RESUMEN
Although sphingomyelin (SM) is the most abundant phospholipid in the plasma, next to phosphatidylcholine (PC), its physiological function in plasma is unclear. Here we employed plasma from various genetic models of mice which naturally differ in their plasma SM/PC ratios, to study the role of SM as a modulator of LCAT, the enzyme responsible for HDL maturation and the synthesis of cholesteryl esters (CE) in normal plasma. Serine palmitoyltransferase deficient mice, and SM synthase deficient mice, both of which have below normal SM/PC ratios, showed significantly elevated LCAT activities when assayed with the endogenous substrates. On the other hand, LDL receptor knockout mice, and apo E knockout mice, both of which have high SM/PC ratios, had markedly reduced (-80%) LCAT activities. The LCAT levels in plasma, as assayed with an exogenous substrate, were similar in all groups, except for a 45% decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 CE all of which are formed by LCAT, and a higher percentage of the atherogenic 18:1 CE which is mainly derived from the action of liver ACAT, showing that in vivo, the contribution of LCAT to plasma CE is reduced while that of liver ACAT is increased. These results show that SM is a physiological modulator of LCAT activity as well as plasma CE composition, and this may contribute to the previously reported pro-atherogenic effect of high plasma SM levels.
Asunto(s)
Ésteres del Colesterol/metabolismo , Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Esfingomielinas/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Colesterol/sangre , Ésteres del Colesterol/sangre , Esterificación , Lípidos/sangre , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolinas/sangre , Fosfatidilcolinas/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina C-Palmitoiltransferasa/deficiencia , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/metabolismo , Esfingomielinas/sangre , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Although the conjugated linoleic acids (CLA) have several isomer-specific biological effects including anti-carcinogenic and anti-adipogenic effects, their mechanisms of action remain unclear. To determine their potential effects on membrane structure and function, we studied the incorporation profiles of four CLA isomers (trans-10 cis-12 (A), trans-9 trans-11 (B), cis-9 trans-11 (C), and cis-9 cis-11 (D)) in CHO and HepG2 cells. All four isomers were incorporated into cellular lipids as efficiently as linoleic acid (LA), with the majority of the incorporated CLA present in membrane rafts. Of the four isomers, only CLA-A increased the cholesterol content of the raft fraction. Over 50% of the incorporated CLAs were recovered in phosphatidylcholine of CHO cells, but in HepG2 the neutral lipids contained the majority of CLA. The desaturation index (18:1/18:0 and 16:1/16:0) was reduced by CLA-A, but increased by CLA-B, the effects being apparent mostly in raft lipids. The Δ9 desaturase activity was inhibited by CLAs A and C. Unlike LA, which was mostly found in the sn-2 position of phospholipids, most CLAs were also incorporated significantly into the sn-1 position in both cell types. These studies show that the incorporation profiles of CLA isomers differ significantly from that of LA, and this could lead to alterations in membrane function, especially in the raft-associated proteins.
Asunto(s)
Ácido Linoleico/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Microdominios de Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Células CHO , Cricetinae , Cricetulus , Células Hep G2 , Humanos , Ácido Linoleico/farmacología , Ácidos Linoleicos Conjugados/farmacología , Proteínas de la Membrana/metabolismoRESUMEN
Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.
Asunto(s)
Fenómenos Bioquímicos/efectos de los fármacos , Fenómenos Biofísicos/efectos de los fármacos , Ácidos Linoleicos Conjugados/química , Ácidos Linoleicos Conjugados/farmacología , Membranas Artificiales , Rastreo Diferencial de Calorimetría , Colesterol/metabolismo , Fluoresceínas/metabolismo , Polarización de Fluorescencia , Humanos , Isomerismo , Ácidos Linoleicos Conjugados/metabolismo , Liposomas/metabolismo , Fluidez de la Membrana/efectos de los fármacos , Modelos Biológicos , Oxidación-Reducción/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfatidilcolinas/metabolismo , Propiedades de Superficie/efectos de los fármacos , Temperatura de Transición/efectos de los fármacosRESUMEN
Diffusible subfibrillar aggregates of amyloid proteins are potent neurotoxins and primary suspects in amyloid diseases including Alzheimer's disease. Despite widespread interest, the molecular structures of the amyloid intermediates and the conformational conversions in amyloid misfolding are poorly understood. Here we present a molecular-level examination of sequence-specific secondary structures and supramolecular structures of a neurotoxic amyloid intermediate of the 40-residue ß-amyloid (Aß) peptide involved in Alzheimer's disease. Using solid-state NMR and electron microscopy, we show that, before fibrillization, natively unstructured monomeric Aß is subject to large conformational changes into a spherical amyloid intermediate of 1535 nm diameter, which has predominantly parallel ß-sheet structures. Structural comparison with Aß fibrils demonstrates that formation of this ß-sheet intermediate I(ß) largely defines conformational transitions in amyloid misfolding. Neurotoxicity assays on PC12 cells show that I(ß) shows higher toxicity than the fibril, indicating that the ß-sheet formation may trigger neurotoxicity.