ABSTRACT
Produtos de glicação avançada (AGE) alteram o metabolismo de lípides e, em especial, o efluxo de colesterol de macrófagos, por meio da redução dos receptores ABCA-1 e ABCG-1. Isto prejudica o transporte reverso de colesterol, sistema que favorece o fluxo de colesterol de macrófagos arteriais ao fígado, permitindo sua excreção na bile e eliminação fecal. Óxidos de colesterol modulam favoravelmente a homeostase lipídica em macrófagos e favorecem o transporte reverso de colesterol, embora o acúmulo de 7-cetocolesterol, 7-hidroxicolesterol e 7-hidroxicolesterol associe-se à aterogênese e morte celular. Neste estudo, avaliou-se o efeito do tratamento com glicolaldeído (GAD; oxoaldeído que induz rápida geração intracelular de AGE), em macrófagos sobrecarregados com LDL oxidada e incubados com HDL ou HDL e indutor de LXR (T0901317) sobre: 1) a distribuição seletiva de óxidos de colesterol e o conteúdo total de esteróis intracelulares e 2) o conteúdo de ABCA-1 e ABCG-1. Colesterol total e os diversos subtipos de óxidos de colesterol foram determinados por cromatografia a gás acoplada à espectrômetro de massa. O conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) foi avaliado por imunoblot. Em macrófagos controles (C), a adição de HDL ou HDL + T0901317 promoveu redução no conteúdo de esteróis totais (colesterol + óxidos de colesterol), colesterol e 7-cetocolesterol. No entanto, isto não foi observado em macrófagos GAD. Nas diversas condições experimentais, não houve diferença no conteúdo intracelular dos outros subtipos de óxidos de colesterol, em células C e GAD. Macrófagos GAD apresentaram menor conteúdo de ABCA-1 (45%), quando comparados aos macrófagos C, mesmo após adição de HDL ou HDL + T0901317. O conteúdo de ABCG-1 foi 36,6% menor em macrófagos GAD, na presença de HDL, em comparação às células C. Em conclusão, em macrófagos sobrecarregados com LDL oxidada, o tratamento com glicolaldeído diminui a exportação celular de colesterol e 7-cetocolesterol...
Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7- hydroxycholesterol and 7- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. In conclusion, in macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus...
Subject(s)
ATP-Binding Cassette Transporters , Cholesterol , Ketocholesterols , Macrophages , Glycation End Products, AdvancedABSTRACT
BACKGROUND: Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The extracellular-signal-regulated kinase (ERK) signaling pathway plays a crucial role in almost all cell functions, including proliferation, differentiation, survival, and death. However, the effect of ERK inhibition on oxysterol-induced apoptosis remains uncertain. METHODS: This study assessed the effect of ERK inhibition on the apoptotic effect of 7-ketocholesterol. RESULTS: Treatment with 7-ketocholesterol increased phosphorylated-ERK1/2 levels in differentiated PC12 cells, while the total amount of ERK was not altered. 7-Ketocholesterol decreased Bid and Bcl-2 levels, increased Bax and p53 levels, and promoted cytochrome c release, which elicits the activation of caspases (-8, -9, and -3), nuclear damage, and cell death. ERK and farnesyltransferase inhibitors inhibited the 7-ketocholesterol-induced phosphorylation of ERK1/2, activation of apoptosis-related proteins, and cell death in PC12 cells. CONCLUSIONS: The ERK and farnesyltransferase inhibitors, which did not exhibit toxicity, may inhibit the 7-ketocholesterol toxicity on differentiated PC12 cells by suppressing the activation of the caspase-8-dependent pathway as well as activation of the mitochondria-mediated cell-death pathway, leading to the activation of caspases. The inhibition of ERK may confer a beneficial protective effect against the neuronal cell injury induced by cholesterol oxidation products.
Subject(s)
Animals , Apoptosis , Caspases , Cell Death , Cholesterol , Cytochromes c , Farnesyltranstransferase , Ketocholesterols , Neurons , PC12 Cells , Phosphorylation , Phosphotransferases , ProteinsABSTRACT
7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorpórate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.
Subject(s)
Animals , Mice , Ketocholesterols/chemistry , Lipoproteins, LDL/chemistry , Nanoparticles , Chromatography, Gel , Emulsions , Ketocholesterols/pharmacokinetics , Lipoproteins, LDL/metabolism , Models, Biological , Nanoparticles/chemistryABSTRACT
BACKGROUND: It has been shown that defects in mitochondrial function are involved in the induction of neuronal cell injury. Prostanoids such as prostaglandin E2 (PGE2) are thought to play an important role in inflammation and neurologic disorders. However, the effect of PGE2 on cholesterol-oxidation-product-induced neuronal cell injury remains uncertain. METHODS: The effect of PGE2 on toxicity of 7-ketocholesterol (7-KCS) was assessed in PC12 cells that were differentiated following treatment with nerve growth factor. The mitochondria-mediated apoptotic process was evaluated by examining the inhibitory effect of PGE2 on 7-KCS-induced toxicity. RESULTS: 7-KCS induced BID cleavage, increased the production of proapoptotic Bax protein, decreased antiapoptotic Bcl-2, increased p53, and promoted cytochrome c release in the cytosolic fraction, which subsequently elicited the activation of caspase-3, DNA fragmentation, and cell death. Treatment with PGE2 inhibited this 7-KCS-induced apoptotic process and cell death. CONCLUSIONS: The results show that PGE2 inhibits 7-KCS-induced toxicity in differentiated PC12 cells by suppressing the mitochondria-mediated apoptotic process. PGE2 may protect against cholesterol-oxidation-product-induced neuronal cell injury.
Subject(s)
Animals , bcl-2-Associated X Protein , Caspase 3 , Cell Death , Cytochromes c , Cytosol , Dinoprostone , DNA Fragmentation , Inflammation , Ketocholesterols , Mitochondria , Nerve Growth Factor , Nervous System Diseases , Neurons , PC12 Cells , ProstaglandinsABSTRACT
BACKGROUND: Formation of cholesterol oxidation products is a suggested mechanism of neurodegenerative disorders. Neuronal cell death is mediated by an increased release of excitotoxic glutamate from the presynaptic nerve endings. Tyrosine-specific protein kinases modulate neurotransmitter release at the nerve terminals. Tyrphostin AG126 has anti-inflammatory and cytoprotective effects. However, it remains uncertain whether tyrphostin AG126 has a preventive effect on the alteration of nerve terminal function induced by cholesterol oxidation products. METHODS: The present study was performed to assess the effect of cholesterol oxidation products against nerve terminal function using synaptosomes isolated from rat cerebrum. We determined the preventive effect of tyrphostin AG126 against oxysterol toxicity by measuring the effects on the glutamate release, depolarization of the membrane potential, changes in Ca2+ levels, and Na+/K+-ATPase activity. RESULTS: Synaptosomes treated with 7-ketocholesterol or 25-hydroxycholesterol exhibited a sustained release of glutamate, depolarization of membrane potential, early rapid increase in cellular Ca2+ levels and decrease in Na+/K+-ATPase activity. Those responses were concentration-dependent. Treatment of tyrphostin AG126 interfered with alteration of synaptosomal functions and decrease in Na+/K+-ATPase activity induced by 7-ketocholesterol or 25-hydroxycholesterol. CONCLUSIONS: The results show that 7-ketocholesterol and 25-hydroxycholesterol seem to cause the release of glutamate by inducing depolarization of the membrane potential and early rapid increase in cellular Ca2+ levels and by inactivating Na+/K+-ATPase in the cerebral synaptosomes. Treatment of tyrphostin AG126 may prevent the oxysterol-induced nerve terminal dysfunction.
Subject(s)
Animals , Rats , Brain , Cell Death , Cerebrum , Cholesterol , Glutamic Acid , Hydroxycholesterols , Ketocholesterols , Membrane Potentials , Neurodegenerative Diseases , Neurons , Neurotransmitter Agents , Presynaptic Terminals , Protein-Tyrosine Kinases , Synaptosomes , TyrphostinsABSTRACT
7-cetocoleterol (7KC) é um oxisterol conhecido por inibir a proliferação celular e por ser citotóxico. Uma nanoemulsão contendo 7KC (LDE/7KC) demonstrou efeito anti-proliferativo sobre as linhagens RPMI 8226 (mieloma) e melanoma (B16F10), in vitro. Sendo preferencialmente captada via receptores de LDL. No presente trabalho, avaliamos, in vivo, a cinética plasmática, biodistribuição e ação anti-tumoral em camundongos portadores de melanoma. A nanoemulsão dirigiu-se principalmente no fígado e ao tumor, demonstrando direcionamento a tecidos com alta expressão de receptores para LDL. LDE/7KC promoveu uma redução superior a cinqüenta por cento do tamanho do tumor, que apresentou maior área de necrose e menor quantidade de vasos e aumentou a sobrevida dos camundongos, sem causar toxicidade.
7-ketocholesterol (7KC) is an oxysterol known to inhibit cell proliferation and to be cytotoxic. A nanoemulsion containing-7KC (LDE/7KC) was shown to had antiproliferative effects on RPMI 8226 myeloma cell line and melanoma (B16F10), in vitro. This particle is taken up mainly by LDL receptors. Here we have evaluated the plasma kinetic, biodistribution and the anti-tumoral action of LDE/7KC in melanoma bearing mice. The nanoemulsion accumulated in the liver and tumor, tissues with a high expression of LDL receptors. LDE/7KC promoted a tumor size reduction over fifty percent. A increased necrosis area and a decreased amount of blood vessels was found. A increased survival rate was observed, together with a lack of toxicicity.
Subject(s)
Emulsions/pharmacokinetics , Ketocholesterols , Skin Neoplasms/immunology , Lipoproteins, LDL , Melanoma, Experimental/immunology , Receptors, LDLABSTRACT
Ovos são importantes como alimento e com matéria-prima industrial. Ao mesmo tempo, têm elevada concentração de colesterol, principalmente quando desidratado. O colesterol, por sua vez, está sujeito à oxidação durante o processamento e/ou estocagem, formando, em conseqüência, derivados oxidados com atividades tóxicas, entre as quais a aterogenicidade. Foi avaliada, neste trabalho, a estabilidade do colesterol em ovo integral em pó comercial, através da ocorrência do 7-cetocolesterol livre, quantificado por cromatografia líqüida de alta eficiência, depois da extração dos lípides totais e separação em coluna de Florisil. O 7-cetocolesterol livre ocorreu em todos os três lotes das cinco marcas analisadas de ovo integral em pó, em teor médio de 84,01±5,34 µg/g de lípides...
Subject(s)
Ketocholesterols/biosynthesis , Cholesterol , Food Preservation/methods , Eggs , Chromatography, High Pressure Liquid/methods , Food SamplesABSTRACT
O ovo é utilizado como matéria-prima na produção de alimentos devido suas propriedades funcionais e nutricionais. Existe, entretanto, a possibilidade de ocorrer a oxidação do colesterol constituinte do ovo em proporções significativas, durante o processamento, estocagem, ou preparo do produto para o consumo. Amostras comerciais de macarrão contendo ovos foram analisadas em relação ao colesterol e à sua estabilidade oxidativa, avaliada pela ocorrência de 7-cetocolesterol livre. A qualificação foi feita por Cromatografia Líquida de Alta Eficiência - CLAE. O colesterol variou bastante, de 11, 0 a 155,88mg/100g. Na maioria das amostras, os valores encontrados estavam abaixo dos declarados pelos fabricantes, indicando menor adição de ovo no produto que a recomendada pela legislação, caracterizando fraude...
Subject(s)
Cholesterol , Eggs , Food Technology , Chromatography, Liquid/methods , KetocholesterolsABSTRACT
The objective of this study was to describe the fresh cooled pink-shrimp (Penaeus brasiliensis and Penaeus paulensis) lipid. The total lipid content was 1.13 +/- 0.09 g/100 g while fatty acid profile showed 32.9 saturated, 20.4 monounsaturated and 40.5 polyunsaturated. Eighteen fatty acids were detected, seven saturated (14:0, 15:0, 16:0, 17:0, 18:0, 20:0, 22:0), four monounsaturated (16:1w7, 17:1w9, 18:1w9, 20:1w9) and seven polyunsaturated (18:2w6, 18:3w3, 20:2w6, 20:4w6, 20:5w3, 22:5w3, 22:6w3). The free cholesterol content was 92 to 136 mg/100 g with average of 118 mg/100 g. In same time, was observed the occurrence of free 7-ketocholesterol, a product of cholesterol oxidation, in levels of 0.185 to 0.366 microgram/g. The fatty acid profile was obtained by gas chromatography with a fused silica column Supelcowax 10. The cholesterol and 7-ketocholesterol was determined by high performance liquid chromatography using a mu-Porasil column, normal phase, and a diode array detector.