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1.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370282

RESUMO

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia.


Assuntos
Autofagia/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Oxigênio/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Hipóxia Celular , Linhagem Celular , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Expressão Gênica/efeitos dos fármacos , Glucose/deficiência , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
2.
World J Gastroenterol ; 25(30): 4172-4180, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31435171

RESUMO

Lysosomal acid lipase (LAL) plays a key role in intracellular lipid metabolism. Reduced LAL activity promotes increased multi-organ lysosomal cholesterol ester storage, as observed in two recessive autosomal genetic diseases, Wolman disease and Cholesterol ester storage disease. Severe liver steatosis and accelerated liver fibrosis are common features in patients with genetic LAL deficiency. By contrast, few reliable data are available on the modulation of LAL activity in vivo and on the epigenetic and metabolic factors capable of regulating its activity in subjects without homozygous mutations of the Lipase A gene. In the last few years, a less severe and non-genetic reduction of LAL activity was reported in children and adults with non-alcoholic fatty liver disease (NAFLD), suggesting a possible role of LAL reduction in the pathogenesis and progression of the disease. Patients with NAFLD show a significant, progressive reduction of LAL activity from simple steatosis to non-alcoholic steatohepatitis and cryptogenic cirrhosis. Among cirrhosis of different etiologies, those with cryptogenic cirrhosis show the most significant reductions of LAL activity. These findings suggest that the modulation of LAL activity may become a possible new therapeutic target for patients with more advanced forms of NAFLD. Moreover, the measurement of LAL activity may represent a possible new marker of disease severity in this clinical setting.


Assuntos
Cirrose Hepática/patologia , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Esterol Esterase/deficiência , Doença de Wolman/patologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Ésteres do Colesterol/metabolismo , Progressão da Doença , Teste em Amostras de Sangue Seco , Terapia de Reposição de Enzimas/métodos , Humanos , Metabolismo dos Lipídeos/genética , Fígado/patologia , Cirrose Hepática/prevenção & controle , Testes de Função Hepática/métodos , Lisossomos/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Índice de Gravidade de Doença , Esterol Esterase/sangue , Esterol Esterase/genética , Esterol Esterase/uso terapêutico , Triglicerídeos/metabolismo , Doença de Wolman/tratamento farmacológico , Doença de Wolman/genética
3.
Invest Ophthalmol Vis Sci ; 60(6): 2286-2293, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31112994

RESUMO

Purpose: Relationships between tear film lipid (TFL) layer composition, structure, and function could provide insight into the etiology of dry eye. The molar ratio of cholesteryl ester (CE)/wax ester (WE) was measured in meibum from normal donors (Mn) and compared with meibum from donors with meibomian gland dysfunction (MMGD). Methods: CE/WE was measured using nuclear magnetic resonance spectroscopy. Results: CE/WE was distributed into two populations with 81% distributed near 0.55 and 19% near 0.3. CE/WE were higher in donors 13 to 19 years old compared with donors 1 to 12 years old and 20 to 88 years old. CE/WE for MMGD was 30% lower, 0.34 ± 0.04, compared with Mn, 0.49 ± 0.04. There were no sex differences in CE/WE. There were no significant racial differences between the CE/WE ratios for Asians and Caucasians. The CE/WE ratio was higher for blacks and lower for Hispanics compared to Caucasians. Due to the small number sampled, confirmation of the later racial results is needed. The packing of CE and WE in the TFL layer was proposed. Conclusions: Although MMGD contains much less CE than Mn, factors other than the CE content, such as the levels of saturation and/or proteins, may be responsible for the higher order of MMGD. In addition to saturation, CE could contribute to the increase in order of Mn between 0 and 20 years of age. Observed changes in the meibum content of CE alone is not likely to influence tear film stability.


Assuntos
Blefarite/metabolismo , Ésteres do Colesterol/metabolismo , Síndromes do Olho Seco/metabolismo , Glândulas Tarsais/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Lipídeos/análise , Masculino , Lágrimas/metabolismo , Adulto Jovem
4.
Int J Mol Sci ; 20(10)2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31121898

RESUMO

Previous studies demonstrated modifications of high-density lipoproteins (HDL) structure and apolipoprotein (apo) A-I catabolism induced by the atorvastatin and fenofibrate combination. However, it remains unknown whether such structural and metabolic changes of HDL were related to an improvement of the HDL-cholesteryl esters (HDL-CE) metabolism. Therefore, we determined the structure of HDL and performed kinetic studies of HDL-CE radiolabeled with tritium in rabbits treated with atorvastatin, fenofibrate, and a combination of both drugs. The atorvastatin and fenofibrate combination increased the HDL size and the cholesterol and phospholipid plasma concentrations of the largest HDL subclasses. Moreover, the relative amount of unsaturated fatty acids contained in HDL increased, in detriment of saturated fatty acids as determined by gas chromatography-mass spectrometry. The transfers of cholesteryl esters (CE) from HDL to very low-density lipoproteins/low-density lipoproteins (VLDL/LDL) and vice versa were enhanced with atorvastatin, alone or in combination. Moreover, the direct elimination of CE from plasma via VLDL/LDL decreased with fenofibrate, whereas the direct elimination of CE via HDL augmented with the combination treatment. Taken together, the rise of unsaturated fatty acid content and the size increase of HDL, suggest that atorvastatin and fenofibrate induce more fluid HDL particles, which in turn favor an enhanced CE exchange between HDL and VLDL/LDL. Our results contribute to a better understanding of the relationship between the structure and function of HDL during the use of anti-dyslipidemic drugs.


Assuntos
Atorvastatina/farmacologia , Ésteres do Colesterol/metabolismo , Fenofibrato/farmacologia , Hipolipemiantes/farmacologia , Lipoproteínas HDL/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Ésteres do Colesterol/análise , Cinética , Lipoproteínas HDL/química , Coelhos
5.
J Biol Chem ; 294(23): 9118-9133, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31023823

RESUMO

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl ester (CE) and retinyl ester (RE) and triglyceride (TG). Mice globally lacking LAL accumulate CE most prominently in the liver. The severity of the CE accumulation phenotype progresses with age and is accompanied by hepatomegaly and hepatic cholesterol crystal deposition. In contrast, hepatic TG accumulation is much less pronounced in these mice, and hepatic RE levels are even decreased. To dissect the functional role of LAL for neutral lipid ester mobilization in the liver, we generated mice specifically lacking LAL in hepatocytes (hep-LAL-ko). On a standard chow diet, hep-LAL-ko mice exhibited increased hepatic CE accumulation but unaltered TG and RE levels. Feeding the hep-LAL-ko mice a vitamin A excess/high-fat diet (VitA/HFD) further increased hepatic cholesterol levels, but hepatic TG and RE levels in these mice were lower than in control mice. Performing in vitro activity assays with lysosome-enriched fractions from livers of mice globally lacking LAL, we detected residual acid hydrolytic activities against TG and RE. Interestingly, this non-LAL acid TG hydrolytic activity was elevated in lysosome-enriched fractions from livers of hep-LAL-ko mice upon VitA/HFD feeding. In conclusion, the neutral lipid ester phenotype in livers from hep-LAL-ko mice indicates that LAL is limiting for CE turnover, but not for TG and RE turnovers. Furthermore, in vitro hydrolase activity assays revealed the existence of non-LAL acid hydrolytic activities for TG and RE. The corresponding acid lipase(s) catalyzing these reactions remains to be identified.


Assuntos
Ésteres do Colesterol/metabolismo , Diterpenos/metabolismo , Fígado/metabolismo , Esterol Esterase/genética , Triglicerídeos/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Colesterol/sangue , Colesterol/metabolismo , Dieta Hiperlipídica , Diterpenos/química , Hepatócitos/citologia , Hepatócitos/metabolismo , Lipídeos/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/análise , Esterol Esterase/deficiência , Esterol Esterase/metabolismo , Vitamina A/administração & dosagem
6.
Clin Chim Acta ; 494: 58-63, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30876856

RESUMO

BACKGROUND: Niemann-Pick disease type C (NPC) is an autosomal recessive inherited disorder with progressive neuronal degeneration. Because conventional diagnostic methods are complicated and invasive, biomarker tests have drawn attention. We aimed to evaluate three urinary conjugated cholesterol metabolites as diagnostic biomarkers for NPC. METHODS: Urine samples from 23 patients with NPC, 28 healthy controls, and 7 patients with inherited metabolic disorders were analyzed. 3ß-Sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine conjugates in urine were quantified by liquid chromatography-tandem mass spectrometry. The diagnostic performance of the three metabolites and their total concentration was evaluated. RESULT: Creatinine-corrected concentrations of three metabolites and their total concentration were all significantly higher in NPC patients (0.0098 < P < .0448). The area under the receiver operating curve for all metabolites exceeded 0.95, the clinical specificity was 92-100%, and the clinical sensitivity was ~95%. In the urine of patients with other inherited metabolic diseases, the concentrations of the metabolites were lower than those in the urine of patients with NPC. CONCLUSION: These conjugated cholesterol metabolites in urine can serve as useful diagnostic markers for noninvasive screening of NPC.


Assuntos
Ésteres do Colesterol/urina , Doença de Niemann-Pick Tipo C/diagnóstico , Doença de Niemann-Pick Tipo C/urina , Adolescente , Adulto , Biomarcadores/metabolismo , Biomarcadores/urina , Criança , Pré-Escolar , Ésteres do Colesterol/metabolismo , Cromatografia Líquida , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Doença de Niemann-Pick Tipo C/metabolismo , Espectrometria de Massas em Tandem , Adulto Jovem
7.
Int J Mol Sci ; 20(4)2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30769921

RESUMO

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 µM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.


Assuntos
Neoplasias do Colo/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Palmíticos/metabolismo , Fosfolipídeos/química , Células CACO-2 , Membrana Celular/química , Membrana Celular/metabolismo , Ésteres do Colesterol/biossíntese , Ésteres do Colesterol/química , Ésteres do Colesterol/metabolismo , Neoplasias do Colo/química , Neoplasias do Colo/patologia , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Graxos Ômega-3/biossíntese , Humanos , Ácidos Linoleicos/química , Ácidos Linoleicos/metabolismo , Ácidos Linoleicos/farmacologia , Linoleoil-CoA Desaturase/química , Microscopia de Fluorescência , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Ácidos Palmíticos/química , Ácidos Palmíticos/farmacologia , Fosfolipídeos/biossíntese
8.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(4): 443-451, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30633988

RESUMO

OBJECTIVE: Since cholesterol is the sole precursor for glucocorticoid synthesis, it is hypothesized that genetic defects in proteins that impact the cellular cholesterol pool may underlie glucocorticoid insufficiency in humans. In the current study, we specifically focused on the cholesterol efflux mediator ATP-binding cassette transporter G1 (ABCG1) as gene candidate. METHODS: The adrenal transcriptional response to fasting stress was measured in wild-type mice to identify putative novel gene candidates. Subsequently, the adrenal glucocorticoid function was compared between ABCG1 knockout mice and wild-type controls. RESULTS: Overnight food deprivation induced a change in relative mRNA expression levels of cholesterol metabolism-related proteins previously linked to steroidogenesis, i.e. scavenger receptor class B type I (+149%; P < 0.001), LDL receptor (-70%; P < 0.001) and apolipoprotein E (-41%; P < 0.01). Strikingly, ABCG1 transcript levels were also markedly decreased (-61%; P < 0.05). In contrast to our hypothesis that decreasing cholesterol efflux would increase the adrenal cholesterol pool and enhance glucocorticoid output, ABCG1 knockout mice as compared to wild-type mice exhibited a reduced ability to secrete corticosterone in response to an ACTH challenge (two-way ANOVA: P < 0.001 for genotype) or fasting stress. As a result, glucocorticoid target gene expression levels in liver and hypothalamus were reduced and blood lymphocyte concentrations and spleen weights increased in ABCG1 knockout mice under fasting stress conditions. This was paralleled by a 48% reduction in adrenal cholesteryl ester stores and stimulation of adrenal NPC intracellular cholesterol transporter 2 (+37%; P < 0.05) and apolipoprotein E (+59%; P < 0.01) mRNA expression. CONCLUSION: ABCG1 deficiency is associated with mild glucocorticoid insufficiency in mice.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Apolipoproteínas E/genética , Glucocorticoides/deficiência , Receptores de LDL/genética , Receptores Depuradores Classe B/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Privação de Alimentos , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Am J Physiol Endocrinol Metab ; 316(4): E578-E589, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30694691

RESUMO

The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.


Assuntos
Colesterol/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Acetatos/metabolismo , Animais , Linhagem Celular , Ésteres do Colesterol/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Cultura Primária de Células
10.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(5): 643-653, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30641224

RESUMO

AIMS: Human plasma lipoproteins are known to contain various glycan structures whose composition and functional importance are starting to be recognized. We assessed N-glycosylation of human plasma HDL and LDL and the role of their glycomes in cellular cholesterol metabolism. METHODS: N-glycomic profiles of native and neuraminidase-treated HDL and LDL were obtained using HILIC-UHPLC-FLD. Relative abundance of the individual chromatographic peaks was quantitatively expressed as a percentage of total integrated area and N-glycan structures present in each peak were elucidated by MALDI-TOF MS. The capacity of HDL to mediate cellular efflux of cholesterol and the capacity of LDL to induce cellular accumulation of cholesteryl esters were evaluated in THP-1 cells. RESULTS: HILIC-UHPLC-FLD analysis of HDL and LDL N-glycans released by PNGase F resulted in 22 and 18 distinct chromatographic peaks, respectively. The majority of N-glycans present in HDL (~70%) and LDL (~60%) were sialylated with one or two sialic acid residues. The most abundant N-glycan structure in both HDL and LDL was a complex type biantennary N-glycan with one sialic acid (A2G2S1). Relative abundances of several N-glycan structures were dramatically altered by the neuraminidase treatment, which selectively removed sialic acid residues. Native HDL displayed significantly greater efficacy in removing cellular cholesterol from THP-1 cells as compared to desialylated HDL (p < 0.05). Cellular accumulation of cholesteryl esters in THP-1 cells was significantly higher after incubations with desialylated LDL particles as compared to native LDL (p < 0.05). CONCLUSIONS: N-glycome of human plasma lipoproteins reveals a high level of diversity, which directly impacts functional properties of the lipoproteins.


Assuntos
Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Linhagem Celular , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Glicosilação , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Lipoproteínas LDL/sangue , Lipoproteínas LDL/química , Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/análise , Polissacarídeos/metabolismo
11.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(2): 137-146, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30448348

RESUMO

SND1 is a putative oncoprotein whose molecular function remains unclear. Its overexpression in hepatocellular carcinoma impairs cholesterol homeostasis due to the altered activation of the sterol regulatory element-binding protein (SREBP) 2, which results in the accumulation of cellular cholesteryl esters (CE). In this work, we explored whether high cholesterol synthesis and esterification originates changes in glycerolipid metabolism that might affect cell growth, given that acetyl-coenzyme A is required for cholesterogenesis and fatty acids (FA) are the substrates of acyl-coenzyme A:cholesterol acyltransferase (ACAT). SND1-overexpressing hepatoma cells show low triglyceride (TG) synthesis, but phospholipid biosynthesis or cell growth is not affected. Limited TG synthesis is not due to low acetyl-coenzyme A or NADPH availability. We demonstrate that the main factor limiting TG synthesis is the utilization of FAs for cholesterol esterification. These metabolic adaptations are linked to high Scd1 expression, needed for the de novo production of oleic acid, the main FA used by ACAT. We conclude that high cholesterogenesis due to SND1 overexpression might determine the channeling of FAs to CEs.


Assuntos
Carcinoma Hepatocelular/metabolismo , Ácidos Graxos/biossíntese , Triglicerídeos/metabolismo , Acil Coenzima A/metabolismo , Aciltransferases/metabolismo , Animais , Linhagem Celular Tumoral , Colesterol/metabolismo , Ésteres do Colesterol/biossíntese , Ésteres do Colesterol/metabolismo , Endonucleases , Esterificação/fisiologia , Hipercolesterolemia/metabolismo , Metabolismo dos Lipídeos , Lipogênese , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácido Oleico/metabolismo , Ratos , Esterol O-Aciltransferase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Triglicerídeos/biossíntese
12.
J Lipid Res ; 60(2): 436-445, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30563909

RESUMO

Atherosclerosis is associated with increased lipid peroxidation, leading to generation of multiple oxidation-specific epitopes (OSEs), contributing to the pathogenesis of atherosclerosis and its clinical manifestation. Oxidized cholesteryl esters (OxCEs) are a major class of OSEs found in human plasma and atherosclerotic tissue. To evaluate OxCEs as a candidate biomarker, we generated a novel mouse monoclonal Ab (mAb) specific to an OxCE modification of proteins. The mAb AG23 (IgG1) was raised in C57BL6 mice immunized with OxCE-modified keyhole limpet hemocyanin, and hybridomas were screened against OxCE-modified BSA. This method ensures mAb specificity to the OxCE modification, independent of a carrier protein. AG23 specifically stained human carotid artery atherosclerotic lesions. An ELISA method, with AG23 as a capture and either anti-apoAI or anti-apoB-100 as the detection Abs, was developed to assay apoAI and apoB-100 lipoproteins that have one or more OxCE epitopes. OxCE-apoA or OxCE-apoB did not correlate with the well-established oxidized phospholipid-apoB biomarker. In a cohort of subjects treated with atorvastatin, OxCE-apoA was significantly lower than in the placebo group, independent of the apoAI levels. These results suggest the potential diagnostic utility of a new biomarker assay to measure OxCE-modified lipoproteins in patients with CVD.


Assuntos
Anticorpos Monoclonais/imunologia , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Ésteres do Colesterol/sangue , Ésteres do Colesterol/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Ésteres do Colesterol/imunologia , Humanos , Camundongos , Oxirredução
13.
Redox Biol ; 21: 101069, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30576926

RESUMO

Dysregulation of cholesterol metabolism represents one of the major risk factors for atherosclerotic cardiovascular disease (CVD). Oxidized cholesterol esters (oxCE) in low-density lipoprotein (LDL) have been implicated in CVD but the underlying mechanisms remain poorly defined. We use a targeted lipidomic approach to demonstrate that levels of oxCEs in human plasma are associated with different types of CVD and significantly elevated in patients with myocardial infarction. We synthesized a major endogenous cholesterol ester hydroperoxide (CEOOH), cholesteryl-13(cis, trans)-hydroperoxy-octadecadienoate (ch-13(c,t)-HpODE) and show that this endogenous compound significantly increases plasma cholesterol level in mice while decrease cholesterol levels in mouse liver and peritoneal macrophages, which is primarily due to the inhibition of cholesterol uptake in macrophages and liver. Further studies indicate that inhibition of cholesterol uptake by ch-13(c,t)-HpODE in macrophages is dependent on LXRα-IDOL-LDLR pathway, whereas inhibition on cholesterol levels in hepatocytes is dependent on LXRα and LDLR. Consistently, these effects on cholesterol levels by ch-13(c,t)-HpODE are diminished in LDLR or LXRα knockout mice. Together, our study provides evidence that elevated plasma cholesterol levels by CEOOHs are primarily due to the inhibition of cholesterol uptake in the liver and macrophages, which may play an important role in the pathogenesis of CVD.


Assuntos
Ésteres do Colesterol/metabolismo , Colesterol/metabolismo , Hepatócitos/metabolismo , Macrófagos/metabolismo , Idoso , Animais , Biomarcadores , Doenças Cardiovasculares , Ésteres do Colesterol/genética , Cromatografia Líquida , Modelos Animais de Doenças , Feminino , Humanos , Metabolismo dos Lipídeos , Receptores X do Fígado/metabolismo , Masculino , Espectrometria de Massas , Metaboloma , Camundongos , Pessoa de Meia-Idade , Receptores de LDL/metabolismo
14.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(3): 260-270, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30557627

RESUMO

Intermediate-density lipoproteins (IDLs), the remnants of very-low-density lipoproteins via lipolysis, are rich in cholesteryl ester and are associated with cardiovascular disease. Despite pharmacological interest in IDLs, their three-dimensional (3D) structure is still undetermined due to their variation in size, composition, and dynamic structure. To explore the 3D structure of IDLs, we reconstructed 3D density maps from individual IDL particles using cryo-electron microscopy (cryo-EM) and individual-particle electron tomography (IPET, without averaging from different molecules). 3D reconstructions of IDLs revealed an unexpected polyhedral structure that deviates from the generally assumed spherical shape model (Frias et al., 2007; Olson, 1998; Shen et al., 1977). The polyhedral-shaped IDL contains a high-density shell formed by flat surfaces that are similar to those of very-low-density lipoproteins but have sharper dihedral angles between nearby surfaces. These flat surfaces would be less hydrophobic than the curved surface of mature spherical high-density lipoprotein (HDL), leading to a lower binding affinity of IDL to hydrophobic proteins (such as cholesteryl ester transfer protein) than HDL. This is the first visualization of the IDL 3D structure, which could provide fundamental clues for delineating the role of IDL in lipid metabolism and cardiovascular disease.


Assuntos
Lipoproteínas IDL/química , Lipoproteínas IDL/fisiologia , Imagem Individual de Molécula/métodos , Apolipoproteína A-I/metabolismo , Apolipoproteínas B/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Voluntários Saudáveis , Humanos , Imagem Tridimensional/métodos , Lipólise/fisiologia , Lipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas IDL/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Plasma/diagnóstico por imagem
15.
Free Radic Biol Med ; 129: 463-472, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30321700

RESUMO

A bulk of cholesteryl esters accumulation in macrophage foam cells drives the occurrence and development of atherosclerosis. Evidence now shows that autophagy plays key roles in the degradation of intracellular lipid droplets via autolysosome, and also in the release of intracellular lipids via cholesterol efflux. In this study, we identified that a mitochondria-targeted antioxidant, Mito-Tempol, has protective effects against cholesteryl esters accumulation by activating autophagy. Mito-Tempol was shown to ameliorate the lipid burden for atherosclerosis, both in vitro and in vivo. In the established in vitro foam cell formation system using oxidized low-density lipoprotein (ox-LDL)-loaded THP-1 macrophages, Mito-Tempol prevented intracellular oxidative stress and attenuated lipid accumulation. Mito-Tempol rescued ox-LDL-impaired autophagic flux, thereby facilitating autophagy-mediated lipid degradation in THP-1 macrophages. Meanwhile, Mito-Tempol also increased the efflux of cholesterol via autophagy-dependent ABCA1 and ABCG1 up-regulation. The classical autophagy pathway of mTOR may be one of the effector for the autophagy restoration of Mito-Tempol. Our findings give the first insight that cardiovascular system disease may benefits more from the treatment of Mito-Tempol for its impact of reversing atherosclerosis via autophagy.


Assuntos
Antioxidantes/farmacologia , Aterosclerose/tratamento farmacológico , Autofagia/genética , Óxidos N-Cíclicos/farmacologia , Hipertensão/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP/agonistas , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/agonistas , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ésteres do Colesterol/metabolismo , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Células Espumosas/patologia , Regulação da Expressão Gênica , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/patologia , Lipoproteínas LDL/farmacologia , Masculino , Mitocôndrias/metabolismo , Estresse Oxidativo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Transdução de Sinais , Marcadores de Spin , Células THP-1 , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
16.
J Lipid Res ; 59(11): 2223-2236, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30279222

RESUMO

The lipid composition of human meibomian gland secretions (meibum) has been analyzed using both targeted and untargeted mass spectrometric approaches, each of which has its advantages and disadvantages. Herein we report the results of shotgun lipidomic profiling of human meibum using a new approach that combines the advantages of targeted and untargeted analyses to yield highly sensitive and comprehensive profiles. Samples containing an estimated 7-13 µg (8-16 nL) of human meibum lipids were analyzed using MS/MSall, an untargeted approach for MS/MS. Using MS/MSall with ESI and successive polarity switching, we obtained tandem mass spectra in both modes at every 1 Da step for all ions in the m/z 200-1,200 range. In approximately 12 min, a total of 2 MS spectra and 2,000 MS/MS spectra were acquired for each sample, from which targeted analysis information was extracted. This approach allowed for the comprehensive and highly sensitive detection of meibum lipids, including species low in abundance. Altogether, more than 600 unique lipid molecular species were identified in meibum, 3 times more than previously reported in untargeted analyses of meibum samples. This untargeted MS and MS/MSall approach may be extended to other biological systems for the detection of lipids with sensitivity comparable to targeted analysis.


Assuntos
Glândulas Tarsais/metabolismo , Espectrometria de Massas em Tandem/métodos , Ésteres do Colesterol/metabolismo , Ácidos Graxos/metabolismo , Humanos , Fosfolipídeos/metabolismo , Triglicerídeos/metabolismo
17.
JCI Insight ; 3(17)2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30185655

RESUMO

Advanced age-related macular degeneration (AMD), the leading cause of blindness among people over 50 years of age, is characterized by atrophic neurodegeneration or pathologic angiogenesis. Early AMD is characterized by extracellular cholesterol-rich deposits underneath the retinal pigment epithelium (RPE) called drusen or in the subretinal space called subretinal drusenoid deposits (SDD) that drive disease progression. However, mechanisms of drusen and SDD biogenesis remain poorly understood. Although human AMD is characterized by abnormalities in cholesterol homeostasis and shares phenotypic features with atherosclerosis, it is unclear whether systemic immunity or local tissue metabolism regulates this homeostasis. Here, we demonstrate that targeted deletion of macrophage cholesterol ABC transporters A1 (ABCA1) and -G1 (ABCG1) leads to age-associated extracellular cholesterol-rich deposits underneath the neurosensory retina similar to SDD seen in early human AMD. These mice also develop impaired dark adaptation, a cardinal feature of RPE cell dysfunction seen in human AMD patients even before central vision is affected. Subretinal deposits in these mice progressively worsen with age, with concomitant accumulation of cholesterol metabolites including several oxysterols and cholesterol esters causing lipotoxicity that manifests as photoreceptor dysfunction and neurodegeneration. These findings suggest that impaired macrophage cholesterol transport initiates several key elements of early human AMD, demonstrating the importance of systemic immunity and aging in promoting disease manifestation. Polymorphisms in genes involved with cholesterol transport and homeostasis are associated with a significantly higher risk of developing AMD, thus making these studies translationally relevant by identifying potential targets for therapy.


Assuntos
Cegueira/induzido quimicamente , Cegueira/metabolismo , Colesterol/metabolismo , Degeneração Macular/induzido quimicamente , Degeneração Macular/metabolismo , Monócitos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Cegueira/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Ésteres do Colesterol/metabolismo , Progressão da Doença , Deleção de Genes , Humanos , Imunidade Inata , Degeneração Macular/imunologia , Degeneração Macular/patologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Oxisteróis/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Receptores Acoplados a Proteínas-G/metabolismo , Retina/anormalidades , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina/anormalidades , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia
18.
PLoS One ; 13(9): e0203464, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30192799

RESUMO

Lipids play very important roles in lung biology, mainly reducing the alveolar surface tension at the air-liquid interface thereby preventing end-expiratory collapse of the alveoli. In the present study we performed an extensive quantitative lipidomic analysis of mouse lung to provide the i) total lipid quantity, ii) distribution pattern of the major lipid classes, iii) composition of individual lipid species and iv) glycerophospholipid distribution pattern according to carbon chain length (total number of carbon atoms) and degree of unsaturation (total number of double bonds). We analysed and quantified 160 glycerophospholipid species, 24 sphingolipid species, 18 cholesteryl esters and cholesterol from lungs of a) newborn (P1), b) 15-day-old (P15) and c) 12-week-old adult mice (P84) to understand the changes occurring during postnatal pulmonary development. Our results revealed an increase in total lipid quantity, correlation of lipid class distribution in lung tissue and significant changes in the individual lipid species composition during postnatal lung development. Interestingly, we observed significant stage-specific alterations during this process. Especially, P1 lungs showed high content of monounsaturated lipid species; P15 lungs exhibited myristic and palmitic acid containing lipid species, whereas adult lungs were enriched with polyunsaturated lipid species. Taken together, our study provides an extensive quantitative lipidome of the postnatal mouse lung development, which may serve as a reference for a better understanding of lipid alterations and their functions in lung development and respiratory diseases associated with lipids.


Assuntos
Lipídeos/análise , Pulmão/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Fatores Etários , Animais , Animais Recém-Nascidos , Colesterol/análise , Colesterol/metabolismo , Ésteres do Colesterol/análise , Ésteres do Colesterol/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Feminino , Pulmão/crescimento & desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , Fosfolipídeos/análise , Fosfolipídeos/metabolismo , Esfingolipídeos/análise , Esfingolipídeos/metabolismo
19.
Sci Signal ; 11(541)2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065028

RESUMO

Although immune responses are essential to protect the body from infection, they can also harm tissues. Certain tissues and organs, including the eye, constitute specialized microenvironments that locally inhibit immune reactivity. Dedicator of cytokinesis protein 2 (DOCK2) is a Rac-specific guanine nucleotide exchange factor (GEF) that is predominantly found in hematopoietic cells. DOCK2 plays a key role in immune surveillance because it is essential for the activation and migration of leukocytes. DOCK2 mutations cause severe immunodeficiency in humans. We found that DOCK2-mediated Rac activation and leukocyte migration were effectively inhibited by cholesterol sulfate (CS), but not by cholesterol or other sulfated steroids. CS bound to the catalytic domain of DOCK2 and suppressed its GEF activity. Mass spectrometric quantification revealed that CS was most abundantly produced in the Harderian gland, which provides the lipids that form the oily layer of the tear film. Sulfation of cholesterol is mediated by the sulfotransferases SULT2B1b and, to a lesser extent, SULT2B1a, which are produced from the same gene through alternative splicing. By genetically inactivating Sult2b1, we showed that the lack of CS in mice augmented ultraviolet- and antigen-induced ocular surface inflammation, which was suppressed by administration of eye drops containing CS. Thus, CS is a naturally occurring DOCK2 inhibitor and contributes to the generation of the immunosuppressive microenvironment in the eye.


Assuntos
Ésteres do Colesterol/metabolismo , Olho/imunologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Evasão da Resposta Imune , Ceratite/prevenção & controle , Transtornos de Fotossensibilidade/prevenção & controle , Animais , Modelos Animais de Doenças , Olho/efeitos dos fármacos , Olho/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Ceratite/etiologia , Ceratite/imunologia , Ceratite/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos de Fotossensibilidade/etiologia , Transtornos de Fotossensibilidade/imunologia , Transtornos de Fotossensibilidade/metabolismo , Inibidores de Serino Proteinase/farmacologia , Sulfotransferases/fisiologia
20.
Sci Rep ; 8(1): 11500, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065281

RESUMO

The skin surface lipids (SSL) result from the blending of sebaceous and epidermal lipids, which derive from the sebaceous gland (SG) secretion and the permeability barrier of the stratum corneum (SC), respectively. In humans, the composition of the SSL is distinctive of the anatomical distribution of the SG. Thus, the abundance of sebum biomarkers is consistent with the density of the SG. Limited evidence on the influence that the SG exerts on the SC lipidome is available. We explored the differential amounts of sebaceous and epidermal lipids in areas at different SG density with lipidomics approaches. SC was sampled with adhesive patches from forearm, chest, and forehead of 10 healthy adults (8F, 2M) after mechanical removal of sebum with absorbing paper. Lipid extracts of SC were analysed by HPLC/(-)ESI-TOF-MS. In the untargeted approach, the naïve molecular features extraction algorithm was used to extract meaningful entities. Aligned and normalized data were evaluated by univariate and multivariate statistics. Quantitative analysis of free fatty acids (FFA) and cholesterol sulfate (CHS) was performed by targeted HPLC/(-)ESI-TOF-MS, whereas cholesterol and squalene were quantified by GC-MS. Untargeted approaches demonstrated that the relative abundance of numerous lipid species was distinctive of SC depending upon the different SG density. The discriminating species included FFA, CHS, and ceramides. Targeted analyses confirmed that sebaceous FFA and epidermal FFA were increased and decreased, respectively, in areas at high SG density. CHS and squalene, which are biomarkers of epidermal and sebaceous lipid matrices, respectively, were both significantly higher in areas at elevated SG density. Overall, results indicated that the SG secretion intervenes in shaping the lipid composition of the epidermal permeability barrier.


Assuntos
Lipídeos/fisiologia , Glândulas Sebáceas/metabolismo , Glândulas Sebáceas/fisiologia , Sebo/metabolismo , Sebo/fisiologia , Pele/metabolismo , Pele/fisiopatologia , Adulto , Ceramidas/metabolismo , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Feminino , Humanos , Masculino , Permeabilidade , Esqualeno/metabolismo
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