RESUMO
Fatty acid binding proteins (FABPs) are responsible for the long-chain fatty acids (FAs) transport inside the cell. However, despite the years, since their structure is known and the many studies published, there is no definitive answer about the stages of the lipid entry-exit mechanism. Their structure forms a ß -barrel of 10 anti-parallel strands with a cap in a helix-turn-helix motif, and there is some consensus on the role of the so-called portal region, involving the second α -helix from the cap ( α 2), ß C- ß D, and ß E- ß F turns in FAs exchange. To test the idea of a lid that opens, we performed a soaking experiment on an h-FABP crystal in which the cap is part of the packing contacts, and its movement is strongly restricted. Even in these conditions, we observed the replacement of palmitic acid by 2-Bromohexadecanoic acid (Br-palmitic acid). Our MD simulations reveal a two-step lipid entry process: (i) The travel of the lipid head through the cavity in the order of tens of nanoseconds, and (ii) The accommodation of its hydrophobic tail in hundreds to thousands of nanoseconds. We observed this even in the cases in which the FAs enter the cavity by their tail. During this process, the FAs do not follow a single trajectory, but multiple ones through which they get into the protein cavity. Thanks to the complementary views between experiment and simulation, we can give an approach to a mechanistic view of the exchange process.
Assuntos
Proteínas de Ligação a Ácido Graxo , Simulação de Dinâmica Molecular , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Raios X , Conformação Proteica , Ácidos Palmíticos/metabolismo , Lipídeos , Ácidos GraxosRESUMO
BACKGROUND: Macamides with a benzylalkylamide nucleus are characteristic and major bioactive compounds in the functional food maca (Lepidium meyenii Walp). The aim of this study was to explore variations in macamide content among maca from China and Peru. Twenty-seven batches of maca hypocotyls with different phenotypes, sampled from different geographical origins, were extracted and profiled by liquid chromatography with ultraviolet detection/tandem mass spectrometry (LC-UV/MS/MS). RESULTS: Twelve macamides were identified by MS operated in multiple scanning modes. Similarity analysis showed that maca samples differed significantly in their macamide fingerprinting. Partial least squares discriminant analysis (PLS-DA) was used to differentiate samples according to their geographical origin and to identify the most relevant variables in the classification model. The prediction accuracy for raw maca was 91% and five macamides were selected and considered as chemical markers for sample classification. CONCLUSION: When combined with a PLS-DA model, characteristic fingerprinting based on macamides could be recommended for labelling for the authentication of maca from different geographical origins. The results provided potential evidence for the relationships between environmental or other factors and distribution of macamides. © 2016 Society of Chemical Industry.
Assuntos
Produtos Agrícolas/química , Suplementos Nutricionais/análise , Qualidade dos Alimentos , Alimento Funcional/análise , Hipocótilo/química , Lepidium/química , Alcamidas Poli-Insaturadas/análise , Biomarcadores/análise , China , Cromatografia Líquida de Alta Pressão , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Análise Discriminante , Inspeção de Alimentos/métodos , Ácidos Heptanoicos/análise , Ácidos Heptanoicos/metabolismo , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Análise dos Mínimos Quadrados , Lepidium/crescimento & desenvolvimento , Lepidium/metabolismo , Ácidos Palmíticos/análise , Ácidos Palmíticos/metabolismo , Peru , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Alcamidas Poli-Insaturadas/metabolismo , Solventes/química , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , Ácidos Esteáricos/análise , Ácidos Esteáricos/metabolismo , Espectrometria de Massas em TandemRESUMO
Mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies are fatty acid oxidation disorders biochemically characterized by tissue accumulation of long-chain fatty acids and derivatives, including the monocarboxylic long-chain 3-hydroxy fatty acids (LCHFAs) 3-hydroxytetradecanoic acid (3HTA) and 3-hydroxypalmitic acid (3HPA). Patients commonly present severe cardiomyopathy for which the pathogenesis is still poorly established. We investigated the effects of 3HTA and 3HPA, the major metabolites accumulating in these disorders, on important parameters of mitochondrial homeostasis in Ca(2+) -loaded heart mitochondria. 3HTA and 3HPA significantly decreased mitochondrial membrane potential, the matrix NAD(P)H pool and Ca(2+) retention capacity, and also induced mitochondrial swelling. These fatty acids also provoked a marked decrease of ATP production reflecting severe energy dysfunction. Furthermore, 3HTA-induced mitochondrial alterations were completely prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca(2+) uptake blocker, indicating that LCHFAs induced Ca(2+)-dependent mPT pore opening. Milder effects only achieved at higher doses of LCHFAs were observed in brain mitochondria, implying a higher vulnerability of heart to these fatty acids. By contrast, 3HTA and docosanoic acids did not change mitochondrial homeostasis, indicating selective effects for monocarboxylic LCHFAs. The present data indicate that the major LCHFAs accumulating in mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies induce mPT pore opening, compromising Ca(2+) homeostasis and oxidative phosphorylation more intensely in the heart. It is proposed that these pathomechanisms may contribute at least in part to the severe cardiac alterations characteristic of patients affected by these diseases.
Assuntos
Sinalização do Cálcio , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ácidos Mirísticos/metabolismo , Fosforilação Oxidativa , Ácidos Palmíticos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cardiomiopatias/enzimologia , Cardiomiopatias/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/metabolismo , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/deficiência , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Miopatias Mitocondriais/enzimologia , Miopatias Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , Proteína Mitocondrial Trifuncional/deficiência , Proteína Mitocondrial Trifuncional/metabolismo , NADP/metabolismo , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/metabolismo , Especificidade de Órgãos , Fosforilação Oxidativa/efeitos dos fármacos , Ratos Wistar , Rabdomiólise/enzimologia , Rabdomiólise/metabolismoRESUMO
Pharmacological synergism has been used to obtain a higher efficacy using drug concentrations at which side effects are minimal. In this study, the pharmacological antinociceptive interaction between N-palmitoylethanolamide (PEA) and tramadol was investigated. The individual concentration-response curves for PEA (0.1-56.2 µg/paw) and tramadol (1-56.2 µg/paw) were evaluated in mice in which nociception was induced by an intraplantar injection of 2% formalin. Isobolographic analysis was used to evaluate the pharmacological interaction between PEA (EC50=23.7±1.6 µg/paw) and tramadol (EC50=26.02±2.96 µg/paw) using the EC50 and a fixed 1:1 ratio combination. The isobologram demonstrated that the combinations investigated in this study produced a synergistic interaction; the experimental values (Zexp=9.5±0.2 µg/paw) were significantly smaller than those calculated theoretically (Zadd=24.8±0.2 µg/paw). The antinociceptive mechanisms of the PEA and tramadol combination involved the opioid receptor, transient receptor potential cation channel subfamily V member 1 (TRPV1), and peroxisome proliferator-activated receptor alpha (PPAR-α). The sedative effect of the combination of PEA and tramadol was less than that generated by individual treatments. These findings suggest that the PEA and tramadol combination produced enhanced antinociceptive efficacy at concentrations at which side effects are minimal.
Assuntos
Analgésicos/administração & dosagem , Etanolaminas/administração & dosagem , Medição da Dor/efeitos dos fármacos , Ácidos Palmíticos/administração & dosagem , Tramadol/administração & dosagem , Amidas , Analgésicos/metabolismo , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Sinergismo Farmacológico , Etanolaminas/metabolismo , Feminino , Conduta do Tratamento Medicamentoso , Camundongos , Medição da Dor/métodos , Ácidos Palmíticos/metabolismo , Tramadol/metabolismoRESUMO
The main monomer of tomato cuticle, 10,16-dihydroxyhexadecanoic acid (10,16-DHPA) and its methyl ester derivative (methyl-10,16-dihydroxyhexadecanote; methyl-10,16-DHHD), were used to study their oligomerization reactions catalyzed by five lipases: Candida antarctica lipase B (CAL-B), Rhizomucor miehei lipase (RM), Thermomyces lanuginosus lipase (TL), Pseudomonas cepacia lipase (PCL) and porcine pancreatic lipase (PPL). For 10,16-DHPA, optimum yields were obtained at 60 °C using toluene and 2-methyl-2-butanol (2M2B) as solvent, while for methyl-10,16-DHHD the bests yields were obtained in toluene and acetonitrile. Both reactions leaded to linear polyesters according to the NMR and FT-IR analysis, and there was no data indicating the presence of branched polymers. Using optimized conditions, poly(10,16-DHPA) and poly(methyl-10,16-DHHD) with Mw = 814 and Mn = 1,206 Da, and Mw = 982 and Mn = 860 Da, respectively, were formed according to their MALDI-TOF MS and ESI-MS data. The self-assembly of the polyesters obtained were analyzed by AFM.
Assuntos
Catálise , Lipase/química , Ácidos Palmíticos/química , Solanum lycopersicum/química , Espectroscopia de Ressonância Magnética , Ácidos Palmíticos/metabolismo , Polímeros/química , Solventes/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Accumulation of long-chain 3-hydroxy fatty acids is the biochemical hallmark of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. These disorders are clinically characterized by neurological symptoms, such as convulsions and lethargy, as well as by cardiomyopathy and muscle weakness. In the present work we investigated the in vitro effect of 3-hydroxydodecanoic (3HDA), 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, which accumulate in these disorders, on important oxidative stress parameters in cerebral cortex of young rats in the hope to clarify the mechanisms leading to the brain damage found in patients affected by these disorders. It was first verified that these compounds significantly induced lipid peroxidation, as determined by increased thiobarbituric acid-reactive substances levels. In addition, carbonyl formation was significantly increased and sulfhydryl content decreased by 3HTA and 3HPA, which indicates that these fatty acids elicit protein oxidative damage. 3HTA and 3HPA also diminished the reduced glutathione (GSH) levels, without affecting nitrate and nitrite production. Finally, we observed that the addition of the antioxidants and free radical scavengers trolox and deferoxamine (DFO) was able to partially prevent lipid oxidative damage, whereas DFO fully prevented the reduction on GSH levels induced by 3HTA. Our present data showing that 3HDA, 3HTA and 3HPA elicit oxidative stress in rat brain indicate that oxidative damage may represent an important pathomechanism involved in the neurologic symptoms manifested by patients affected by LCHAD and MTP deficiencies.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Encefalopatias Metabólicas/metabolismo , Encéfalo/metabolismo , Transtornos do Metabolismo dos Lipídeos/metabolismo , Complexos Multienzimáticos/deficiência , Ácidos Mirísticos/toxicidade , Estresse Oxidativo/fisiologia , Ácidos Palmíticos/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encefalopatias Metabólicas/induzido quimicamente , Ácidos Decanoicos/metabolismo , Ácidos Decanoicos/toxicidade , Ácidos Graxos/metabolismo , Ácidos Graxos/toxicidade , Transtornos do Metabolismo dos Lipídeos/induzido quimicamente , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa , Masculino , Proteína Mitocondrial Trifuncional , Ácidos Mirísticos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácidos Palmíticos/metabolismo , Ratos , Ratos WistarRESUMO
OBJECTIVE: Because meconium directly inhibits surfactant function, we sought to determine the effect of meconium on endogenous surfactant synthesis and clearance. STUDY DESIGN: We studied surfactant phosphatidylcholine kinetics with the use of stable isotopes in 11 newborn infants with meconium aspiration syndrome (MAS) who required extracorporeal membrane oxygenation (ECMO). For comparison we studied 6 neonates with persistent pulmonary hypertension (PPHN) on ECMO and 10 term neonates ventilated for non-pulmonary indications and not on ECMO. All patients received a 24-hour [U- 13C]glucose infusion as precursor for the palmitic acid in surfactant phosphatidylcholine. RESULTS: In the meconium group, the maximal 13C-incorporation in phosphatidylcholine (PC) was half of that in controls (0.09 +/- 0.01 vs 0.18 +/- 0.03 atom percent excess [APE], P = .027). There was a trend toward lower surfactant synthesis in the MAS group (3.3 +/- 0.7%/day) and PPHN group (2.6 +/- 0.3%/day) compared with controls 8.0 +/- 2.4%/day, P = .058). Significantly lower PC concentrations in tracheal aspirates were found in the MAS group (4.4 +/- 2.6 mg/mL) and PPHN group (3.6 +/- 2.0 mg/mL) compared with controls (12.8 +/- 2.6 mg/mL, P = .01). Endogenously synthesized surfactant had a similar half-life in all groups, ranging from 63 to 98 hours. CONCLUSION: We conclude that surfactant synthesis is disturbed and that surfactant PC concentrations are low in infants with MAS on ECMO.
Assuntos
Oxigenação por Membrana Extracorpórea , Síndrome de Aspiração de Mecônio/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/uso terapêutico , Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/uso terapêutico , Análise de Variância , Estudos de Casos e Controles , Terapia Combinada , Feminino , Glucose/administração & dosagem , Glucose/análogos & derivados , Meia-Vida , Humanos , Recém-Nascido , Infusões Intravenosas , Masculino , Síndrome de Aspiração de Mecônio/fisiopatologia , Síndrome de Aspiração de Mecônio/terapia , Ácidos Palmíticos/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Respiração Artificial , Fatores de Tempo , Traqueia/metabolismo , Resultado do Tratamento , Ureia/administração & dosagem , Ureia/análogos & derivados , Ureia/sangueRESUMO
CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.
Assuntos
Proteínas de Plantas , Proteínas Quinases/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Membrana Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Ácidos Palmíticos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/enzimologia , Solanum tuberosum/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
Calcium-dependent protein kinases (CDPKs), the most abundant serine/threonine kinases in plants, are found in various subcellular localizations, which suggests that this family of kinases may be involved in multiple signal transduction pathways. A complete analysis to try to understand the molecular basis of the presence of CDPKs in various localizations in the cell has not been accomplished yet. It has been suggested that myristoylation may be responsible for membrane association of CDPKs. In this study, we used a rice CDPK, OSCPK2, which has a consensus sequence for myristoylation at the N-terminus, to address this question. We expressed wild-type OSCPK2 and various mutants in different heterologous systems to investigate the factors that affect its membrane association. The results show that OSCPK2 is myristoylated and palmitoylated and targeted to the membrane fraction. Both modifications are required, myristoylation being essential for membrane localization and palmitoylation for its full association. The fact that palmitoylation is a reversible modification may provide a mechanism for regulation of the subcellular localization. OSCPK2 is the first CDPK shown to be targeted to membranes by an src homology domain 4 (SH4) located at the N-terminus of the molecule.
Assuntos
Membranas/enzimologia , Ácido Mirístico/metabolismo , Oryza/enzimologia , Ácidos Palmíticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Biológico , Cálcio/farmacologia , DNA Recombinante/genética , DNA Recombinante/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Protoplastos/metabolismo , Zea mays/genéticaRESUMO
The alteration in the fluorescence spectra observed for the polyene antibiotics: nystatin and amphotericin B in the presence of human serum albumin is due to a decrease in the polar character of the antibiotic environment when these are bound to the protein. Amphotericin B showed two types of binding sites, the first having very high affinity (5.8 10(7) M(-1]) and a secondary binding site with an affinity one order lower than the primary sites. This secondary binding site was very sensitive to temperature change. Nystatin yielded only one type of binding sites with an affinity of 1.1 10(6) M(-1). An electrostatic component was found in the binding of both ligands, as well as an important disorder at the protein binding sites. However the secondary binding site for AMP showed negative entropic change value, which suggests different mechanism of binding respect to the primary one. Conformational change induced by the temperature in the albumin molecule was detected by nystatin binding. Fatty acids produced an interference in the binding of both antibiotics to albumin.
Assuntos
Anfotericina B/química , Anfotericina B/metabolismo , Nistatina/química , Nistatina/metabolismo , Albumina Sérica/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Sítios de Ligação , Humanos , Ácido Linoleico/química , Ácido Linoleico/metabolismo , Ácido Linoleico/farmacologia , Ácidos Palmíticos/química , Ácidos Palmíticos/metabolismo , Ácidos Palmíticos/farmacologia , Albumina Sérica/química , Albumina Sérica/efeitos dos fármacos , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
In vivo labeling experiments with [3H]palmitic acid, [3H]inositol, and [3H]glucose allowed the identification of two main classes of inositolphospholipids (IPLs) from the trypomastigote stage of Trypanosoma cruzi. Purification of these compounds was achieved by ion-exchange chromatography, high performance liquid chromatography and thin layer chromatography. Specific phosphatidyl-inositol phospholipase C digestion, dephosphorylation and acid methanolysis showed a ceramide structure for the lower migrating IPL1. Palmitoyldihydrosphingosine and palmitoylsphingosine were detected by reverse-phase thin-layer chromatography. On the other hand, IPL2 showed to be a mixture of diacylglycero- and alkylacylglycero-phospholipids in a 1:1 ratio. After PI-PLC digestion, the lipids were separated by preparative TLC and individually analysed. The diacylglycerol contained mainly C18:0 fatty acid together with a low amount of C16:0. Hexadecylglycerol esterified with the C18:0 fatty acid was the only alkylacylglycerol detected. The C18:2 and C18:1 fatty acids, preponderant in the PI molecules of epimastigote forms, were not detected in trypomastigote forms. This is the first report on inositol phospholipids, putative precursors of lipid anchors in the infective stage of T. cruzi.
Assuntos
Glicosilfosfatidilinositóis/análise , Fosfatidilinositóis/análise , Trypanosoma cruzi/química , Animais , Ceramidas/análise , Ceramidas/química , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Diglicerídeos/análise , Ácidos Graxos/análise , Ácidos Graxos/química , Glicosilfosfatidilinositóis/química , Fosfatos de Inositol/análise , Estrutura Molecular , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/isolamento & purificação , Esfingosina/análiseRESUMO
We report here the isolation of a fatty acid-binding protein (FABP) from the liver of the catfish Rhamdia sapo. The purification procedure involves gel filtration, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The purified protein is basic (pI > 8.7) and migrates on sodium dodecyl sulfate-gel electrophoresis as a single entity of about 15 kDa. Its amino acid composition resembles those of FABPs isolated from other animals. Unlike mammalian liver FABPs, catfish liver FABP contains at least one tryptophan residue per molecule. No significant cross-reactivity was observed between the purified protein and polyclonal antibodies against either rat liver FABP or rat heart FABP. Amino acid sequencing of peptides obtained by digestion with Lys-C revealed that the catfish protein is structurally more similar to chicken liver FABP (69% identity in a 67-residue overlap) than to human liver FABPs (36%), nurse shark (Ginglymostoma cirratum) liver FABP (30%) and human heart FABP (31%). Taken together, these results suggest that catfish liver FABP is far more closely related to chicken liver FABP than to the FABPs isolated from the liver of mammals or elasmobranchs.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Peixes-Gato/metabolismo , Fígado/metabolismo , Proteína P2 de Mielina/química , Proteína P2 de Mielina/isolamento & purificação , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Ácidos Palmíticos/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Galinhas , Citosol/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Dados de Sequência Molecular , Proteína P2 de Mielina/metabolismo , Ácido Palmítico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
It is well-known that ethanol alters fatty acid and glycerolipid metabolism in liver, but most of the studies have been developed on rats, so little is known about the corresponding effects on human liver. We have chosen the Hep G2 human hepatoma cell line, which appears to be an excellent in vitro model system. Cells were incubated in ethanol containing medium (0-400 mM) for 48 h. Incorporation and metabolism of radioactive substrates (14C(U) glycerol,[1-14C] palmitic acid and [1-14C] eicosatrienoic acid (n-6) were analyzed in cellular and conditioned medium lipids. Cellular growth rate and lipid composition of control and ethanol-treated cells were also studied. The results showed that ethanol inhibited logarithmic cellular growth rate in a concentration dependent manner, without affecting viability. Ethanol (400 mM) did not modify cellular major lipid composition except for an increase of cholesteryl esters, but produced a decrease in the proportions of myristic, palmitic and palmitoleic acids. Ethanol enhanced the incorporation of radioactive fatty acids into cellular glycerolipids but did not alter the rate of incorporation of 14C(U) glycerol. This was attributed to an isotopic solution of the radioactive glycerol as a result of increased alpha-glycerophosphate biosynthesis. Incorporation of radioactive fatty acids and glycerol into conditioned medium glycerolipids were increased in cells incubated in presence of ethanol. The increased incorporation of 14C glycerol into conditioned medium together with a simultaneous diminution in labeling cellular glycerides suggest that there would be a stimulation of the export of these lipid classes to conditioned medium. Conversion of [1-14C] palmitic to oleic acid and eicosatrienoic to arachidonic acid were inhibited in 400 mM ethanol treated cells suggesting an inhibition of delta 9 and delta 5 desaturase activity.
Assuntos
Carcinoma Hepatocelular/metabolismo , Ácidos Eicosanoicos/metabolismo , Etanol/farmacologia , Ácidos Graxos/metabolismo , Glicolipídeos/metabolismo , Inibidores do Crescimento/farmacologia , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/patologia , Ácidos Eicosanoicos/análise , Glicerol/metabolismo , Glicolipídeos/antagonistas & inibidores , Humanos , Neoplasias Hepáticas/patologia , Ácidos Palmíticos/metabolismo , Células Tumorais CultivadasRESUMO
It is well-known that ethanol alters fatty acid and glycerolipid metabolism in liver, but most of the studies have been deleloped on rats, so little known about the corresponding effects on human liver. We have chosen the Hep G2 human hepatoma cell line, which appears be an excellent in vitro model system. Cells were incubated in ethanol containing medium (0-400 mM) for 48 h. Incorporation and metabolism of radioactive substrates (14C(U) glycerol, [1-(14)C] palmitic acid and [1-(14)C] eicosatrienoic acid (n-6) were analyzed in cellular and conditioned medium lipids. Cellular growth rate and lipid composition of control and ethanol-treated cells were also studied. The results showed that ethanol inhibited logarithmic cellular growth rate in a concentration dependent manner, without affecting viability. Ethanol (400 mM) did not modify cellular major lipid composition except for an increase of cholesteryl esters, but produced a decrease in the proportions of myristic, palmitic and palmitoleic acids. Ethanol enchanced the incorporation of radioactive fatty acids into cellular glycerolipids but did not alter the rate of incorporation of 14C(U) glycerol. This was attributed to an isotopic solution of the radioactive glycerol as a result of increasde alfa-glycerophosphate biosynthesis. Incorporation of radioactive fatty acids and glycerol into conditioned medium glycerolipids were increased in cells incubated in presence of ethanol. The increased incorporation of 14C glycerol into conditioned medium together with a simultaneous diminuition in labeling cellular glycerides suggest that there would be a simulation of the export of these lipid classes to conditioned medium. Conversion of [1-(14)C] palmitic to oleic acid and eicosatrienoic to arachidonic acid were inhibited in 400 mM ethanol treated cells suggesting an inhibition of delta 9 and delta 5 desaturase activity.
Assuntos
Humanos , /análogos & derivados , Ácidos Graxos/metabolismo , Carcinoma Hepatocelular/metabolismo , Etanol/farmacologia , Glicolipídeos/metabolismo , Neoplasias Hepáticas/metabolismo , /metabolismo , Ácidos Palmíticos/metabolismo , Carcinoma Hepatocelular/patologia , Glicerol/metabolismo , Glicolipídeos/antagonistas & inibidores , Neoplasias Hepáticas/patologia , Células Tumorais CultivadasRESUMO
It is well-known that ethanol alters fatty acid and glycerolipid metabolism in liver, but most of the studies have been deleloped on rats, so little known about the corresponding effects on human liver. We have chosen the Hep G2 human hepatoma cell line, which appears be an excellent in vitro model system. Cells were incubated in ethanol containing medium (0-400 mM) for 48 h. Incorporation and metabolism of radioactive substrates (14C(U) glycerol, [1-(14)C] palmitic acid and [1-(14)C] eicosatrienoic acid (n-6) were analyzed in cellular and conditioned medium lipids. Cellular growth rate and lipid composition of control and ethanol-treated cells were also studied. The results showed that ethanol inhibited logarithmic cellular growth rate in a concentration dependent manner, without affecting viability. Ethanol (400 mM) did not modify cellular major lipid composition except for an increase of cholesteryl esters, but produced a decrease in the proportions of myristic, palmitic and palmitoleic acids. Ethanol enchanced the incorporation of radioactive fatty acids into cellular glycerolipids but did not alter the rate of incorporation of 14C(U) glycerol. This was attributed to an isotopic solution of the radioactive glycerol as a result of increasde alfa-glycerophosphate biosynthesis. Incorporation of radioactive fatty acids and glycerol into conditioned medium glycerolipids were increased in cells incubated in presence of ethanol. The increased incorporation of 14C glycerol into conditioned medium together with a simultaneous diminuition in labeling cellular glycerides suggest that there would be a simulation of the export of these lipid classes to conditioned medium. Conversion of [1-(14)C] palmitic to oleic acid and eicosatrienoic to arachidonic acid were inhibited in 400 mM ethanol treated cells suggesting an inhibition of delta 9 and delta 5 desaturase activity. (AU)
Assuntos
Humanos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Glicolipídeos/metabolismo , Ácidos Graxos/metabolismo , Etanol/farmacologia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Glicolipídeos/antagonistas & inibidores , Glicerol/metabolismo , Ácidos Palmíticos/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Células Tumorais CultivadasRESUMO
1. This study examines the effect of propionate, normally produced in the gut, on lipid metabolism of resident macrophage. This cell is very abundant in the epithelial lining of the gut. 2. The activity of propionyl-CoA synthetase in macrophages was shown to be 0.39 nmol/min per mg protein, so this cell presents the ability to use propionate. Propionate at concentrations varying from 0.5 to 5 mM did not affect the activities of carnitine acetyltransferase, ATP-citrate lyase, acetoacetyl-CoA thiolase and 3-oxoacid-CoA transferase. 3. Thus this short chain fatty acid did not alter the capacity for transferring acetyl-CoA from mitochondria to cytosol and for ketone bodies formation and oxidation. However, propionate (40 mM) inhibited the incorporation of [1-14C]-palmitate into phospholipids, cholesterol, cholesterol ester and triacylglycerol and the incorporation of [3-14C]-pyruvate into phospholipids. 4. These findings suggest that fibre-rich diet by generating propionate may regulate macrophage lipid metabolism.
Assuntos
Lipídeos/biossíntese , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Propionatos/farmacologia , ATP Citrato (pro-S)-Liase/metabolismo , Animais , Radioisótopos de Carbono , Carnitina O-Acetiltransferase/metabolismo , Células Cultivadas , Coenzima A/metabolismo , Coenzima A Ligases/metabolismo , Hidrolases/metabolismo , Macrófagos Peritoneais/enzimologia , Masculino , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Piruvatos/metabolismo , Ácido Pirúvico , Ratos , Fatores de TempoAssuntos
Bioensaio , Hansenostáticos/farmacocinética , Hansenostáticos/farmacologia , Hanseníase/microbiologia , Mycobacterium bovis , Mycobacterium bovis/metabolismo , Nervo Tibial/metabolismo , Oxirredução , Rifampina/farmacocinética , Rifampina/farmacologia , Tecido Nervoso/metabolismo , Ácidos Palmíticos/metabolismoRESUMO
Fat digestion and absorption in the infant is a multistep process. An initial gastric phase of lipolysis generates modest amounts of diglycerides, monoglycerides, and free fatty acids. These initial digestion products, as well as bile salts, are required for optimal activity of the intestinal phase of lipolysis. Colipase-dependent pancreatic lipase catalyzes the intraduodenal phase of triglyceride digestion in formula-fed infants; in breast-fed infants this process is also mediated by bile salt-stimulated lipase. Triglyceride fatty acid positional distribution may modulate the efficiency of nutrient absorption. Human milk contains palmitic acid (C16:0) primarily in the sn-2 position; infant formula fat blends contain palmitic acid predominantly in the sn-1 and sn-3 positions. Because pancreatic lipase selectively hydrolyzes triglycerides at the sn-1 and sn-3 positions, free fatty acids and 2-monoglycerides are produced. Free palmitic acid, but not 2-monopalmitin (which is efficiently absorbed), may be lost as a calcium-fatty acid soap in the feces. As a result, many infant formulas contain substantial levels of well-absorbed saturated fatty acids of shorter chain lengths (e.g., C12:0) in place of palmitic acid. Means of increasing the proportion of 2-palmitic acid in infant formula may make possible fat blends closer to that of human milk with acceptable absorption characteristics.
Assuntos
Gorduras na Dieta/farmacocinética , Ácidos Graxos Insaturados/farmacocinética , Ácidos Graxos/farmacocinética , Alimentos Infantis , Fenômenos Fisiológicos da Nutrição do Lactente , Lipase/metabolismo , Leite Humano/química , Ácidos Palmíticos/metabolismo , Pâncreas/metabolismo , Triglicerídeos/metabolismo , Animais , Animais Recém-Nascidos , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Humanos , Lactente , Recém-Nascido , Absorção Intestinal , Ácido Palmítico , RatosRESUMO
In the studies described here rat liver microsomes containing labeled palmitic, stearic, oleic or linoleic acids were incubated with fatty acid binding protein (FABP) and the rate of removal of 14C-labeled fatty acids from the membrane by the soluble protein was measured using a model system. More unsaturated than saturated fatty acids were removed from native liver mircrosomes incubated with similar amounts of FABP. The in vitro peroxidation of microsomal membranes mediated by ascorbate-Fe++, modified its fatty acid composition with a considerable decrease of the peroxidizability index. These changes in the microsomes facilitated the removal of oleic and linoeic acids by FABP, but the removal of palmitic and stearic acids was not modified. This effect is proposed to result from a perturbation of membrane structure following peroxidation with release of free fatty acids from susceptible domains.
Assuntos
Proteínas de Transporte/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos Insaturados/metabolismo , Peroxidação de Lipídeos , Microssomos Hepáticos/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Animais , Ácido Ascórbico/farmacologia , Radioisótopos de Carbono , Fracionamento Celular , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Cinética , Ácido Linoleico , Ácidos Linoleicos/metabolismo , Medições Luminescentes , Microssomos Hepáticos/efeitos dos fármacos , Ácido Oleico , Ácidos Oleicos/metabolismo , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Ratos , Ácidos Esteáricos/metabolismoRESUMO
Rats were orally administered separately one dose of three labelled long-chain fatty acids: [1-14C] oleic, [1-14C] palmitic or [1-14C] stearic. Samples of lymph were obtained from previously cannulated thoracic ducts and analyzed for the composition and content of labelled fatty acids. Most of the newly recovered and labelled fatty acids were qualitatively and quantitatively similar regardless of which 14C-fatty acid had been administered. Over 20% of the administered fat were interconverted in six hours. The results suggest that the mucosa of the small intestine, the first sites of fatty acid absorption, is also one of the sites of various metabolic processes such as beta oxidation and synthesis which are responsible for some of the changes observed. These processes indicated that by shortening or lengthening the fatty acid composition, the content of the lymph became approximately the same regardless of the precursor fatty acid. The intestinal mucosa became the first tissue to help maintain lipid homeostasis in the rat.