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1.
Nat Commun ; 11(1): 4279, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855410

RESUMO

Plasma and tumor caveolin-1 (Cav-1) are linked with disease progression in prostate cancer. Here we report that metabolomic profiling of longitudinal plasmas from a prospective cohort of 491 active surveillance (AS) participants indicates prominent elevations in plasma sphingolipids in AS progressors that, together with plasma Cav-1, yield a prognostic signature for disease progression. Mechanistic studies of the underlying tumor supportive onco-metabolism reveal coordinated activities through which Cav-1 enables rewiring of cancer cell lipid metabolism towards a program of 1) exogenous sphingolipid scavenging independent of cholesterol, 2) increased cancer cell catabolism of sphingomyelins to ceramide derivatives and 3) altered ceramide metabolism that results in increased glycosphingolipid synthesis and efflux of Cav-1-sphingolipid particles containing mitochondrial proteins and lipids. We also demonstrate, using a prostate cancer syngeneic RM-9 mouse model and established cell lines, that this Cav-1-sphingolipid program evidences a metabolic vulnerability that is targetable to induce lethal mitophagy as an anti-tumor therapy.


Assuntos
Caveolina 1/metabolismo , Neoplasias da Próstata/metabolismo , Esfingolipídeos/metabolismo , Idoso , Animais , Caveolina 1/sangue , Caveolina 1/genética , Linhagem Celular Tumoral , Ceramidas/metabolismo , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica , Glicoesfingolipídeos/biossíntese , Humanos , Lipídeos/sangue , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estudos Prospectivos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Pirrolidinas/farmacologia , Esfingomielinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Food Chem ; 332: 127384, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615384

RESUMO

Dairy polar lipids (PL) seem to exhibit antiplatelet effects. However, it is not known what molecular species may be responsible. In this study, we confirmed using C30 reversed-phase (C30RP) ultra-high-performance liquid chromatography (UHPLC) coupled to high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) that fermentation of yoghurts from ovine milk using specific starter cultures altered the PL composition. These lipid alterations occurred concomitant with increased antithrombotic properties of the yoghurts PL fractions against platelet-activating factor (PAF) and thrombin-induced platelet aggregation. Specifically, elevation in phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylcholine (PC) and their molecular species were observed following yoghurt fermentation. Furthermore, PC(18:0/18:1), PE(18:1/18:2), SM(d18:0/22:0) and several other molecular species were significantly inversely correlated with the inhibition of PAF and thrombin. These molecular species were abundant in the most bioactive yoghurts fermented by S. thermophilus and L. acidophilus, which suggest that fermentation by these microorganisms increases the antithrombotic properties of ovine milk PL.


Assuntos
Lipídeos/análise , Leite/metabolismo , Inibidores da Agregação de Plaquetas/análise , Iogurte/análise , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Fermentação , Lipídeos/farmacologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Ovinos , Esfingomielinas/metabolismo , Espectrometria de Massas em Tandem , Trombina/farmacologia
3.
Sci Rep ; 10(1): 6124, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273521

RESUMO

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat colorectal cancer. 5-FU is known to gradually lose its efficacy in treating colorectal cancer following the acquisition of resistance. We investigated the mechanism of 5-FU resistance using comprehensive lipidomic approaches. We performed lipidomic analysis on 5-FU-resistant (DLD-1/5-FU) and -sensitive (DLD-1) colorectal cancer cells using MALDI-MS and LC-MRM-MS. In particular, sphingomyelin (SM) species were significantly up-regulated in 5-FU-resistant cells in MALDI-TOF analysis. Further, we quantified sphingolipids including SM and Ceramide (Cer) using Multiple Reaction Monitoring (MRM), as they play a vital role in drug resistance. We found that 5-FU resistance in DLD-1/5-FU colorectal cancer cells was mainly associated with SM increase and Cer decrease, which are controlled by acid sphingomyelinase (SMPD1). In addition, reduction of SMPD1 expression was confirmed by LC-MRM-MS analysis and the effect of SMPD1 in drug resistance was assessed by treating DLD-1 cells with siRNA-SMPD1. Furthermore, clinical colorectal cancer data set analysis showed that down-regulation of SMPD1 was associated with resistance to chemotherapy regimens that include 5-FU. Thus, from our study, we propose that SM/Cer and SMPD1 are new potential target molecules for therapeutic strategies to overcome 5-FU resistance.


Assuntos
Ceramidas/metabolismo , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Regulação para Baixo , Fluoruracila/toxicidade , Humanos , Esfingomielina Fosfodiesterase/metabolismo
4.
Sci Rep ; 10(1): 5576, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221338

RESUMO

Lipids play a significant role in regulation of health and disease. To enhance our understanding of the role of lipids in regulation of lifespan and healthspan additional studies are required. Here, UHPLC-MS/MS lipidomics was used to measure dynamic changes in lipid composition as a function of age and gender in genetically identical male and female Daphnia magna with different average lifespans. We demonstrate statistically significant age-related changes in triglycerides (TG), diglycerides (DG), phosphatidylcholine, phosphatidylethanolamine, ceramide and sphingomyelin lipid groups, for example, in males, 17.04% of TG lipid species decline with age whilst 37.86% increase in relative intensity with age. In females, 23.16% decrease and 25.31% increase in relative intensity with age. Most interestingly, the rate and direction of change can differ between genetically identical female and male Daphnia magna, which could be the cause and/or the consequence of the different average lifespans between the two genetically identical genders. This study provides a benchmark dataset to understand how lipids alter as a function of age in genetically identical female and male species with different average lifespan and ageing rate.


Assuntos
Envelhecimento/metabolismo , Daphnia/metabolismo , Daphnia/fisiologia , Metabolismo dos Lipídeos/fisiologia , Longevidade/fisiologia , Animais , Diglicerídeos/metabolismo , Feminino , Masculino , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Triglicerídeos/metabolismo
5.
Sci Rep ; 10(1): 1837, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32020000

RESUMO

Liposomes or biological vesicles can be created from cholesterol, phospholipid, and water. Their stability is affected by their phospholipid composition which can influence disease treatment and drug delivery efficacy. In this study, the effect of phospholipid type on the formation and stability of liposomes using coarse-grained molecular dynamics simulations is investigated. For this purpose, the simulation study of the DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and DPSM (Egg sphingomyelin) lipids were considered. All simulations were carried out using the Gromacs software and Martini force field 2.2. Energy minimization (3000 steps) model, equilibrium at constant volume to adjust the temperature at 400 Kelvin and equilibrium at constant pressure to adjust the pressure, at atmospheric pressure (1 bar) have been validated. Microsecond simulations, as well as formation analysis including density, radial distribution function, and solvent accessible surface area, demonstrated spherical nanodisc structures for the DPSM and DSPC liposomes. The results revealed that due to the cylindrical geometric structure and small-size head group, the DSPC lipid maintained its perfectly spherical structure. However, the DPSM lipid showed a conical geometric structure with larger head group than other lipids, which allows the liposome to form a micelle structure. Although the DSPC and DPSM lipids used in the laboratory tests exhibit liposome and micelle behaviors, the simulation results revealed their nanodisc structures. Energy analysis including overall energy, Van der Waals interaction energy, and electrostatic interaction energy showed that DPSM liposome is more stable than DSPC liposome.


Assuntos
Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Pressão do Ar , Metabolismo Energético , Nanoestruturas , Eletricidade Estática , Temperatura
6.
Int J Mol Sci ; 21(3)2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32033121

RESUMO

Bioactive sphingolipids are emerging as key regulators of vascular function and homeostasis. While most of the clinical studies have been devoted to profile circulating sphingolipids in maternal plasma, little is known about the role of the sphingolipid at the feto-placental vasculature, which is in direct contact with the offspring circulation. Our study aims to compare the sphingolipid profile of normal with preeclamptic (PE) placental chorionic arteries and isolated endothelial cells, with the goal of unveiling potential underlying pathomechanisms in the vasculature. Dihydrosphingosine and sphingomyelin (SM) concentrations (C16:0-, C18:0-, and C24:0- sphingomyelin) were significantly increased in chorionic arteries of preeclamptic placentas, whereas total ceramide, although showing a downward trend, were not statistically different. Moreover, RNA and immunofluorescence analysis showed impaired sphingosine-1-phosphate (S1P) synthesis and signaling in PE vessels. Our data reveal that the exposure to a deranged maternal intrauterine environment during PE alters the sphingolipid signature and gene expression on the fetal side of the placental vasculature. This pathological remodeling consists in increased serine palmitoyltransferase (SPT) activity and SM accrual in PE chorionic arteries, with concomitance impairment endothelial S1P signaling in the endothelium of these vessels. The increase of endothelial S1P phosphatase, lyase and S1PR2, and blunted S1PR1 expression support the onset of the pathological phenotype in chorionic arteries.


Assuntos
Feto/metabolismo , Troca Materno-Fetal/fisiologia , Placenta/metabolismo , Circulação Placentária/fisiologia , Pré-Eclâmpsia/metabolismo , Esfingolipídeos/metabolismo , Artérias/metabolismo , Artérias/fisiopatologia , Ceramidas/metabolismo , Córion/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Feto/fisiopatologia , Expressão Gênica/fisiologia , Humanos , Lisofosfolipídeos/metabolismo , Placenta/fisiologia , Pré-Eclâmpsia/fisiopatologia , Gravidez , Transdução de Sinais/fisiologia , Esfingomielinas/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
7.
Biochem Biophys Res Commun ; 524(1): 135-141, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980165

RESUMO

Entamoeba invadens is the protozoan which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment/excystment in vitro. Here we report that EinCerS2 knockdown promoted decrease in sphingomyelin (SM) subspecies with long-chain fatty acids (24:0) down to 50% but increase sphingolipids with short-chain fatty acids (16:0) up to three times in both trophozoites and cysts of E. invadens. EinCerS2 silencing also resulted in decreased trophozoites' movement, proliferation, cysts formation, and trophozoites hatched after excystment. By immunofluorescence assays, a polyclonal antibody against EinCerS2 detected the enzyme in the cytoplasm of E. invadens trophozoites, colocalizing with Endoplasmic Reticulum-resident cognate EiSERCA. Interestingly, EinCerS2 was redistributed close to the plasma membrane during encystation, suggesting that the generation of diacylglycerol (DAG) via synthesis of sphingolipids and the activation protein kinase C might participate in the encystment process of E. invadens.


Assuntos
Movimento Celular , Entamoeba/citologia , Entamoeba/enzimologia , Técnicas de Silenciamento de Genes , Oxirredutases/metabolismo , Trofozoítos/enzimologia , Trofozoítos/crescimento & desenvolvimento , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo/genética , Entamoeba/genética , Amplificação de Genes , Estágios do Ciclo de Vida , Oxirredutases/genética , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esfingomielinas/metabolismo
8.
Cardiovasc Res ; 116(5): 1006-1020, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31399738

RESUMO

AIMS: Adaptive immunity contributes to the pathogenesis of cardiovascular metabolic disorders (CVMD). The omega-3 polyunsaturated fatty acids (n-3PUFA) are beneficial for cardiovascular health, with potential to improve the dysregulated adaptive immune responses associated with metabolic imbalance. We aimed to explore the mechanisms through which n-3PUFA may alter T cell motility and tissue distribution to promote a less inflammatory environment and improve lymphocyte function in CVMD. METHODS AND RESULTS: Using mass spectrometry lipidomics, cellular, biochemical, and in vivo and ex vivo analyses, we investigated how eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the main n-3PUFA, modify the trafficking patterns of activated CD4+ T cells. In mice subjected to allogeneic immunization, a 3-week n-3PUFA-enriched diet reduced the number of effector memory CD4+ T cells found in adipose tissue, and changed the profiles of eicosanoids, octadecanoids, docosanoids, endocannabinoids, 2-monoacylglycerols, N-acyl ethanolamines, and ceramides, in plasma, lymphoid organs, and fat tissues. These bioactive lipids exhibited differing chemotactic properties when tested in chemotaxis assays with activated CD4+ T cells in vitro. Furthermore, CD4+ T cells treated with EPA and DHA showed a significant reduction in chemokinesis, as assessed by trans-endothelial migration assays, and, when implanted in recipient mice, demonstrated less efficient migration to the inflamed peritoneum. Finally, EPA and DHA treatments reduced the number of polarized CD4+ T cells in vitro, altered the phospholipid composition of membrane microdomains and decreased the activity of small Rho GTPases, Rhoα, and Rac1 instrumental in cytoskeletal dynamics. CONCLUSIONS: Our findings suggest that EPA and DHA affect the motility of CD4+ T cells and modify their ability to reach target tissues by interfering with the cytoskeletal rearrangements required for cell migration. This can explain, at least in part, the anti-inflammatory effects of n-3PUFA supporting their potential use in interventions aiming to address adipocyte low-grade inflammation associated with cardiovascular metabolic disease.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Células Cultivadas , Microambiente Celular , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Feminino , Glicerofosfolipídeos/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Transdução de Sinais , Esfingomielinas/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
10.
Mol Cell Biochem ; 463(1-2): 57-66, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31541354

RESUMO

Membrane lipids regulate the structure and function of G protein-coupled receptors (GPCRs). Previously we have shown that membrane cholesterol regulates the signaling of two human bitter taste receptors (T2Rs), T2R4 and T2R14. Another major plasma membrane lipid known to influence the function of membrane proteins including GPCRs is sphingomyelin. The role of sphingomyelin in T2R function is unexplored thus far. In this work, we examined the significance of sphingomyelin in T2R14 signaling. Results suggest that unavailability of membrane sphingomyelin did not affect the agonist-promoted T2R14 Ca2+ signaling in heterologous expression system and also in primary airway smooth muscle cells (HASM cells). In addition, T2R14 mediated downstream AMPK activation was also unaffected in sphingomyelin-depleted condition; however, cholesterol depletion impaired the T2R14-mediated AMPK activation. Angiotensin II type1A receptor (AT1R) expressed in HASM cells and signals through Ca2+ and AMPK was used as a control. Results suggest that similar to T2R14, membrane sphingomyelin depletion did not affect AT1R signaling. However, membrane cholesterol depletion impaired AT1R mediated Ca2+ signaling and AMPK activation. Interestingly, amino acid sequence analysis revealed the presence of putative sphingolipid binding motif in both T2R14 and AT1R suggesting that the presence of a motif alone might not be suggestive of sphingomyelin sensitivity. In conclusion, these results demonstrate that in contrast to membrane cholesterol, sphingomyelin does not affect the agonist-induced T2R14 signaling, however it may play a role in other aspects of T2R14 function.


Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Colesterol/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Esfingomielinas/metabolismo , Linhagem Celular , Membrana Celular/genética , Colesterol/genética , Humanos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas-G/genética , Esfingomielinas/genética
11.
FEBS Lett ; 594(3): 519-529, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31596951

RESUMO

The obligate intracellular bacterium Chlamydia trachomatis proliferates in the membranous compartment inclusion formed in host cells. The host ceramide transport protein CERT delivers ceramide from the endoplasmic reticulum to the Golgi complex for the synthesis of sphingomyelin (SM). Chlamydia trachomatis has been suggested to employ CERT to produce SM in the inclusion by host SM synthases (SMSs). Here, we found that C. trachomatis proliferates and produces infective progeny even in SMS1 and SMS2 double-knockout HeLa cells, but not in the SMS1/SMS2/CERT triple-knockout cells. Interestingly, infected cells convert ceramide to SM without host SMSs. These results suggest that C. trachomatis-infected cells can convert ceramide to SM without host SMSs after CERT-mediated transfer of ceramide to the inclusions.


Assuntos
Ceramidas/metabolismo , Chlamydia trachomatis/fisiologia , Esfingomielinas/metabolismo , Sequência de Bases , Técnicas de Inativação de Genes , Células HeLa , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
12.
Int J Cancer ; 146(3): 720-730, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951192

RESUMO

Metabolomics may reveal novel insights into the etiology of prostate cancer, for which few risk factors are established. We investigated the association between patterns in baseline plasma metabolite profile and subsequent prostate cancer risk, using data from 3,057 matched case-control sets from the European Prospective Investigation into Cancer and Nutrition (EPIC). We measured 119 metabolite concentrations in plasma samples, collected on average 9.4 years before diagnosis, by mass spectrometry (AbsoluteIDQ p180 Kit, Biocrates Life Sciences AG). Metabolite patterns were identified using treelet transform, a statistical method for identification of groups of correlated metabolites. Associations of metabolite patterns with prostate cancer risk (OR1SD ) were estimated by conditional logistic regression. Supplementary analyses were conducted for metabolite patterns derived using principal component analysis and for individual metabolites. Men with metabolite profiles characterized by higher concentrations of either phosphatidylcholines or hydroxysphingomyelins (OR1SD = 0.77, 95% confidence interval 0.66-0.89), acylcarnitines C18:1 and C18:2, glutamate, ornithine and taurine (OR1SD = 0.72, 0.57-0.90), or lysophosphatidylcholines (OR1SD = 0.81, 0.69-0.95) had lower risk of advanced stage prostate cancer at diagnosis, with no evidence of heterogeneity by follow-up time. Similar associations were observed for the two former patterns with aggressive disease risk (the more aggressive subset of advanced stage), while the latter pattern was inversely related to risk of prostate cancer death (OR1SD = 0.77, 0.61-0.96). No associations were observed for prostate cancer overall or less aggressive tumor subtypes. In conclusion, metabolite patterns may be related to lower risk of more aggressive prostate tumors and prostate cancer death, and might be relevant to etiology of advanced stage prostate cancer.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Próstata/patologia , Idoso , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Seguimentos , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Avaliação Nutricional , Fosfatidilcolinas/sangue , Fosfatidilcolinas/metabolismo , Estudos Prospectivos , Próstata/patologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/metabolismo , Fatores de Risco , Esfingomielinas/sangue , Esfingomielinas/metabolismo
13.
Gut ; 69(3): 487-501, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31189655

RESUMO

OBJECTIVE: To investigate whether milk polar lipids (PL) impact human intestinal lipid absorption, metabolism, microbiota and associated markers of cardiometabolic health. DESIGN: A double-blind, randomised controlled 4-week study involving 58 postmenopausal women was used to assess the chronic effects of milk PL consumption (0, 3 or 5 g-PL/day) on lipid metabolism and gut microbiota. The acute effects of milk PL on intestinal absorption and metabolism of cholesterol were assessed in a randomised controlled crossover study using tracers in ileostomy patients. RESULTS: Over 4 weeks, milk PL significantly reduced fasting and postprandial plasma concentrations of cholesterol and surrogate lipid markers of cardiovascular disease risk, including total/high-density lipoprotein-cholesterol and apolipoprotein (Apo)B/ApoA1 ratios. The highest PL dose preferentially induced a decreased number of intestine-derived chylomicron particles. Also, milk PL increased faecal loss of coprostanol, a gut-derived metabolite of cholesterol, but major bacterial populations and faecal short-chain fatty acids were not affected by milk PL, regardless of the dose. Acute ingestion of milk PL by ileostomy patients shows that milk PL decreased cholesterol absorption and increased cholesterol-ileal efflux, which can be explained by the observed co-excretion with milk sphingomyelin in the gut. CONCLUSION: The present data demonstrate for the first time in humans that milk PL can improve the cardiometabolic health by decreasing several lipid cardiovascular markers, notably through a reduced intestinal cholesterol absorption involving specific interactions in the gut, without disturbing the major bacterial phyla of gut microbiota. TRIAL REGISTRATION NUMBER: NCT02099032 and NCT02146339; Results.


Assuntos
Doenças Cardiovasculares/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/farmacologia , Sobrepeso/metabolismo , Esfingomielinas/metabolismo , Animais , Apolipoproteína A-I/sangue , Apolipoproteína B-100/sangue , Colestanol/metabolismo , Colesterol/metabolismo , HDL-Colesterol/sangue , Estudos Cross-Over , Suplementos Nutricionais , Método Duplo-Cego , Emulsificantes/farmacologia , Fezes/química , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Ileostomia , Absorção Intestinal/efeitos dos fármacos , Lipídeos/administração & dosagem , Lipídeos/análise , Pessoa de Meia-Idade , Leite/química , Pós-Menopausa , Fatores de Risco
14.
Artigo em Inglês | MEDLINE | ID: mdl-31678513

RESUMO

Bacterial lipopolysaccharide (LPS) is recognized by CD14 protein and the Toll-like receptor (TLR)4/MD2 complex localized in the plasma membrane of immune cells. TLR4 triggers two signaling pathways engaging the MyD88 and TRIF adaptor proteins which lead to production of various pro-inflammatory cytokines. These processes are likely to be modulated by sphingomyelin, as the CD14 - TLR4 interaction takes place in plasma membrane rafts enriched in this lipid. To verify this assumption, we analyzed the influence of tricyclodecane-9-yl xanthogenate (D609), which was proven here to be an SMS inhibitor, and silencing of sphingomyelin synthase (SMS) 1 and/or SMS2 on LPS-induced signaling in macrophages. LPS up-regulated the expression and activity of SMS while exposure to D609 or silencing of SMS1 and SMS2 counteracted this action and led (except for SMS2 silencing) to a depletion of sphingomyelin in cells. Concomitantly, the MyD88- and TRIF-dependent signaling pathways of TLR4 were inhibited with the latter being especially sensitive to the reduction of the SMS1 and/or SMS2 activity. The D609 treatment and SMS1 and/or SMS2 depletion all reduced the level of CD14 protein in cells, which likely was an important determinant of the reduction of the LPS-induced pro-inflammatory responses.


Assuntos
Transdução de Sinais/imunologia , Esfingomielinas/metabolismo , Receptor 4 Toll-Like/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Norbornanos , Cultura Primária de Células , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiocarbamatos , Tionas/farmacologia , Receptor 4 Toll-Like/genética , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-31672573

RESUMO

Mimetic peptides are promising therapeutic agents for atherosclerosis prevention. A 10-residue class G* peptide from apolipoprotein J (apoJ), namely, D-[113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties. This prompted us to determine its effect on the aggregation process of low-density lipoprotein (LDL) particles, an early event in the development of atherosclerosis. LDL particles with and without [113-122]apoJ peptide were incubated at 37 °C with sphingomyelinase (SMase) or were left to aggregate spontaneously at room temperature. The aggregation process was analyzed by size-exclusion chromatography (SEC), native gradient gel electrophoresis (GGE), absorbance at 405 nm, dynamic light scattering (DLS), and transmission electronic microscopy (TEM). In addition, circular dichroism was used to determine changes in the secondary structure of apoB, and SDS-PAGE was performed to assess apoB degradation. At an equimolar ratio of [113-122]apoJ peptide to apoB-100, [113-122]apoJ inhibited both SMase-induced or spontaneous LDL aggregation. All methods showed that [113-122]apoJ retarded the progression of SMase-induced LDL aggregation at long incubation times. No effect of [113-122]apoJ on apoB secondary structure was observed. Binding experiments showed that [113-122]apoJ presents low affinity for native LDL but binds readily to LDL during the first stages of aggregation. Laurdan fluorescence experiments showed that mild aggregation of LDL resulted in looser lipid packaging, which was partially prevented by D-[113-122]apoJ. These results demonstrate that [113-122]apoJ peptide prevents SMase-induced LDL aggregation at an equimolar ratio and opens the possibility for the use of this peptide as a therapeutic tool.


Assuntos
Clusterina/farmacologia , Lipoproteínas LDL/metabolismo , Fragmentos de Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Esfingomielinas/metabolismo , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Clusterina/química , Clusterina/uso terapêutico , Voluntários Saudáveis , Humanos , Lipoproteínas LDL/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/sangue
16.
Biochim Biophys Acta Biomembr ; 1862(2): 183077, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31805269

RESUMO

Multiple sclerosis (MS) is correlated with increased deimination of myelin basic protein (MBP) in the central nervous system. Here, the interaction of MBP C1 (charge: +19) and MBP C8 (charge: +13) with the major lipids of the cytoplasmic side of the oligodendrocyte membrane is analysed using monolayer adsorption experiments and epifluorescence microscopy. Our findings show that the electrostatic attraction between the positively charged proteins and negatively charged lipids in the myelin-like monolayers competes with the incorporation of MBP into regions directly bordering cholesterol-rich domains. The latter is favoured to avoid additional lipid condensation and reduction in fluidity of the phospholipid layer. We find that MBP C1 does not incorporate at the cholesterol-rich domains if sphingomyelin (SM) is absent from the lipid composition. In contrast, MBP C8 is still incorporated near cholesterol-enriched regions without SM. Thus, the highly charged C1 variant needs a specific interaction with SM, whereas for C8 the incorporation at the cholesterol-rich regions is ensured due to its reduced net positive charge. This phenomenon may be relevant for the correlation of higher amounts of MBP C8 in brains of adult MS patients and healthy children, in which the amount of SM is reduced compared to healthy adults.


Assuntos
Proteína Básica da Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Esfingomielinas/metabolismo , Lipossomas Unilamelares/metabolismo , Adulto , Criança , Colesterol/metabolismo , Humanos , Íons , Modelos Biológicos , Proteína Básica da Mielina/química , Oligodendroglia , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Eletricidade Estática
17.
Int J Mol Sci ; 20(23)2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31766565

RESUMO

Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide-sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.


Assuntos
Ataxina-2/genética , Ceramidas/metabolismo , Esfingomielinas/metabolismo , Ataxias Espinocerebelares/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Ataxina-2/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismo , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
18.
J Physiol Pharmacol ; 70(4)2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31741460

RESUMO

There is an increasing attention to the role that sex/gender plays in health, behavior and outcomes. Even though we know that males and females are not the same, experiments have sometimes been carried out without considering sex in scientific research. It is essential for scientists and clinicians to consider sex differences as one of the underlying physiological determinants of health and disease to provide the building blocks for evidence-based, individualized medicine. Our work aimed to reveal sex-associated differences in lipid metabolite levels of adult female (n = 10) and male (n = 10) Wistar rats, aged 60 days. Partial least square determination analysis (PLS-DA) method and a variance importance in projection (VIP) score was used to identify the key sex-specific metabolites. Our results show that all groups of lipid metabolites: lysophosphatidylcholines (lysoPCs), phosphatidylcholines (PCs), and sphingomyelins (SMs) show a significant sex-dependent pattern. According to our results, more than a half of lysoPCs studied showed sex-specific features. PCs and lysoPCs tend to be significantly elevated in the blood plasma of females. The most distinct increase in more than 90% of SMs has been revealed in female blood plasma, compared with males. According to VIP score, the most important feature was the metabolite PC aa C38:4. Our study points out a sex dimorphism in lipid metabolism. The identification of main lipid features may play a key role in preclinical and clinical practice.


Assuntos
Metabolismo dos Lipídeos , Caracteres Sexuais , Animais , Feminino , Masculino , Fosfatidilcolinas/sangue , Fosfatidilcolinas/metabolismo , Ratos Wistar , Esfingomielinas/sangue , Esfingomielinas/metabolismo
19.
J Chem Inf Model ; 59(12): 5207-5217, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31738555

RESUMO

Using replica exchange with solute tempering all-atom molecular dynamics, we studied the equilibrium binding of Aß25-35 peptide to the ternary bilayer composed of an equimolar mixture of dimyristoylphosphatidylcholine (DMPC), N-palmitoylsphingomyelin (PSM), and cholesterol. Binding of the same peptide to the pure DMPC bilayer served as a control. Due to significant C-terminal hydrophobic moment, binding to the ternary and DMPC bilayers promotes helical structure in the peptide. For both bilayers a polarized binding profile is observed, in which the N-terminus anchors to the bilayer surface, whereas the C-terminus alternates between unbound and inserted states. Both ternary and DMPC bilayers feature two Aß25-35 bound states, surface bound, S, and inserted, I, separated by modest free energy barriers. Experimental data are in agreement with our results but indicate that cholesterol impact is Aß fragment dependent. For Aß25-35, we predict that its binding mechanism is independent of the inclusion of PSM and cholesterol into the bilayer.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Colesterol/metabolismo , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Esfingomielinas/metabolismo , Peptídeos beta-Amiloides/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica
20.
Elife ; 82019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31724953

RESUMO

Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Células COS , Proteínas de Transporte/química , Membrana Celular/efeitos dos fármacos , Chlorocebus aethiops , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Proteínas Mutantes/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Sirolimo/farmacologia , Esfingomielinas/metabolismo
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