2 was increased. Long-term pretreatment with a high insulin concentration downregulated insulin binding capacity and when combined with high glucose, it produced a pronounced reduction of cellular IRS-1 and 2 content together with insensitivity to insulin’s effect to activate PKB and a decrease in glucose uptake capacity. A common denominator for a decrease in glucose uptake capacity in our rat adipocyte studies seems to be a decrease in IRS-1 content. Adipocytes from type 2 diabetes patients are insulin-resistant, but in our work the insulin resistance could be reversed by incubation of the cells at a physiological glucose level for 24 h. Insulin resistance in fresh adipocytes from type 2 diabetes patients was associated with in vivo insulin resistance and glycemic level and with adipocyte cell size and waist-hip ratio (WHR). As a potential mechanism for postprandial dyslipidemia in type 2 diabetes, we examined the nutritional regulation of subcutaneous adipose tissue lipoprotein lipase (LPL) activity. It was upregulated by ~40-50 % after a standardised lipid-enriched meal and this was very similar in type 2 diabetes patients and control subjects, suggesting that the postprandial hypertriglyceridemia found in type 2 diabetes is not explained by an altered nutritional regulation of LPL in subcutaneous fat. In conclusion, the present work provides evidence for novel interactions between glucocorticoids and insulin in the regulation of glucose metabolism that may potentially contribute to the development of insulin resistance. High levels of glucose and insulin produce perturbations in the insulin signalling pathway that may be of relevance for human type 2 diabetes. Cellular insulin resistance may be secondary to the diabetic state in vivo, e.g. via glucotoxicity. This is supported by our finding that insulin resistance in adipocytes from type 2 diabetes patients can be reversed after incubation at a physiological glucose level. Key words: adipocyte, insulin resistance, type 2 diabetes, insulin signalling, glucose uptake, insulin, glucose, dexamethasone, insulin receptor substrate, protein kinase B, GLUT4, lipoprotein lipase.

Graduation date: 1993