Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
J Lipid Res ; 60(9): 1516-1534, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239285

RESUMO

NMR-based quantification of human lipoprotein (sub)classes is a powerful high-throughput method for medical diagnostics. We evaluated select proton NMR signals of serum lipoproteins for elucidating the physicochemical features and the absolute NMR visibility of their lipids. We separated human lipoproteins of different subclasses by ultracentrifugation and analyzed them by 1H NMR spectroscopy at different temperatures (283-323 K) and pressures (0.1-200 MPa). In parallel, we determined the total lipid content by extraction with chloroform/methanol. The visibility of different lipids in the 1H NMR spectra strongly depends on temperature and pressure: it increases with increasing temperatures but decreases with increasing pressures. Even at 313 K, only part of the lipoprotein is detected quantitatively. In LDL and in HDL subclasses HDL2 and HDL3, only 39%, 62%, and 90% of the total cholesterol and only 73%, 70%, and 87% of the FAs are detected, respectively. The choline head groups show visibilities of 43%, 75%, and 87% for LDL, HDL2, and HDL3, respectively. The description of the NMR visibility of lipid signals requires a minimum model of three different compartments, A, B, and C. The thermodynamic analysis of compartment B leads to melting temperatures between 282 K and 308 K and to enthalpy differences that vary for the different lipoproteins as well as for the reporter groups selected. In summary, we describe differences in NMR visibility of lipoproteins and variations in biophysical responses of functional groups that are crucial for the accuracy of absolute NMR quantification.


Assuntos
Lipoproteínas/análise , Espectroscopia de Ressonância Magnética/métodos , Lipoproteínas IDL/análise , Lipoproteínas LDL/análise , Lipoproteínas VLDL/análise , Metabolômica , Pressão , Temperatura
2.
Cytometry A ; 95(8): 869-884, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30994973

RESUMO

Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.


Assuntos
Antígenos CD13/ultraestrutura , Colesterol/isolamento & purificação , Citometria de Fluxo , Proteínas de Membrana Transportadoras/genética , Receptores de IgG/ultraestrutura , Aterosclerose/genética , Transporte Biológico/efeitos dos fármacos , Antígenos CD13/antagonistas & inibidores , Colesterol/metabolismo , Ezetimiba/farmacologia , Glucuronatos/genética , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/metabolismo , Monócitos/metabolismo , Monócitos/ultraestrutura , Receptores de IgG/antagonistas & inibidores
3.
Biochim Biophys Acta ; 1861(8 Pt A): 681-7, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27112638

RESUMO

Lipoprotein X (Lp-X) is an abnormal lipoprotein that may typically be formed in intra- and extrahepatic cholestasis and potentially interfere with lipid analysis in the routine lab. To gain insight into lipid class and species composition, Lp-X, LDL and HDL from cholestatic and control serum samples were subjected to mass spectrometric analysis including phospholipids (PL), sphingolipids, free cholesterol (FC), cholesteryl esters (CE) and bile acids. Our analysis of Lp-X revealed a content of 46% FC, 49% PL with 34% phosphatidylcholine (PC) as main PL component. The lipid species pattern of Lp-X showed remarkable high fractions of mono-unsaturated species including PC 32:1 and PC 34:1 and phosphatidylethanolamine (PE) 32:1 and 34:1. LDL and HDL lipid composition in the same specimens strongly reflected the lipid composition of Lp-X with increased PC 32:1, PC 34:1, PE 32:1, PE 34:1 and FC accompanied by decreased CE compared to controls. Comparison of Lp-X and biliary lipid composition clearly indicates that Lp-X does not originate from a sole release of bile lipids. Moreover, these data present evidence for increased hepatic fatty acid and PL synthesis which may represent a reaction to high hepatic FC level observed during cholestasis.


Assuntos
Bile/metabolismo , Colestase/metabolismo , Dislipidemias/metabolismo , Lipoproteína-X/metabolismo , Bile/química , Humanos , Lipoproteína-X/química
4.
Transfusion ; 57(1): 144-156, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28026009

RESUMO

BACKGROUND: Platelets (PLTs) are derived from megakaryocytes during PLT shedding. Senescent or activated PLTs are expanded in vascular and neurological diseases and release PLT extracellular vesicles (PL-EVs). A systematic analysis of regular messenger RNA (mRNA) and small RNA composition in PLTs and PL-EVs during in vitro PLT senescence has not yet been published. STUDY DESIGN AND METHODS: We isolated PLTs, total PL-EVs, and PL-EV subsets on Days 0 and 5 from human stored donor platelet concentrates. Isolated mRNA species and microRNA (miRNA) species were analyzed by microarrays and deep sequencing. Correlation of mRNA and miRNA species (miR) and miRNA target analyses were performed using bioinformatics. RESULTS: During in vitro PLT senescence, residual PLT mRNA species were decreased and partially converted to miRNA species. Residual mRNAs included encoded genes relevant for atherosclerosis, inflammation (matrix metallopeptidase 14 [MMP-14], granulin [GRN], angiopoietin like 2 [ANGPTL2]), and neurotransmission (dopamine receptor 2 [DRD2], γ-aminobutyric acid type A receptor ρ3 [GABRR3]). Compared with senescent PLTs, PL-EVs have up-regulated their miRNA species involved in "diabesity" and in vascular and metabolic disease (miR-144-3p, miR-486-5p, miR-142-5p, miR-451a, miR-25-3p, miR-145-5p, and let-7f-5p). The 100 highest expressed PL-EV miRNA species determined by microarrays were compared with the 100 highest expressed PL-EV miRNA species detected by deep sequencing. This approach resulted in 66 overlaps. The regulated miRNAs (assessed by both methods) were related to neurological disorders, including targets for Alzheimer's disease (e.g., ß-site amyloid precursor protein APP-cleaving enzyme 1 [BACE1], translocase of outer mitochondrial membrane 40 homolog [TOMM40], neuron navigator 3 [NAV3]). CONCLUSION: During in vitro senescence, PLTs degrade large RNA species. Concomitantly, they up-regulate a distinct set of known small RNA species involved in atherosclerosis, inflammation, and neurodegeneration. PL-EVs enrich miRNA species, likely supporting the role of PLTs and PL-EVs in vascular homeostasis and as carriers of neurodegenerative disease-related miRNA cargo.


Assuntos
Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Senescência Celular , Perfilação da Expressão Gênica , Transcriptoma , Feminino , Humanos , Masculino , MicroRNAs/biossíntese , RNA Mensageiro/biossíntese
5.
Biochim Biophys Acta ; 1851(5): 527-36, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25625330

RESUMO

The syntrophins alpha (SNTA) and beta 2 (SNTB2) are molecular adaptor proteins shown to stabilize ABCA1, an essential regulator of HDL cholesterol. Furthermore, SNTB2 is involved in glucose stimulated insulin release. Hyperglycemia and dyslipidemia are characteristic features of the metabolic syndrome, a serious public health problem with rising prevalence. Therefore, it is important to understand the role of the syntrophins herein. Mice deficient for both syntrophins (SNTA/B2-/-) have normal insulin and glucose tolerance, hepatic ABCA1 protein and cholesterol. When challenged with a HFD, wild type and SNTA/B2-/- mice have similar weight gain, adiposity, serum and liver triglycerides. Hepatic ABCA1, serum insulin and insulin sensitivity are normal while glucose tolerance is impaired. Liver cholesterol is reduced, and expression of SREBP2 and HMG-CoA-R is increased in the knockout mice. Scavenger receptor-BI (SR-BI) protein is strongly diminished in the liver of SNTA/B2-/- mice while SR-BI binding protein NHERF1 is not changed and PDZK1 is even induced. Knock-down of SNTA, SNTB2 or both has no effect on hepatocyte SR-BI and PDZK1 proteins. Further, SR-BI levels are not reduced in brown adipose tissue of SNTA/B2-/- mice excluding that syntrophins directly stabilize SR-BI. SR-BI stability is regulated by MAPK and phosphorylated ERK2 is induced in the liver of the knock-out mice. Blockage of ERK activity upregulates hepatocyte SR-BI showing that increased MAPK activity contributes to low SR-BI. Sphingomyelin which is well described to regulate cholesterol metabolism is reduced in the liver and serum of the knock-out mice while the size of serum lipoproteins is not affected. Current data exclude a major function of these syntrophins in ABCA1 activity and insulin release but suggest a role in regulating glucose uptake, ERK and SR-BI levels, and sphingomyelin metabolism in obesity.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Dieta Hiperlipídica , Proteínas Associadas à Distrofina/deficiência , Lipídeos/sangue , Fígado/metabolismo , Obesidade/metabolismo , Tecido Adiposo Marrom/metabolismo , Adiposidade , Animais , Glicemia/metabolismo , Linhagem Celular Tumoral , Colesterol/sangue , Modelos Animais de Doenças , Proteínas Associadas à Distrofina/genética , Ativação Enzimática , Genótipo , Intolerância à Glucose/sangue , Intolerância à Glucose/genética , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Obesidade/sangue , Obesidade/genética , Obesidade/fisiopatologia , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Receptores Depuradores Classe B/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Esfingomielinas/sangue , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Triglicerídeos/sangue , Aumento de Peso
6.
Transfusion ; 53(3): 612-26, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22804622

RESUMO

BACKGROUND: Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC-EVs), and plasma. STUDY DESIGN AND METHODS: Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC-EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC-EV protein expression. RESULTS: During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC-EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine-1-phosphate (-53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC-EVs were enriched in lysophosphatidic acid (60-fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A-I, E, and J/clusterin. CONCLUSION: This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC-EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease.


Assuntos
Plaquetas/metabolismo , Preservação de Sangue , Glicerofosfolipídeos/metabolismo , Vesículas Secretórias/metabolismo , Esfingolipídeos/metabolismo , Adulto , Transporte Biológico , Plaquetas/fisiologia , Preservação de Sangue/métodos , Senescência Celular/fisiologia , Espaço Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plaquetoferese , Adulto Jovem
7.
J Pharmacol Exp Ther ; 343(3): 556-67, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22918042

RESUMO

Farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, plays an important role in the regulation of cholesterol and more specifically high-density lipoprotein (HDL) homeostasis. Activation of FXR is reported to lead to both pro- and anti-atherosclerotic effects. In the present study we analyzed the impact of different FXR agonists on cholesterol homeostasis, plasma lipoprotein profiles, and transhepatic cholesterol efflux in C57BL/6J mice and cynomolgus monkeys and atherosclerosis development in cholesteryl ester transfer protein transgenic (CETPtg) low-density lipoprotein receptor (LDLR) (-/-) mice. In C57BL/6J mice on a high-fat diet the synthetic FXR agonists isopropyl 3-(3,4-difluorobenzoyl)-1,1-dimethyl-1,2,3,6-tetrahydroazepino[4,5-b]indole-5-carboxylate (FXR-450) and 4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl]benzoic acid (PX20606) demonstrated potent plasma cholesterol-lowering activity that affected all lipoprotein species, whereas 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) and 6-ethyl chenodeoxycholic acid (6-ECDCA) showed only limited effects. In FXR wild-type mice, but not FXR(-/-) mice, the more efficacious FXR agonists increased fecal cholesterol excretion and reduced intestinal cholesterol (re)uptake. In CETPtg-LDLR(-/-) mice PX20606 potently lowered total cholesterol and, despite the observed HDL cholesterol (HDLc) reduction, caused a highly significant decrease in atherosclerotic plaque size. In normolipidemic cynomolgus monkeys PX20606 and 6-ECDCA both reduced total cholesterol, and PX20606 specifically lowered HDL(2c) but not HDL(3c) or apolipoprotein A1. That pharmacological FXR activation specifically affects this cholesterol-rich HDL(2) subclass is a new and highly interesting finding and sheds new light on FXR-dependent HDLc lowering, which has been perceived as a major limitation for the clinical development of FXR agonists.


Assuntos
Anticolesterolemiantes/farmacologia , Aterosclerose/prevenção & controle , Benzoatos/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Colesterol/sangue , Isoxazóis/farmacologia , Lipoproteínas HDL/sangue , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores de LDL/metabolismo , Animais , Anticolesterolemiantes/química , Anticolesterolemiantes/uso terapêutico , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/metabolismo , Benzoatos/química , Benzoatos/uso terapêutico , Transporte Biológico , Colesterol/administração & dosagem , Colesterol/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Fezes/química , Feminino , Humanos , Isoxazóis/química , Isoxazóis/uso terapêutico , Fígado/metabolismo , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Receptores de LDL/genética , Especificidade da Espécie , Relação Estrutura-Atividade
8.
Transfusion ; 50(8): 1665-76, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20158687

RESUMO

BACKGROUND: A hallmark of platelet (PLT) storage lesion is the loss of PLT lipids. Due to technical limitations a detailed lipidomic analysis of plateletpheresis products during storage was so far not available. STUDY DESIGN AND METHODS: Fifty plateletpheresis products were stored for 5 days at 22°C under agitation. Each day plasma and PLTs were isolated by gel filtration and lipid species analyzed by electrospray ionization tandem mass spectrometry. RESULTS: During 5 days of storage the total lipid content decreased by 10% in PLTs and increased by 5% in plasma. We observed the following changes in lipid class fractions during storage relative to the day of preparation: increases of 69% ceramide (Cer), 32% lysophosphatidylcholine (LPC), and 49% cholesteryl esters (CE) and a decrease of 10% free cholesterol (FC) in PLTs and elevation of 43% LPC and 14% CE and a decline of 20% phosphatidylcholine (PC) and 24% FC in plasma. Significant lipid species shifts were observed for phosphatidylserine, Cer, and LPC. Correlation analysis of lipid changes in plasma indicated that lecithin-cholesterol-acyltransferase (LCAT) activity may be responsible for the shift in plasma lipid composition. These lipid changes correlated between plasma and PLTs for LPC, FC, and CE fractions. CONCLUSIONS: This study presents for the first time detailed lipid species profiles of PLTs and plasma during storage of PLT concentrates. These data provide clear evidence for LCAT-mediated esterification of FC and LPC generation in the plasma of PLT concentrates. Moreover, we showed evidence that these changes also impact PLT lipid composition.


Assuntos
Plaquetas/química , Preservação de Sangue , Senescência Celular , Lipídeos/sangue , Adulto , Plaquetas/fisiologia , Cromatografia em Gel , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plaquetoferese , Espectrometria de Massas por Ionização por Electrospray
9.
Biochim Biophys Acta ; 1762(3): 341-50, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16439103

RESUMO

Inflammatory bowel disease (IBD) constitutes a severe intestinal disorder in developed countries with increasing incidence worldwide. Upcoming evidence indicates an important role of intestinal epithelial barrier function in the development of IBD. Fatty acids exert nutritional and protective effects on enterocytes, serve as activators of transcription and constitute precursors of inflammatory mediators. The aim of this study was to investigate differential regulation of genes involved in fatty acid uptake and endogenous fatty acid biosynthesis in IBD. Mucosal biopsy specimens from non-affected regions of the intestine were subjected to DNA microarray analysis. Gene array analysis revealed a variety of genes involved in fatty acid uptake and synthesis to be differentially expressed in ileum and colon of selected IBD patients. To verify these results, real-time RT-PCR was performed for selected regulated candidate genes in larger IBD sample numbers. In single biopsy analysis long chain acyl-CoA synthetase (ACSL) 1 and 4 were upregulated in IBD (P<0.05), while a significant decrease in fatty acid synthase expression was found in ileum and colon of ulcerative colitis patients (P<0.001). Expression of the transcription factor liver X receptor (LXR) which was previously shown to induce fatty acid synthase gene expression was not altered on mRNA level in IBD. However, in cell culture experiments using the human intestinal cell line LS174T induction of fatty acid synthase by the LXR ligand T0901317 was inhibited by TNFalpha. Moreover, these experiments indicated a decrease of LXR protein levels by TNFalpha treatment. These data suggest that the decrease of fatty acid synthase expression in ulcerative colitis patients could be at least partially due to a loss of LXR expression and function in the presence of pro-inflammatory cytokines. Observed alterations in expression of genes of fatty acid metabolism may contribute to the pathophysiology of ulcerative colitis.


Assuntos
Ácidos Graxos/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Animais , Biópsia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Mucosa Intestinal/citologia , Receptores X do Fígado , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Nucleares Órfãos , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
10.
Mol Biol Cell ; 15(12): 5399-407, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15469992

RESUMO

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Membrana/metabolismo , Fagocitose , Doença de Tangier/metabolismo , Doença de Tangier/patologia , Transportador 1 de Cassete de Ligação de ATP , Adulto , Células Cultivadas , Humanos , Macrófagos/metabolismo , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Monócitos/metabolismo , Ligação Proteica , Proteínas Qa-SNARE , RNA Interferente Pequeno
11.
PLoS One ; 12(5): e0178368, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28542499

RESUMO

BACKGROUND: Sequence variants near the human gene for P4-type ATPase, class V, type 10D (ATP10D) were shown to significantly associate with circulating hexosylceramide d18:1/16:0 and d18:1/24:1 levels, obesity, insulin resistance, plasma high density lipoprotein (HDL), coronary stenotic index and intracranial atherosclerotic index. In mice Atp10d is associated with HDL modulation and C57BL/6 mice expressing a truncated, non-functional form of ATP10D easily develop obesity and insulin resistance on high-fat diet. RESULTS: We analyzed metabolic differences of ATP10D deficient C57BL/6J wild type and ATP10D transgenic C57BL/6J BAC129 mice. ATP10D transgenic mice gain 25% less weight on high-fat diet concomitant with a reduced increase in fat cell mass but independent of adipocyte size change. ATP10D transgenic mice also had 26% lower triacylglycerol levels with approximately 76% bound to very low density lipoprotein while in ATP10D deficient wild type mice 57% are bound to low density lipoprotein. Furthermore increased oxygen consumption and CO2 production, 38% lower glucose and 69% lower insulin levels and better insulin sensitivity were observed in ATP10D transgenic mice. Besides decreased hexosylceramide species levels were detected. Part of these effects may be due to reduced hepatic stearoyl-CoA desaturase 1 (SCD1) expression in ATP10D transgenic mice, which was reflected by altered fatty acid and lipid species patterns. There was a significant decrease in the hepatic 18:1 to 18:0 free fatty acid ratio in transgenic mice. The ratio of 16:1 to 16:0 was not significantly different. Interestingly both ratios were significantly reduced in plasma total fatty acids. SUMMARY: In summary we found that ATP10D reduces high-fat diet induced obesity and improves insulin sensitivity. ATP10D transgenic mice showed altered hepatic expression of lipid-metabolism associated genes, including Scd1, along with changes in hepatic and plasma lipid species and plasma lipoprotein pattern.


Assuntos
Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/metabolismo , Metabolismo dos Lipídeos/fisiologia , Adipócitos/metabolismo , Animais , Dieta Hiperlipídica/métodos , Ácidos Graxos/metabolismo , Fígado Gorduroso/metabolismo , Insulina/metabolismo , Resistência à Insulina/fisiologia , Lipoproteínas HDL/sangue , Lipoproteínas VLDL/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/sangue
12.
FASEB J ; 18(3): 574-6, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14734645

RESUMO

Cholesterol efflux to apolipoprotein A-1 (apoA-1) from cholesterol-loaded macrophages is an important anti-atherosclerotic mechanism in reverse cholesterol transport. We recently provided kinetic evidence for two distinct pathways for cholesterol efflux to apoA-1 [Gaus et al. (2001) Biochemistry 40, 9363]. Cholesterol efflux from two membrane pools occurs sequentially with different kinetics; a small pool rapidly effluxed over the first hour, followed by progressive release from a major, slow efflux pool over several hours. In the present study, we propose that the rapid and slow cholesterol efflux pools represent cholesterol derived from lipid raft and nonraft domains of the plasma membrane, respectively. We provide direct evidence that apoA-1 binds to both lipid raft and nonraft domains of the macrophage plasma membrane. Conditions that selectively deplete plasma membrane lipid raft cholesterol, such as incorporation of 7-ketocholesterol or rapid exposure to cyclodextrins, block apoA-1 binding to these domains but also inhibit cholesterol efflux from the major, slow pool. We propose that cholesterol exported to apoA-1 from this major slow efflux pool derives from nonraft regions of the plasma membrane but that the interaction of apoA-1 with lipid rafts is necessary to stimulate this efflux.


Assuntos
Apolipoproteína A-I/fisiologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Cetocolesteróis/metabolismo , Macrófagos/metabolismo , Microdomínios da Membrana/metabolismo , beta-Ciclodextrinas , 2-Hidroxipropil-beta-Ciclodextrina , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP , Transporte Biológico , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , LDL-Colesterol/farmacologia , Ciclodextrinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos
13.
J Photochem Photobiol B ; 116: 79-88, 2012 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-22982209

RESUMO

Many ATP binding cassette (ABC) transporters are important regulators of lipid homeostasis and have been implicated in keratinocyte lipid transport. Ultraviolet (UV) light exposure is a known epidermal stressor, which amongst other effects causes lipid alterations and defective lamellar body biogenesis. To elucidate the background of these lipid changes we studied the effect of UVB light on ABC transporter expression. The effect of UVB treatment on the levels of 47 known human ABC transporter mRNAs was analyzed in normal human epidermal keratinocytes. Immunoblots and promoter assays were carried out for ABCA1 and ABCG1. The mRNA levels of cholesterol transport regulators ABCA1 and ABCG1 were markedly downregulated by UVB, parallel to the lamellar ichthyosis related glucosylceramide transporter ABCA12 and the suspected sphingosine-1-phosphate and cholesterol sulfate transporter ABCC1. The long but not the short alternative splice variant of the ABCF2 was found to be markedly upregulated rapidly after UVB irradiation. Immunoblot confirmed ABCA1 and ABCG1 protein downregulation, and luciferase assays showed suppression of their promoters by UVB. These proteins mostly transport lipids, which account for the integrity of the epidermal barrier; therefore our findings on the UVB regulation of ABC transporters may explain the appearance of barrier dysfunction after UVB exposure.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação da Expressão Gênica/efeitos da radiação , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Transporte Biológico , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Células Epidérmicas , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos da radiação , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Protetores contra Radiação/farmacologia
14.
J Lipid Res ; 50(3): 574-585, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18832345

RESUMO

Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 microl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort.


Assuntos
Lipídeos/sangue , Lipoproteínas/sangue , Lipoproteínas/química , Adulto , Análise Química do Sangue , Colesterol/sangue , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Feminino , Glicerofosfolipídeos/sangue , Humanos , Lipídeos/isolamento & purificação , Lipoproteínas/classificação , Masculino , Espectrometria de Massas por Ionização por Electrospray , Esfingolipídeos/sangue , Triglicerídeos/sangue , Adulto Jovem
15.
Cytometry A ; 69(3): 196-9, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16479606

RESUMO

BACKGROUND: Platelet activation by atherogenic lipoproteins can be antagonized by high density lipoprotein (HDL), probably via interaction with the ATP-binding cassette transporter A1 (ABCA1). METHODS: ABCA1 expression and its association with cholesterol rich membrane domains was analyzed by mRNA and Western blot analysis. HDL effects on platelet receptor clustering were analyzed by flow cytometric analysis of fluorescence resonance energy transfer between fluorochrome-labeled antibodies. RESULTS: ABCA1 expression increased upon megakaryocytic differentiation of human stem cells and ABCA1 protein partially associated to LubroIWX-resistant membrane domains. Plasma HDL-cholesterol in healthy donors negatively correlated to the platelet membrane cholesterol content. Receptor cluster analysis revealed a decrease in the association of Gplb and FcgammaRII upon incubation of platelets with HDL3. CONCLUSION: Our results suggest that HDL modulates platelet reactivity by altering lipid raft associated receptor clustering.


Assuntos
Plaquetas/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos CD34/análise , Plaquetas/química , Plaquetas/metabolismo , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Sangue Fetal/citologia , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica/efeitos dos fármacos , Humanos , Integrina beta3/análise , Lipoproteínas HDL3 , Megacariócitos/química , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Glicoproteínas de Membrana , Microdomínios da Membrana/química , Proteínas de Membrana/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteína IIb da Membrana de Plaquetas/análise , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de IgG/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
16.
Cytometry A ; 69(3): 192-5, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16479617

RESUMO

Rafts resemble cholesterol- and glycosphingolipid-enriched, liquid-ordered plasma membrane microdomains, showing resistance to nonionic detergents, and are involved in various cellular processes. In the present study, we have tested surface antigens on resting and lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes for their detergent resistance (i.e. raft-association), by flow cytometry. Constitutive (CD14, CD32, CD55), or LPS-induced (CD81) raft-association, and detergent solubility (i.e. exclusion of rafts) (CD71) of monocyte antigens in the presence of 0.01% Triton X-100 are clearly demonstrated. Flow cytometric detergent insolubility is a powerful tool for rapid screening the raft-association of monocyte antigens in a whole-blood assay.


Assuntos
Antígenos de Superfície/análise , Detergentes/química , Citometria de Fluxo/métodos , Microdomínios da Membrana/química , Monócitos/química , Antígenos CD/análise , Antígenos de Superfície/metabolismo , Antígenos CD55/análise , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Cinética , Receptores de Lipopolissacarídeos/análise , Lipopolissacarídeos/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Octoxinol/química , Receptores de IgG/análise , Receptores da Transferrina/análise , Solubilidade , Tetraspanina 28
17.
Biochem Biophys Res Commun ; 342(4): 1083-7, 2006 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-16513087

RESUMO

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway.


Assuntos
Proteína de Ligação a Androgênios/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Diferenciação Celular , Linhagem Celular , Humanos , Fagócitos/citologia , Fagócitos/metabolismo , Proteína de Ligação a Fosfatidiletanolamina
18.
Am J Respir Crit Care Med ; 174(5): 571-80, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16728712

RESUMO

RATIONALE: ABCA3 mutations are known to cause fatal surfactant deficiency. OBJECTIVE: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. METHODS: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. RESULTS: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. CONCLUSION: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Mutação/genética , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Lavagem Broncoalveolar , Estudos de Casos e Controles , Feminino , Humanos , Recém-Nascido , Masculino , Linhagem , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Síndrome do Desconforto Respiratório do Recém-Nascido/patologia , Nascimento a Termo
19.
Biochem Biophys Res Commun ; 293(2): 759-65, 2002 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-12054535

RESUMO

Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues. ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1. Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains. Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex. ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains. Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Adulto , Sequência de Aminoácidos , Membrana Celular/química , Células Cultivadas , Vesículas Citoplasmáticas/química , Proteínas Associadas à Distrofina , Fibroblastos/metabolismo , Humanos , Substâncias Macromoleculares , Pessoa de Meia-Idade , Dados de Sequência Molecular , Testes de Precipitina , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Utrofina
20.
Genomics ; 79(5): 693-702, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11991719

RESUMO

Apolipoprotein A-I (apoA-I) is the major apolipoprotein of high-density lipoproteins (HDL) and has an important role in the regulation of the stability, lipid transport, and metabolism of HDL particles. To identify novel proteins that are involved in HDL metabolism, we used mature apoA-I (amino acids 25-267) as a bait for the screening of a human liver two-hybrid cDNA library. Among the identified genes, several encoded known proteins, including serum amyloid A(2a) (SAA(2a)), apoC-I, and phosphodiesterase HCAM1 (PDE1A), found to interact with apoA-I. In addition, we have cloned a novel 29 kDa apoA-I interacting protein, which we named AI-BP (apoA-I binding protein). The AI-BP encoding gene, APOA1BP, which is located on chromosome 1q21, is composed of six exons and five introns and spans 2.5 kb. Northern blot analysis demonstrated ubiquitous expression of the APOA1BP mRNA with the highest expression in kidney, heart, liver, thyroid gland, adrenal gland, and testis. AI-BP protein is not detectable in serum of healthy probands, but serum samples of patients with septic syndromes may contain elevated levels of AI-BP. Significant amounts of AI-BP protein are found in cerebrospinal fluid and urine of healthy probands. The stimulation of cells derived from the kidney proximal tubules with apoA-I or HDL induces a concentration-dependent secretion of AI-BP indicating an important role for AI-BP, in the renal tubular degradation or resorption of apoA-I.


Assuntos
Apolipoproteína A-I/farmacologia , Proteínas de Transporte/genética , Túbulos Renais Proximais/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Proteínas da Gravidez , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Sequência de Bases , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Expressão Gênica , Genes/genética , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Racemases e Epimerases , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa