RESUMO
Although Cholesteryl Ester Transfer Protein (CETP) mediates the transfer of cholesteryl esters and triglycerides between lipoprotein particles and thus plays a crucial role in reverse cholesterol transport, the association of variations in the CETP gene with acute myocardial infarction (MI) remains unclear. In this study we examined whether common genetic variation in the CETP gene is related to early-onset non-fatal MI risk in a population-based case-control study from western Washington State. Genotyping for the CETP -2708 G/A, -971 A/G, -629 A/C, Intron-I TaqI G/A and exon-14 A/G (I405V) SNPs was performed in 578 cases with first acute non-fatal MI and in 666 demographically similar controls, free of clinical cardiovascular disease, identified randomly from the community. In-person interviews and non-fasting blood specimens provided data on coronary heart disease risk factors. In men, there was little evidence for an association between single SNPs and MI risk, but in women the age- and race-adjusted OR was found to be significant in 4 out of the 5 CETP single variants. Haplotype analysis revealed two haplotypes associated with MI risk among men. As compared to men homozygous for the most common haplotype D (-2708 G, -971 G, -629 C, TaqI G and exon-14 A), the fully-adjusted multiplicative model identified haplotype G (-2708 G, -971 A, -629 A, TaqI G and exon-14 G) was associated with a 4.0-6.0-fold increased risk of MI for each additional copy; [95%CI 2.4-14.8] and haplotype B (-2708 G, -971 G, -629 A, TaqI A and exon-14 A) showed a significant decreased risk for early onset MI [OR = 0.18; 95%CI 0.04 - 0.75]. An evolutionary-based haplotype analysis indicated that the two haplotypes associated with the MI risk are most evolutionarily divergent from the other haplotypes. Variation at the CETP gene locus is associated with the risk of early-onset non-fatal MI. This association was found to be independent of HDL-C levels. These data and the sex-specific findings require confirmation in other populations.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/genética , Predisposição Genética para Doença , Infarto do Miocárdio/genética , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Fatores Sexuais , Washington/epidemiologiaRESUMO
Familial hypercholesterolemia (FH) has a frequency of 0.2% in most populations of the world. In selected populations such as the Afrikaners in South Africa, the Christian Lebanese, and the French Canadians, the disease is more frequent due to the founder effect. Previous studies demonstrated that a single mutation at the LDL receptor locus, the so-called French Canadian deletion, makes up 60% of the mutant genes responsible for FH in the French Canadian population. In this study, efforts were directed to determine if there were other common LDL receptor mutations in this population. Three missense mutations were identified and each mutation was reproduced and expressed in vitro. Two of the three mutations result in the production of an LDL receptor protein that is not processed to its mature form at a normal rate. Molecular assays were developed to detect the mutations directly, and the LDL receptor genes of 130 French Canadian FH heterozygotes were screened for the presence of the three missense mutations as well as two deletions. LDL receptor mutations were detected in 76% of individuals and 14% had one of the three missense mutations.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Sequência de Bases , Canadá/epidemiologia , DNA/análise , França/etnologia , Haplótipos , Humanos , Hiperlipoproteinemia Tipo II/epidemiologia , Hiperlipoproteinemia Tipo II/etiologia , Dados de Sequência Molecular , Mutação , Receptores de LDL/análiseRESUMO
Familial hypercholesterolemia (FH), an autosomal dominant disease caused by mutations in the LDL receptor gene, is five times more frequent in the Afrikaner population of South Africa than it is in the population of the United States and Europe. It has been proposed that the high frequency is due to a founder effect. In this paper, we characterized 24 mutant LDL receptor alleles from 12 Afrikaner individuals homozygous for FH. We identified two mutations that together makeup greater than 95% of the mutant LDL receptor genes represented in our sample. Both mutations were basepair substitutions that result in single-amino acid changes. Each mutation can be detected readily with the polymerase chain reaction and restriction analysis. The finding of two common LDL receptor mutations in the Afrikaner FH homozygotes predicts that these mutations will predominate in the Afrikaner population and that the high frequency of FH is due to a founder effect. The increased incidence of ischemic heart disease in the Afrikaner population may in part be due to the high frequency of these two mutations in the LDL receptor gene.
Assuntos
Genes , Hiperlipoproteinemia Tipo II/genética , Mutação , Receptores de LDL/genética , População Branca/genética , Alelos , Aminoácidos/genética , Composição de Bases , Etnicidade/genética , Amplificação de Genes , Haplótipos , Homozigoto , Humanos , Hiperlipoproteinemia Tipo II/etiologia , Países Baixos/etnologia , Polimorfismo de Fragmento de Restrição , Receptores de LDL/biossíntese , África do SulRESUMO
The low density lipoprotein (LDL) receptors in fibroblasts from 132 subjects with the clinical syndrome of homozygous familial hypercholesterolemia were analyzed by immunoprecipitation with an anti-LDL receptor monoclonal antibody. 16 of the 132 cell strains (12%) synthesized no immunodetectable LDL receptor protein, indicating the presence of two mutant genes that failed to produce cross-reacting material (crm- mutations). DNA and mRNA from 15 of the 16 crm- patients, representing 30 crm- genes, were available for further study. Haplotype analysis based on 10 restriction fragment length polymorphisms (RFLPs) suggested that the 30 crm- genes represent 13 mutant alleles. Four of the alleles produced no mRNA. Three of these four mRNA- alleles had large deletions ranging from 6 to 20 kb that eliminated the promoter region of the gene. The fourth mRNA- allele did not contain any deletion or alteration in the promoter sequence; the reason for the mRNA- phenotype was not apparent. Nine alleles were positive for mRNAs, of which three encoded mRNAs of abnormal size. One of the abnormal mRNAs was produced by a gene harboring a deletion, and another was produced by a gene with a complex rearrangement. The third abnormal-sized mRNA (3.1 kb larger than normal) was produced by an allele that had no detectable alterations as judged by Southern blotting. The other six mRNA+ alleles appeared normal by Southern blotting and produced normal-sized mRNA but no receptor protein. The current studies demonstrate that mRNA analysis coupled with haplotype determination by Southern blot analysis can be used to classify crm- mutations at a genetic locus where multiple alleles exist.
Assuntos
Alelos , Deleção Cromossômica , Hiperlipoproteinemia Tipo II/genética , Mutação , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Clonagem Molecular , Reações Cruzadas , Feminino , Fibroblastos/análise , Homozigoto , Humanos , Hiperlipoproteinemia Tipo II/imunologia , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Receptores de LDL/genética , Receptores de LDL/imunologiaRESUMO
This paper describes an unusual kindred with familial hypercholesterolemia in which one-third of the relatives with a mutant LDL receptor gene have normal plasma cholesterol concentrations. The proband, a 9-yr-old boy with a plasma cholesterol value greater than 500 mg/dl, is homozygous for a point mutation that changes Ser156 to Leu in the LDL receptor. This substitution in the fourth repeat of the ligand binding domain slows the transport of the protein to the cell surface. The defective receptor cannot bind LDL, which contains apo B-100, but it does bind beta-migrating VLDL, which contains apo E in addition to apo B-100. Although the mother is heterozygous for this mutation, her LDL-cholesterol concentration is consistently in the 28th percentile for the population. Through direct examination of genomic DNA, we identified the mutant gene in heterozygous form in 17 of the mother's relatives, five of whom had normal LDL-cholesterol values. The pedigree was consistent with dominant transmission of a single gene that ameliorates or suppresses the hypercholesterolemic effect of the LDL receptor mutation. Through linkage analysis, we excluded the possibility that this suppressor gene was an allele at the LDL receptor locus. We also excluded the genes for the two ligands for the LDL receptor, apo B-100 and apo E. The existence of this putative suppressor gene may explain the occasional observation of normal LDL-cholesterol concentrations in heterozygotes for LDL receptor mutations.
Assuntos
Genes Dominantes , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Supressão Genética , Adolescente , Adulto , Idoso , Apolipoproteínas B/genética , Apolipoproteínas E/genética , Criança , Feminino , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Receptores de LDL/biossínteseRESUMO
The sterol 27-hydroxylase (EC 1.14.13.15) catalyzes steps in the oxidation of sterol intermediates that form bile acids. Mutations in this gene give rise to the autosomal recessive disease cerebrotendinous xanthomatosis (CTX). CTX is characterized by tendon xanthomas, cataracts, a multitude of neurological manifestations, and premature atherosclerosis. A relatively high prevalence of the disease has been noted in Jews originating from Morocco. The major objectives of the present investigation were to determine the gene structure and characterize the common mutant alleles that cause CTX in Moroccan Jews. The gene contains nine exons and eight introns and encompasses at least 18.6 kb of DNA. The putative promoter region is rich in guanidine and cytosine residues and contains potential binding sites for the transcription factor Sp1 and the liver transcription factor, LF-B1. Blotting analysis revealed that the mutant alleles do not produce any detectable sterol 27-hydroxylase mRNA. No major gene rearrangements were found and single-strand conformational polymorphism followed by sequence analysis identified two underlying mutations: deletion of thymidine in exon 4 and a guanosine to adenosine substitution at the 3' splice acceptor site of intron 4 of the gene. The molecular characterization of CTX in Jews of Moroccan origin provides a definitive diagnosis of this treatable disease.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Mutação da Fase de Leitura/genética , Genes Recessivos/genética , Judeus/genética , Splicing de RNA/genética , Esteroide Hidroxilases/genética , Xantomatose/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Colestanotriol 26-Mono-Oxigenase , Mapeamento Cromossômico , Éxons/genética , Feminino , Biblioteca Gênica , Genoma Humano , Heterozigoto , Humanos , Íntrons/genética , Israel , Dados de Sequência Molecular , Marrocos/etnologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Xantomatose/diagnóstico , Xantomatose/fisiopatologiaRESUMO
OBJECTIVE: To report a case of recurrent tako-tsubo syndrome that developed despite treatment with calcium channel antagonists. CASE SUMMARY: A 76-year-old woman with past medical history of ischemic heart disease and mild chronic asthma presented in 2001 with clinical characteristics and laboratory markers consistent with myocardial ischemia. Coronary angiogram was done with successful balloon angioplasty to LAD stenosis. Ventriculogram and echocardiography demonstrated apical ballooning believed to represent aneurysm formation. Several months later, a follow-up echocardiogram (ECG) revealed normal LV size and function with no wall motion abnormalities. ECG was unremarkable. In 2004, the patient was admitted with dyspnea, chest pain and ST elevation in ECG with normal troponin. Coronary angiogram demonstrated patent coronary tree. Left ventriculogram revealed apical ballooning sparing the base of the heart. Medically controlling the asthma attack led to clinical, echocardiographic and remarkable electrocardiographic normalization within days. Rest thallium perfusion scan done within 48 h demonstrated isolated fully reversible defect in the apex after 24 h suggesting a microvessel etiology. CONCLUSION: Tako-tsubo cardiomyopathy is an increasingly recognized condition. We report here the first case of tako-tsubo recurrence despite treatment with verapamil, and suggest a microvessel pathophysiology supported by rest thallium scan.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Cardiomiopatias/diagnóstico , Infarto do Miocárdio/diagnóstico , Disfunção Ventricular Esquerda/diagnóstico , Verapamil/uso terapêutico , Idoso , Cardiomiopatias/etiologia , Angiografia Coronária , Diagnóstico Diferencial , Eletrocardiografia , Feminino , Aneurisma Cardíaco/etiologia , Aneurisma Cardíaco/prevenção & controle , Sistema de Condução Cardíaco , Humanos , Infarto do Miocárdio/complicações , Isquemia Miocárdica/diagnóstico , Isquemia Miocárdica/tratamento farmacológico , RecidivaRESUMO
BACKGROUND AND AIM: Hypercholesterolaemia is a major risk factor for atherosclerosis. Cholesterol is modulated by genetic and environmental factors. An important regulatory pathway is controlled by the sterol-regulatory element-binding proteins (SREBPs) and the SREBP cleavage-activating protein (SCAP). Both SREBP-2 and SCAP are candidates to contribute to the development of atherosclerosis. We investigated the possible effects of the variability of proteins involved in this regulatory pathway on plasma lipids among familial hypercholesterolaemia patients. METHODS AND RESULTS: Single nucleotide polymorphisms (SNPs) in the genes encoding SREBP-2 and SCAP causing amino acid changes at positions 595 (595G/A) and 796 (796I/V), respectively, were genotyped in 801 FH individuals originating from Israel, The Netherlands, and Switzerland. A linear regression model to examine the associations between SREBP-2 and SCAP isoforms and lipid and lipoprotein levels was used. In females, homozygosity either for the SREBP-2-595A or for the SCAP-796I isoform was associated with higher LDL-cholesterol plasma concentrations (14.7 mg/dl and 20.3 mg/dl, respectively). Surprisingly, heterozygosity for the combination SREBP-2-595A/SCAP-796I was associated with a decrease of 30.28 mg/dl in LDL-C (p-value for gene-gene interaction=0.09). No such effect was observed among FH males. Subgroup analysis considering the most frequent (N>/=24) LDL receptor mutations (del191-2, ins313+1-2, C660X, E207K, S285L) revealed further gene-dosage- and gender-dependent effects of the SCAP mutations on LDL-cholesterol concentrations (p=0.0345). These effects were, however, not present when less frequent LDL receptor mutations were investigated. CONCLUSIONS: These results suggest a possible gene-gene interaction between the genes encoding SREBP-2 and SCAP that modulate plasma lipids in a strictly gender-specific fashion. Further investigation is needed to confirm this effect. A study in a larger FH group or in non-FH hypercholesterolaemic subjects may further define the role of this regulatory mechanism in determining plasma lipid concentration.
Assuntos
DNA/genética , Hiperlipoproteinemia Tipo II/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipídeos/sangue , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Aterosclerose/sangue , Aterosclerose/etiologia , Aterosclerose/genética , Feminino , Genótipo , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/complicações , Israel , Masculino , Mutação , Países Baixos , Reação em Cadeia da Polimerase , Fatores Sexuais , SuíçaRESUMO
Bovine adrenal cells were isolated from the subcapsular region of the gland to obtain cultures enriched in cells of the zona glomerulosa. The cells kept in primary cultures were shown to respond to angiotensin II and adrenocorticorticotropin (ACTH) by a significant increase in aldosterone production. These primary adrenal cultures were used to study the effect of angiotensin II on LDL metabolism. Addition of angiotensin II for 48 h to the culture medium resulted in a 200-300% increase in LDL metabolism, and the lowest effective concentration was 10(-8) -10(-9) M. The angiotensin II effect became evident after 12-16 h of incubation. To compare the metabolism of the 125I-labeled protein moiety to that of cholesteryl ester of LDL, the lipoprotein was labeled also with cholesteryl linoleyl ether, a nonhydrolyzable analog of cholesteryl ester. Under basal conditions and in the presence of angiotensin II or ACTH the ratio of [3H]cholesteryl linoleyl ether to 125I indicate some preferential uptake of the cholesteryl ester moiety. Stimulation of specific LDL binding at 4 degrees C and LDL metabolism at 37 degrees C by 10(-7) M angiotensin II occurred at all concentrations of LDL studied. Linearization of the kinetic data showed that angiotensin II increased the LDL receptor number significantly but not the affinity of the LDL receptor for its ligand. The present findings indicate that in analogy to ACTH, angiotensin II can influence receptor-mediated uptake of LDL by adrenal cortical cells. It remains to be shown whether the angiotensin II effect on LDL metabolism is limited to adrenal cells or will affect other cells which express the angiotensin II receptor.
Assuntos
Córtex Suprarrenal/metabolismo , Angiotensina II/farmacologia , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Córtex Suprarrenal/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Aldosterona/metabolismo , Animais , Bovinos , Células Cultivadas , Humanos , Hidrocortisona/metabolismo , Radioisótopos do Iodo , Lipoproteínas LDL/sangue , Receptores de LDL/efeitos dos fármacosRESUMO
Rat hepatocytes isolated by collagenase perfusion were cultured for 48-72 h and examined for synthesis and secretion of hepatic triacylglycerol lipase activity. Low levels of enzyme activity found in the culture medium increased with time of incubation, and a 3-10-fold rise was encountered in the presence of optimal concentrations of heparin (5 U/ml). After interruption of enzyme synthesis by cycloheximide, plateauing of enzyme activity in the medium occurred, indicating that addition of heparin may not only stabilize but also enhance hepatic triacylglycerol lipase secretion. Synthesis and secretion of hepatic triacylglycerol lipase was not related to cell density, and enzyme secretion was encountered in subconfluent cultures. Release of enzyme activity into the medium was not sensitive to chlorpromazine, a lysosomal enzyme inhibitor, but was completely inhibited by treatment with tunicamycin, an inhibitor of glycosylation. As release of enzyme activity could be maintained for 12 h in the absence of serum, possible hormonal regulation was sought. Under the present experimental conditions, no modulation of hepatic triacylglycerol lipase was encountered by either gonadal or thyroid hormones. Addition of cyclic AMP to the culture medium resulted in a 30% decrease in enzyme activity. The dependence of hepatic triacylglycerol lipase secretion on the intactness of the Golgi apparatus and on vesicular transport was demonstrated by the treatment with monensin. The present results show that cultured rat hepatocytes provide a good model system by which the regulation of synthesis and secretion of hepatic triacylglycerol lipase can be studied.
Assuntos
Lipase/biossíntese , Fígado/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Células Cultivadas , Clorpromazina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Cicloeximida/farmacologia , Heparina/farmacologia , Lipólise/efeitos dos fármacos , Masculino , Monensin/farmacologia , Ratos , Tionucleotídeos/farmacologia , Fatores de Tempo , Tunicamicina/farmacologiaRESUMO
Membranes isolated from bovine adrenal cortex, incubated with human high-density lipoproteins (HDL3), labeled with 125I and [3H]cholesteryl linoleyl ether, showed preferential binding of [3H]cholesteryl linoleyl ether. The preferential binding was Ca2+ independent, temperature sensitive and was slightly increased after phospholipase C or pronase treatment. Reduction of membrane phosphatidylcholine by phospholipase A2 resulted in a marked increase in the binding of the entire HDL3 particle and a relative decrease in preferential binding of [3H]cholesteryl linoleyl ether. These findings suggest that the presence of intact phospholipid in the membrane plays an important role in the magnitude of the preferential binding.
Assuntos
Córtex Suprarrenal/metabolismo , Colesterol/análogos & derivados , Lipoproteínas HDL/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Colesterol/metabolismo , Humanos , Lipoproteínas HDL3 , Fosfatidilcolinas/metabolismo , Fosfolipases A/farmacologia , Fosfolipases A2 , Pronase/farmacologia , Temperatura , Fosfolipases Tipo C/farmacologiaRESUMO
Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.
Assuntos
Glândulas Suprarrenais/metabolismo , Colesterol/análogos & derivados , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Colesterol/metabolismo , Radioisótopos do Iodo , Cinética , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/sangue , Masculino , Ratos , TrítioRESUMO
Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.
Assuntos
Glândulas Suprarrenais/metabolismo , Colesterol/análogos & derivados , Lipoproteínas HDL/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Animais , Bovinos , Células Cultivadas , Colesterol/metabolismo , Heparina/farmacologia , Lipoproteínas HDL/farmacologia , Lipossomos , Masculino , Fosfatidilcolinas/farmacologia , RatosRESUMO
To synthesize the ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1-O-cis-9'- octadecenyl -2-O-cis-9'-[9',10'(n)-3H] ocatadecenyl -sn-glycero-3- phosphocholine, we have adapted available methodology and have obtained a product of high specific activity and purity. The labelled dioleyl ether phosphatidylcholine ( DOEPC ) was used to prepare 250-350 A unilamellar liposomes, which contained also PC and free cholesterol. Following intravenous injection into rats, labelled PC was cleared from the plasma at a faster rate than DOEPC . The uptake of both labelled compounds by the liver increased up to 3 h, at which time there was about 40% of injected PC and 60% of DOEPC . The PC disappeared more rapidly than the DOEPC , so that 17 and 48% of injected label were present in the liver 24 h after injection of PC and DOEPC , respectively. Ten days after injection of DOEPC , about 10% of the label was still present in the liver. During the first 5 days after injection of DOEPC , 10% of radioactivity was found in the gastrointestinal tract and about 20% in the carcass; no increase in carcass radioactivity occurred during the loss of label from the liver. 24 and 48 h after injection of DOEPC , 40% of liver radioactivity was present in a neutral lipid, which on TLC comigrated with triacylglycerol. Since after alkaline hydrolysis this compound comigrated with diacylglycerol, it appears that the ether bond of DOEPC was not hydrolyzed, but after removal of phosphocholine, presumably by phospholipase C, the diether glycerol was reacylated . In experiments in vitro, the rate of exchange of labelled PC with red blood cell phospholipids exceeded that of DOEPC . Incubation of cultured hepatocytes with liposomes containing PC and/or DOEPC resulted in uptake of both phospholipids and metabolism of DOEPC to neutral lipids. The present findings indicate that DOEPC undergoes slow metabolism and can be eliminated from the body. These properties could prove advantageous for the use of DOEPC as a carrier of drugs and possibly as a carrier of free cholesterol in reverse cholesterol transport.
Assuntos
Fígado/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Transporte Biológico , Colesterol/metabolismo , Éteres , Lipossomos , Masculino , Taxa de Depuração Metabólica , RatosRESUMO
Lipoprotein lipase mediated transfer of cholesteryl ester and its ether analog, cholesteryl linoleyl ether, from unilamellar liposomes, prepared from a nonhydrolyzable ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleyl ether-sn-glycero-3-phosphocholine (DOEPC), was studied in various cells in culture. It was found that lipoprotein lipase enhanced the uptake of cholesteryl linoleyl ether and of DOEPC. These findings provided a definitive proof that hydrolysis of liposomal PC is not needed for the lipoprotein lipase catalyzed transfer of cholesteryl linoleyl ether and cholesteryl ester to cells. The lipids transferred by lipoprotein lipase to cells were localized in three compartments, trypsin-releasable, resistant and metabolic; the latter was a chloroquine-sensitive pool as evidenced by inhibition of cholesteryl ester hydrolysis. Labeled PC and, to a lesser extent DOEPC, in the trypsin-releasable pool was able to return to the medium, while cholesteryl linoleyl ether and cholesteryl ester required cholesteryl ester transfer protein for release. The transfer of cholesteryl linoleyl ether and cholesteryl ester into a trypsin-resistant compartment did not require metabolic energy and occurred also in formaldehyde-fixed cells. Metabolic energy was needed for the translocation of cholesteryl linoleyl ether and cholesteryl ester into the lysosomal compartment, presumably by a process of endocytosis. The physiological relevance of the present findings is that as intravascular hydrolysis of triacylglycerol-rich lipoproteins is mediated by lipoprotein lipase attached to endothelial cells, the latter can provide a very extensive surface for removal and metabolism of phospholipids and cholesteryl ester by a mechanism mediated by lipoprotein lipase.
Assuntos
Ésteres do Colesterol/metabolismo , Lipase Lipoproteica/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Transporte Biológico , Bovinos , Células Cultivadas , Cianetos/farmacologia , Desoxiglucose/farmacologia , Fibroblastos/metabolismo , Humanos , Hidrólise , Lipossomos , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Músculo Liso/metabolismo , Ratos , Pele/metabolismoRESUMO
South Africans of Indian origin have a high frequency of Familial Hypercholesterolemia (FH). Fibroblasts from a South African Indian FH homozygote, D, expressed about 30% of the normal number of LDL receptors. These receptors showed defective LDL binding. Sequence and haplotype analysis revealed that D had two different mutant LDL receptor alleles: FH Durban-1 is a point mutation [asp69(GAT) to tyr(TAT)] in ligand-binding repeat 2 and FH Durban-2 is a point mutation [glu119(GAG) to lys(AAG)] in ligand-binding repeat three of the LDL receptor. Single-strand conformational polymorphism analysis, which was used in the initial detection of these mutations, was also employed for subsequent population screening assays. These mutations were not detected in any of the South African Indian FH or hypercholesterolemic patients that were screened.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Sequência de Bases , Fibroblastos/metabolismo , Testes Genéticos , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Índia/etnologia , Dados de Sequência Molecular , Linhagem , Mutação Puntual , Receptores de LDL/química , Receptores de LDL/metabolismo , África do SulRESUMO
Regulation of lipoprotein lipase was studied in mesenchymal rat heart-cell cultures. Treatment of the cultures with dibutyryl cyclic AMP or with cholera toxin resulted in an increase in LPL activity and a comparable increase in LPL mRNA. When the cells were exposed to 100 mM Hepes for 24 h, total enzyme activity rose 2-fold and LPL mRNA increased 2.4-fold. After 72 h, there was a 3-fold increase in LPL mRNA and a 4-fold rise in cellular LPL activity, while medium activity increased 20-fold. Exposure of the cultures to heparin for 24 h resulted in a 3.2-fold increase in total activity and a 36-fold increase in medium activity. This increase was not accompanied by any rise in LPL mRNA. Addition of actinomycin D to control dishes for 24 h resulted in a 33% reduction in LPL mRNA and a 43% reduction in enzyme activity. These values were 71% and 56%, respectively, in Hepes-treated cells, indicating that no stabilization of LPL mRNA occurred under these conditions. It can be concluded that in mesenchymal rat heart-cells in culture cAMP and cholera toxin upregulate lipoprotein lipase at the level of transcription. The increase in LPL activity after 24 h exposure to Hepes could be compatible with transcriptional regulation, while exposure to heparin is not accompanied by a change in LPL mRNA.
Assuntos
Bucladesina/farmacologia , Toxina da Cólera/farmacologia , HEPES/farmacologia , Heparina/farmacologia , Lipase Lipoproteica/metabolismo , Miocárdio/enzimologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Dactinomicina/farmacologia , Lipase Lipoproteica/genética , Miocárdio/citologia , RNA/genética , Ratos , Transcrição GênicaRESUMO
Cerebrotendinous xanthomatosis is characterized by the accumulation of cholestanol and cholesterol in xanthomas and brain causing a number of severe symptoms. More than 20 different mutations have been identified in the gene encoding sterol 27-hydroxylase. Defects in the gene lead to reduced bile acid biosynthesis, with accumulation of 7 alpha-hydroxylated intermediates, one of which is a precursor to cholestanol. The disease can be treated successfully with chenodeoxycholic acid, which reduces the upregulation of cholesterol 7 alpha-hydroxylase and, therefore, the formation of cholestanol. Disruption of the gene encoding sterol 27-hydroxylase in mice does not have the same metabolic consequences as in humans.
Assuntos
Sistema Enzimático do Citocromo P-450/deficiência , Erros Inatos do Metabolismo/complicações , Esteroide Hidroxilases/deficiência , Xantomatose Cerebrotendinosa/etiologia , Animais , Colestanotriol 26-Mono-Oxigenase , Colestanol/metabolismo , Colesterol/sangue , Colesterol/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Modelos Animais de Doenças , Humanos , Mutação , Valores de Referência , Esteroide Hidroxilases/genética , Xantomatose Cerebrotendinosa/diagnóstico , Xantomatose Cerebrotendinosa/genética , Xantomatose Cerebrotendinosa/terapiaRESUMO
The peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARalpha activators. This incited us to screen for PPARalpha variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARalpha mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPARalpha protein (PPARalphatr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARalphatr mRNA is expressed in several human tissues and cells, representing between 20-50% of total PPARalpha mRNA. By contrast, PPARalphatr mRNA could not be detected in rodent tissues. Western blot analysis using PPARalpha-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARalphatr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARalphatr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARalphawt was mainly nuclear localized, the majority of PPARalphatr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARalphatr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARalphawt transactivation function, PPARalphatr interfered with PPARalphatr transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARalphawt by PPARalphatr, suggesting that the dominant negative effect of PPARalphatr might occur through competition for essential coactivators. In addition, PPARalphatr interfered with transcriptional activity of other nuclear receptors such as PPARgamma, hepatic nuclear factor-4, and glucocorticoid receptor-alpha, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARalpha splice variant that may negatively interfere with PPARalphawt function. Factors regulating either the ratio of PPARalphawt vs. PPARalphatr mRNA or the nuclear entry of PPARalphatr protein should therefore lead to altered signaling via the PPARalpha and, possibly also, other nuclear receptor pathways.
Assuntos
Splicing de RNA , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Animais , Apolipoproteína A-II/genética , Apolipoproteína A-II/metabolismo , Sequência de Bases , Sítios de Ligação , Células COS , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Between 1978 and 1985, we conducted a prospective study of 21 patients who survived several attacks of pancreatitis and were diagnosed as having primary hyperlipidemia. None of the patients suffered from chronic alcoholism, primary diabetes, or cholelithiasis or was receiving prolonged steroid therapy. Lowering of plasma lipid values toward normal was achieved in all patients following a program of combined dietary and drug (bezafibrate) therapy. Five patients had recurrent episodes of pancreatitis during the treatment program. These patients were diagnosed subsequently as suffering from bulimia and were all given cognitive behavioral therapy. One patient died following an attack of pancreatitis. An underlying eating disorder should be suspected in patients who relapse after treatment for pancreatitis and hyperlipidemia. Multidisciplinary treatment should be used in these patients to improve therapeutic efficacy and uncover behavioral patterns that have a direct impact on their life expectancy.