O desenvolvimento de resistência a múltiplas drogas na terapêutica do câncer é um importante obstáculo para o tratamento efetivo. Os mecanismos de resistência a múltiplas drogas ocasionam a redução intracelular de agentes quimioterápicos e, por conseqüência, estão envolvidos no fracasso no tratamento do câncer. Os principais genes envolvidos neste fenômeno são: o gene MDR1(multiple drug resisctance), que codifica uma glicoproteína de alto peso molecular, a P-gp; o gene MRP1, que codifica uma glicoproteína de 190 Kda, denominada proteína associada à resistência a múltiplas drogas; e o gene da LRP (proteína relacionada à resistência de pulmão). Alguns estudos sugerem que o colesterol pode estar envolvido diretamente com o fenômeno de resistência a múltiplas drogas, e que os lipídeos podem influenciar várias e complexas funções no MDR, por afetarem o transporte de drogas através da membrana plasmática. Além disso, células tumorais tem maior necessidade de colesterol devido a uma taxa de multiplicação mais elevada que as células normais. Neste estudo analisou-se a expressão dos genes MDR1, MRP1 e LRP em células de sarcoma uterino resistentes à doxorrubicina, e a influência de lipoproteínas. Houve aumento da expressão dos genes MDR1, MRP1 e LRP nas células tratadas com a LDL, sendo mais expressivo o gene MDR1. A HDL diminuiu a expressão dos genes MRP1 e LRP. No entanto, o gene MDR1 teve sua expressão diminuída somente em concentrações maiores. As células cultivadas em meio sem soro fetal apresentaram um elevado aumento na expressão destes genes. Em conclusão, as lipoproteínas podem modular a expressão dos genes MDR1, MRP1 e LRP e, assim, atuar na resistência a múltiplas drogas. / The development of multidrug resistance in anticancer therapy is an obstacle in the efficiency of the treatment. The multidrug resistance mechanism causes reduction of intracellular chemotherapeutical drugs. Therefore, it leads to treatment failure. There are three main multidrug resistance genes: MDR1, which codifies the P-gp (a high weight glycoprotein); MRP1, which codifies a 190 Kda glycoprotein; and, the LRP (lung resistance related protein) gene. Several reports suggest that cholesterol may be directly involved with the multidrug resistance phenomenon and that lipids may affect many complex functions in this regard, as the activity of the drug transport across the plasmatic membrane. Moreover, tumor cells have great cholesterol necessity due to the high cell multiplication rate. Here we described the MDR, MRP, LRP gene expression of a doxorubicin-resistant uterine sarcoma cell line under the influence of lipoproteins. LDL increased the expression of all genes, mainly MDR1. Treatment with HDL led to reduction of MRP and LRP expression. However, the MDR gene expression decreased only by higher concentrations of HDL. Cells grown in serumdeprived medium led to an increased expression of all the studied genes. Therefore, lipoproteins may modulate the MDR, MRP, LRP gene expression and, consequently, the cell resistance to drugs.

Insulin resistance and type 2 diabetes are pathogenetically linked to increased risk of cardiovascular disease. While insulin resistance is defined by a dysregulation in hepatic insulin signaling, it is unclear how this impairment relates to the development of cardiovascular disease. Recently, there has been evidence showing that in insulin resistant individuals, cardiovascular disease is associated with a defect in reverse cholesterol transport – the cardioprotective process by which excess cholesterol is removed from the periphery, and returned to the liver for biliary excretion. Reverse cholesterol transport is facilitated by high-density lipoprotein (HDL) metabolism. Thus, malfunction in HDL turnover during reverse cholesterol transport may contribute to the buildup of atherosclerotic plaques, and subsequent cardiovascular disease in insulin resistant individuals. In this thesis, we seek to establish a better understanding of HDL metabolism and reverse cholesterol transport, as they relate to key transcription factors that mediate hepatic insulin signaling, namely the insulin-repressible forkhead transcription factors, FoxO1, FoxO3, and FoxO4 (FoxOs). We demonstrate that mice with liver-specific triple FoxO knockout (L-FoxO1,3,4) have increased HDL-cholesterol (HDL-C), associated with decreased expression of HDL-C clearance factors, scavenger receptor class B type I (SR-BI) and hepatic lipase, and defective selective uptake of HDL-cholesteryl ester by the liver. As such, we uncover a novel mechanism by which HDL-mediated reverse cholesterol transport to the liver is regulated by the hepatic insulin-->FoxO signaling pathway.

Estudos clínicos e epidemiológicos indicam que baixas concentrações plasmáticas da lipoproteína de alta densidade (HDL) estão forte e independentemente associadas a uma maior incidência de doença arterial coronária (DAC). Entretanto, o insucesso dos agentes que são capazes de aumentar a concentração de HDL-C sugere que a funcionalidade da HDL pode representar um alvo terapêutico mais apropriado. Para a avaliação de um dos aspectos funcionais da HDL, o presente trabalho descreve o desenvolvimento de um método de grande praticidade que permite uma visão integrada de uma etapa fundamental do metabolismo que é a transferência de lípides entre as diferentes classes de lipoproteínas. A avaliação deste fenômeno nas subfrações de HDL, aspecto ainda não explorado, poderá fornecer novas informações a respeito da fisiopatologia da DAC. O método descrito no presente trabalho permite a avaliação da transferência simultânea das quatro principais classes lipídicas doadas por uma nanoemulsão semelhante à LDL para a HDL3. Foi realizada análise dos possíveis interferentes neste método. Verificou-se que a elevação da temperatura de 0 a 40 °C resultou em aumento progressivo na transferência de todos os lipídeos para a HDL3. A variação de pH entre 6,5 e 8,5 e o aumento na concentração de albumina não alteraram os valores de transferência. O aumento no tempo de incubação acima de 60 minutos promoveu diminuição na transferência de colesterol esterificado para a HDL3 e aumento na transferência de fosfolipídeos. O método apresentou boa precisão intra e inter-ensaio, sendo o coeficiente de variação menor que 5% para todos os lipídeos. A porcentagem média de transferência de colesterol livre, fosfolipídeos, triacilglicerol e colesterol em 45 invíduos saudáveis foi de respectivamente de 1,1±0,06; 13,5±0,15; 2±0,05 e 0,84±0,04% e em 45 portadores de doença arterial coronária foi respectimente 1,0±0,04; 15,8±0,44; 1,77±0,04 e 1,0±0,06%. Não houve diferença nos valores de idade, IMC, colesterol total, HDL-C, LDL-C, triacilglicerol, apo A-1, apo B, CETP, PLTP e LCAT, mas os indivíduos portadores de doença arterial coronária apresentaram valores maiores de colesterol livre e colesterol total em relação aos indivíduos saudáveis. O método desenvolvido no presente estudo é prático, preciso e de potencial relevância como ferramenta no estudo dos distúrbios de função da HDL.. / Clinical and epidemiological studies show that low concentrations of high density lipoproteins (HDL) are strongly and independently associated to an increased incidence of coronary artery disease (CAD). However, the lack of success of some drugs developed to increase HDL cholesterol concentrations (HDL-C) suggests that the functional aspects of HDL may represent a more appropriate therapeutic target. To study one of the functional aspects of HDL, the present work describes the development of a practical method that provides an integrated view of a fundamental step of lipid metabolism, namely, the lipid transfer among different lipoprotein classes. This phenomenon in the HDL subfractions is yet unexplored, and could provide new insights on the pathophysiology of CAD. The method described here allows the measurement of the ability of HDL3 to receive the major lipid classes from a radioactively labeled nanoparticle that resemble LDL. The possible interfering factors at the lipid transfer to HDL3 were studied. The increase in the assay temperature from 0 to 40 °C results in a progressive increase in the net transfer of all lipids to HDL3. The increase in incubation time above 60 minutes resulted in a reduced transfer of cholesterol esters to HDL3 with a concomitant increase in the transfer of phospholipids to the latter. The method presented adequate intra and inter-assay precision, with a coefficient of variation smaller than 5% for all lipids. The average percentage of free cholesterol, phospholipids, triacilglycerol an cholesterol transfer to HDL3 was respectively of 1,1±0,06; 13,5±0,15; 2±0,05 e 0,84±0,04% in 45 healthy individuals and 1,0±0,04; 15,8±0,44; 1,77±0,04 e 1,0±0,06% in 45 CAD patients. There was no difference in the age, BMI, total cholesterol, HDL-C, LDL-C, triacilglycerol, apo A-1, apo B, CETP, PLTP and LCAT but the CAD patients had higher levels of total cholesterol and free cholesterol. The method described here is practical, precise and potentially relevant as a tool to study HDL function.

by Chan Chi Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 119-137). / Chapter SECTION 1 --- INTRODUCTION / Chapter 1.1 --- Overview on lipids --- p.2 / Chapter 1.1.1 --- Definition and Classification of Lipids --- p.2 / Chapter 1.1.2 --- Lipoproteins and Apolipoproteins --- p.4 / Chapter 1.1.3 --- Outline of Lipoprotein Metabolism --- p.9 / Chapter 1.1.4 --- LDL Metabolism --- p.12 / Chapter 1.2 --- Dyslipidaemia and Cardiovascular Disease (CVD) --- p.16 / Chapter 1.2.1 --- Definition --- p.16 / Chapter 1.2.2 --- Dyslipidaemia and CAD --- p.16 / Chapter 1.2.3 --- Dyslipidaemia in Non-Insulin Dependent Diabetes Millitus Patients --- p.18 / Chapter 1.2.4 --- Claasification of Dyslipidaemia --- p.24 / Chapter 1.2.5 --- Causes of Hyperlipidaemia --- p.26 / Chapter 1.3 --- Dyslipidaemia and Atherosclerosis --- p.29 / Chapter 1.3.1 --- Pathogenesis of Atherosclerosis --- p.29 / Chapter 1.3.2 --- Mechanism of Atherogenesis --- p.31 / Chapter 1.3.3 --- Intrinsic Roles of LDL in Atherogenesis --- p.33 / Chapter (1) --- LDL Oxidizability --- p.33 / Chapter (2) --- LDL Particle Size Heterogeneity --- p.39 / Chapter 1.4 --- Management of Dyslipidaemia --- p.42 / Chapter 1.5 --- Aims of This Study --- p.49 / Chapter SECTION 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.52 / Chapter 2.1.1 --- Patients and Controls --- p.52 / Chapter 2.1.2 --- Drug Administration Trials --- p.54 / Chapter 2.1.3 --- Blood Samples --- p.55 / Chapter 2.1.4 --- Biochemicals --- p.56 / Chapter 2.1.5 --- Solutions and Buffers --- p.56 / Chapter 2.1.6 --- Apparatus and Equipment --- p.60 / Chapter 2.2 --- Methods --- p.62 / Chapter 2.2.1 --- General Clinical Biochemistry Tests --- p.62 / Chapter 2.2.2 --- Apolipoprotein Assays --- p.62 / Chapter 2.2.3 --- Ultracentrifugation of LDL Fraction --- p.63 / Chapter 2.2.4 --- De-Salting of LDL Fraction --- p.64 / Chapter 2.2.5 --- Qualitative Determination of LDL-Cholesterol and Protein Fractions --- p.64 / Chapter 2.2.6 --- In Vitro Assessment of LDL Oxidizability --- p.65 / Chapter 2.2.7 --- Electrophoretic Gel Pattern of LDL Fraction During In Vitro Oxidizability --- p.65 / Chapter 2.2.8 --- Study of LDL Particle Size --- p.66 / Chapter 2.2.9 --- Statistical Analysis --- p.67 / Chapter SECTION 3 --- RESULTS / Chapter 3.1 --- Quantitative Determination and Standardization of LDL Fractions --- p.69 / Chapter 3.2 --- In Vitro Assessment of LDL Oxidizability --- p.72 / Chapter 3.3 --- Electrophoretic Patterns of LDL during In Vitro Oxidizability --- p.72 / Chapter 3.4 --- LDL Sizing --- p.73 / Chapter 3.5 --- "Correlations of Triglycerides Concentration, LDL Particle Size and Oxidizability" --- p.76 / Chapter 3.6 --- Diabetes Millitus --- p.83 / Chapter 3.6.1 --- NIDDM Patients & Controls --- p.83 / Chapter 3.6.2 --- Effect of Drug Treatment on Serum Lipid-Lipoprotein- Apolipoprotein Profile --- p.86 / Chapter 3.7 --- Hypertriglyceridaemic Patients --- p.90 / Chapter 3.7.1 --- Patients & Controls --- p.90 / Chapter 3.7.2 --- Bezafibrate Treatment --- p.91 / Chapter 3.8 --- CAD Patients --- p.97 / Chapter 3.8.1 --- CAD Patients & Controls --- p.97 / Chapter SECTION 4 --- DISCUSSION / Chapter 4.1 --- Patients and Controls --- p.101 / Chapter 4.2 --- Ultracentrifugation of LDL Fractions --- p.102 / Chapter 4.3 --- In Vitro LDL Oxidizability --- p.103 / Chapter 4.4 --- "Association of TG, LDL Oxidizability and Particle Size" --- p.105 / Chapter 4.5 --- LDL Sizing --- p.106 / Chapter 4.6 --- Comparsion of Patients and Controls in Lipid-Lipoprotein- Apolipoprotein Profiles --- p.107 / Chapter 4.7 --- The Effect of Lovastatin and Acipimox on NIDDM Patients --- p.111 / Chapter 4.8 --- The Effect of Bezafibrate on Hypertriglyceridaemic Patients --- p.114 / Chapter SECTION 5 --- CONCLUSION --- p.116 / References --- p.119 / Appendices --- p.138

Orientador: César Eduardo Fernades / Banca: Eliana Petri Nahas / Banca: Paulo Traiman / Banca: Luciano de Melo Pompei / Banca: Paula Andrea de Albuquerque Salles Navarro / Resumo: Avaliar comparativamente a eficácia da terapêutica de reposição hormonal (TRH) de baixa dose isolada ou associada à sinvastatina no comportamento de marcadores de risco cardiovasculares e do perfil lipídico e lipoprotéico em mulheres sintomáticas e com dislipidemia na pós-menopausa. Duzentas e quarenta e duas mulheres na pós-menopausa, sintomáticas e com dislipidemia foram randomizadas em três grupos de tratamento: A) estradiol (E2) 1mg/acetato de noretisterona (NETA) 0,5mg [E2/NETA] + sinvastativa 20mg; B) E2/NETA + placebo; e C) sinvastatina 20mg + placebo. A eficácia de cada tratamento foi avaliada pela melhora do perfil lipídico e lipoprotéico e dos sintomas climatéricos ao final de 16 semanas de tratamento. O colesterol total, o LDL-C, o colesterol não-HDL e a Apo B diminuíram de forma significativa (p<0,0001) ao final de 16 semanas no grupo que utilizou E2/NETA + sinvastatina e naquele tratado com sinvastatina + placebo. A relação Apo B/Apo A1 também apresentou redução significativa nestes dois grupos (p<0,0001 e p=0,0026 respectivamente). A Apo A1 diminuiu apenas no grupo que recebeu E2/NETA + sinvastatina (p=0,0055). O grupo E2/NETA + placebo não apresentou alterações significativas no perfil lipídico e lipoprotéico entre as visitas basal e final. Aquele que utilizou E2/NETA + sinvastatina apresentou redução significativa do HDL-C e da Apo A1 quando comparado às usuárias de sinvastatina + placebo (p=0,0233 e p=0,0231 respectivamente). No alívio dos sintomas climatéricos, os grupos que utilizaram E2/NETA foram superiores a sinvastatina + placebo. Em mulheres na pós-menopausa com dislipidemia, a associação de E2/NETA em baixa dose com sinvastatina aliviou os sintomas climatéricos de forma semelhante à observada com a E2/NETA isolada e melhorou o perfil lipídico e lipoprotéico de modo semelhante ao uso isolado da sinvastatina. O uso de E2/NETA sem ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: To evaluate low-dose hormone therapy (HT) + simvastatin for vasomotor symptoms and cardiovascular risk markers. Symptomatic postmenopausal women (n=242) with dyslipidemia were randomized to one of three treatment groups: A) 1mg estradiol/0.5mg norethisterone acetate (E2/NETA) + 20mg simvastatin; B) E2/NETA + placebo; or C) 20mg simvastatin + placebo. Lipid and lipoprotein profiles and menopausal symptoms were evaluated after 16 weeks. Total cholesterol, LDL cholesterol, non-HDL cholesterol and Apo-B decreased (p<0.0001) in groups A and C, as did Apo-B/Apo-A1 (p<0.0001 and p=0.0026, respectively). Apo-A1 decreased only in group A (p=0.0055). HDL cholesterol and Apo-A1 were lower in A than C (p=0.0233 and p=0.0231, respectively). Relief of menopausal symptoms was better in A and B compared to C. HT + simvastatin were effective for the treatment of symptomatic postmenopausal women and improved the lipid profile similar to simvastatin alone. It also delivered an improvement in the simultaneous treatment of menopausal symptoms and dyslipidemia / Doutor

A leptospirose é a zoonose mais disseminada no mundo e uma das principais causas de perda econômica no agronegócio. O estudo de novos antígenos de superfície de Leptospira interrogans, é intrigante e pode fornecer conhecimento na interação inicial patógeno-hospedeiro. Os genes LIC13059 e LIC10879, escolhidos por bioinformática, com predição de localização na superfície celular, foram clonados e as proteínas recombinantes expressas em E. coli, para avaliar a interação com componentes do hospedeiro. Após purificação, as proteínas encontravam-se estruturadas e foram reconhecidas por soro de indivíduos infectados. As proteínas recombinantes interagem com plasminogênio, fibrinogênio e laminina. rLIC13059, nomeada Lsa25.6, quando ligada ao fibrinogênio é capaz de inibir a formação de coágulo de fibrina e rLIC10879, nomeada Lsa16, interage com e-caderina, sugerido envolvimento na cascata de coagulação e ligação com o hospedeiro, respectivamente. O plasminogênio ligado às proteínas é convertido em plasmina, o que poderia ajudar a penetração bacteriana no hospedeiro. / Leptospirosis is the most widespread zoonosis and also a major cause of economic loss in animal production worldwide. The study of new surface antigens of Leptospira interrogans is intriguing and may shed light into the initial pathogen-host interactions. We set out to study two novel coding sequences LIC13059 and LIC10879 predicted to be located at the cell surface. The genes were cloned and the recombinant proteins were expressed in E. coli. The purified recombinant proteins presented secondary structures, and interacted with plasminogen, fibrinogen and laminin human components. rLIC13059, named Lsa25.6, when bound to fibrinogen was capable of inhibiting the formation of fibrin clot, while rLIC10879, named Lsa16, interacted with e-cadherin, a mammalian cell receptor, suggesting participation in coagulation pathway and host-cell binding, respectively. The plasminogen captured by both recombinant proteins could be converted into plasmin, a mechanism that could help bacterial penetration in the host.

Alpha-tocopherol, the most abundant form of vitamin E in man, is transported in the circulation by plasma lipoproteins. It plays important roles, not only in preventing lipid peroxidation, but also in modulating several cell functions such as cell signaling and gene expression. While chylomicrons transport dietary alpha-tocopherol after intestinal absorption, LDL and HDL are the major carriers of alpha-tocopherol in fasting plasma and largely contribute to its delivery to cells and tissues. Exchanges of alpha-tocopherol occur between plasma lipoproteins. In addition, alpha-tocopherol transfers have also been observed, in both directions, between plasma lipoproteins and artificial chylomicrons such as intravenous lipid emulsion particles used in parenteral nutrition. In acute conditions, intravenous supply of vitamin E via lipid emulsions, which bypasses the intestinal tract, may offer some advantages over oral administration to rapidly increase alpha-tocopherol plasma concentration. However, many questions remain unanswered regarding kinetics and factors facilitating vitamin E exchanges between lipid emulsions and plasma lipoproteins. The first part of this work aimed at characterizing alpha-tocopherol transfers between alpha-tocopherol rich emulsion particles and plasma lipoproteins as well as the potential for plasma proteins to modulate such transfers. An in vitro model of incubation was used in which emulsion triglyceride concentration was relatively low and lipoprotein levels comparable to those commonly found in the circulation. Results indicate a high capacity for LDL and HDL to acquire extra-amounts of alpha-tocopherol by rapid mass transfers from alpha-tocopherol-rich emulsion particles. Data further shows that, at a fixed alpha-tocopherol concentration provided by emulsion particles, the limiting factor for alpha-tocopherol enrichment is not the capacity of plasma lipoproteins to accommodate extra-amounts of alpha-tocopherol but the facilitating effect of plasma proteins on alpha-tocopherol transfer, the duration of the incubation and possibly the competition between different acceptor particles. Two lipid transfer proteins, PLTP and CETP, appear to largely mediate facilitation of alpha-tocopherol transfer; however, other plasma proteins may be involved. Data further shows that alpha-tocopherol enriched LDL and HDL can readily transfer newly acquired alpha-tocopherol to cells, without any regulation by plasma proteins. Short-term prophylactic vitamin E supplementation has been suggested to be beneficial in some patients in acute conditions who present reduced plasma vitamin E concentrations in association with important changes in plasma lipids and severe oxidative stress. However, it was not clear whether low plasma vitamin E concentration in critically ill patients is related to changes in the composition of plasma lipoproteins or to a decrease in the number of alpha-tocopherol carriers. In the second part of this work, two clinical studies were conducted to analyze changes of lipoprotein concentration and composition in relation to inflammatory reaction and oxidative stress in selected subgroups of critically ill patients, namely patients undergoing cardiac surgery with different procedures. Important changes in LDL and HDL lipid content were observed, some of which contrast with previous observations made in critically ill septic patients. The reduced plasma level of alpha-tocopherol measured after cardiac surgery is entirely due to a reduced number of circulating LDL and HDL particles. Data suggests that such reduced number in alpha-tocopherol carriers post-surgery may impede the delivery of alpha-tocopherol to cells in conditions of increased requirements due to oxidative stress. Avoidance of extracorporeal circulation during cardiac surgery does not reduce inflammation-related changes in plasma lipids but largely prevents oxidative stress. This data on changes occurring in plasma lipoproteins may help to better define strategies against pro-inflammatory changes or oxidative stress. If further studies would confirm a clinical benefit with evidence-based rationale, alpha-tocopherol enriched lipid emulsions may be used to guarantee a sufficient alpha-tocopherol supply in acute conditions associated with fewer alpha-tocopherol transporters and increased requirements due to high risk of oxidative tissue injury.

A common theme in amyloid disorders is the deposition of disease-specific protein aggregates in tissues. Amyloid proteins bind to heparan sulfate (HS), a sulfated glycosaminoglycan, and HS has been found to promote the aggregation process. The present work relates to HS mediated mechanisms of amyloidosis, particularly transthyretin (TTR) amyloidosis, AA-amyloidosis and Alzheimer’s disease (AD). TTR is a transport protein present in the blood and cerebrospinal fluid, which under unclear circumstances can deposit as amyloid in the myocardium of elderly individuals. Examination of cardiac tissue from a 70 year old patient with reported cardiomyopathy reveald co-deposition of TTR amyloid and HS. Studies revealed that HS promotes TTR fibrillization through interaction with a basic motif in the protein. Empolyment of a cell model demonstrated that cell surface HS mediates internalization of TTR, an effect likely facilitated by HS-binding to the basic motif on TTR. Collectively, HS-TTR interactions at the cell surface may have dual outcomes, resulting in either fibrillization or internalization, respectively. During inflammatory conditions, serum amyloid A (SAA), an acute-phase protein associated with the high-density lipoprotein (HDL), can assemble into insoluble amyloid fibrils, causing AA-amyloidosis. We found that HS structures exceeding 12-14 sugar units in length separates SAA from HDL and induces subsequent aggregation of the polypeptide. Our result proposes a novel role for HS in AA-amyloidosis in which a critical length of HS is required for separation of SAA from HDL. Late-onset AD patients show reduced ability to clear cerebral amyloid-β (Aβ) aggregates, a pathological hallmark of the disease. Althought the pathway of Aβ clearance is still unclear, several cell-surface receptors are implicated in Aβ internalization. We found that ApoE facilitated Aβ uptake through interactions with HS-proteoglycans and low-density lipoprotein receptor-related protein 1. The ApoE interaction with Aβ likely promotes Aβ clearance in the brain, but, if unbalanced, may contribute to the pathology of AD.     These findings are in accord with the concept of HS as a promoter of amyloid protein aggregation, but also point to more complex relationship.

Apolipoprotein B100 (apoB), the structural protein component of triglyceride-rich very low density lipoprotein (VLDL) and atherogenic low density lipoprotein, is considered an important risk indicator of atherosclerosis. In insulin resistant states, hepatic overproduction of apoB leads to metabolic dyslipidemia, characterized by high circulating VLDL and hypertriglyceridemia. Since the mitogen-activated protein kinases (MAPKs) and the inhibitor of NFkappaB kinase (IKK)-NFkappaB cascades are perturbed in insulin resistance, we hypothesized that the MAPKs (ERK, p38 and JNK) and the IKK-NFkappaB pathways regulate hepatic apoB output. We modulated these pathways in HepG2, a human hepatoma cell line, and primary hamster hepatocytes using chemical inhibitors and protein overexpression. ApoB synthesis and secretion were examined by metabolic pulse labeling. HepG2 is typically defective in secreting apoB as large VLDL particles and secretes smaller triglyceride-poor apoB-particles. Under continuous pulse labeling, ERK inhibition not only increased apoB secretion, it enabled HepG2 to secrete VLDL-sized particles in the presence of exogenous fatty acid (oleate). Concomitant with the increased apoB-particle size, ERK inhibition raised intracellular triglyceride level and diacylglycerol acyltransferase (DGAT) 1 and DGAT2 mRNA levels. Conversely, ERK activation decreased VLDL-apoB secretion from primary hepatocytes. In contrast to ERK, p38 or JNK inhibition decreased apoB secretion without affecting apoB-particle size from oleate-treated HepG2 cells. JNK inhibition also modulated apoB levels in primary hamster hepatocytes. Interestingly, the development of diet-induced hepatic insulin resistance was associated with decreased ERK, and enhanced p38 and NFkappaB activities. Thus we investigated the role of the NFkappaB pathway in regulating hepatic apoB production. IKK inhibition decreased and IKK overexpression increased apoB levels by modulating apoB mRNA translation and protein stability. IKK inhibition also suppressed hepatic apoB overproduction in an insulin resistance model, the fructose-fed hamster. Altogether, our results suggest that among the MAPK cascades, the MEK-ERK pathway is crucial in regulating apoB-lipoprotein assembly, possibly by modulating lipid availability to newly-synthesized apoB. The inflammatory IKK-NFkappaB cascade is also involved in regulating apoB synthesis and secretion. We postulate that dysregulation in the MAPK or NFkappaB cascades in insulin resistant and inflammatory states may contribute to hepatic apoB overproduction, and the common phenotype of hypertriglyceridemia and dyslipidemia.

Apolipoprotein B100 (apoB), the structural protein component of triglyceride-rich very low density lipoprotein (VLDL) and atherogenic low density lipoprotein, is considered an important risk indicator of atherosclerosis. In insulin resistant states, hepatic overproduction of apoB leads to metabolic dyslipidemia, characterized by high circulating VLDL and hypertriglyceridemia. Since the mitogen-activated protein kinases (MAPKs) and the inhibitor of NFkappaB kinase (IKK)-NFkappaB cascades are perturbed in insulin resistance, we hypothesized that the MAPKs (ERK, p38 and JNK) and the IKK-NFkappaB pathways regulate hepatic apoB output. We modulated these pathways in HepG2, a human hepatoma cell line, and primary hamster hepatocytes using chemical inhibitors and protein overexpression. ApoB synthesis and secretion were examined by metabolic pulse labeling. HepG2 is typically defective in secreting apoB as large VLDL particles and secretes smaller triglyceride-poor apoB-particles. Under continuous pulse labeling, ERK inhibition not only increased apoB secretion, it enabled HepG2 to secrete VLDL-sized particles in the presence of exogenous fatty acid (oleate). Concomitant with the increased apoB-particle size, ERK inhibition raised intracellular triglyceride level and diacylglycerol acyltransferase (DGAT) 1 and DGAT2 mRNA levels. Conversely, ERK activation decreased VLDL-apoB secretion from primary hepatocytes. In contrast to ERK, p38 or JNK inhibition decreased apoB secretion without affecting apoB-particle size from oleate-treated HepG2 cells. JNK inhibition also modulated apoB levels in primary hamster hepatocytes. Interestingly, the development of diet-induced hepatic insulin resistance was associated with decreased ERK, and enhanced p38 and NFkappaB activities. Thus we investigated the role of the NFkappaB pathway in regulating hepatic apoB production. IKK inhibition decreased and IKK overexpression increased apoB levels by modulating apoB mRNA translation and protein stability. IKK inhibition also suppressed hepatic apoB overproduction in an insulin resistance model, the fructose-fed hamster. Altogether, our results suggest that among the MAPK cascades, the MEK-ERK pathway is crucial in regulating apoB-lipoprotein assembly, possibly by modulating lipid availability to newly-synthesized apoB. The inflammatory IKK-NFkappaB cascade is also involved in regulating apoB synthesis and secretion. We postulate that dysregulation in the MAPK or NFkappaB cascades in insulin resistant and inflammatory states may contribute to hepatic apoB overproduction, and the common phenotype of hypertriglyceridemia and dyslipidemia.