RESUMO
Myoglobin, besides its role in oxygen turnover, has gained recognition as a potential regulator of lipid metabolism. Previously, we confirmed the interaction of fatty acids and acylcarnitines with Oxy-Myoglobin, using both molecular dynamic simulations and Isothermal Titration Calorimetry studies. However, those studies were limited to testing only the binding sites derived from homology to fatty acid binding proteins and predictions using automated docking. To explore the entry mechanisms of the lipid ligands into myoglobin, we conducted molecular dynamic simulations of murine Oxy- and Deoxy-Mb structures with palmitate or palmitoylcarnitine starting at different positions near the protein surface. The simulations indicated that both ligands readily (under â¼10-20 ns) enter the Oxy-Mb structure through a dynamic area ("portal region") near heme, known to be the entry point for small molecule gaseous ligands like O2, CO and NO. The entry is not observed with Deoxy-Mb where lipid ligands move away from protein surface, due to a compaction of the entry portal and the heme-containing crevice in the Mb protein upon O2 removal. The results suggest quick spontaneous binding of lipids to Mb driven by hydrophobic interactions, strongly enhanced by oxygenation, and consistent with the emergent role of Mb in lipid metabolism.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Mioglobina/metabolismo , Ácido Palmítico/metabolismo , Palmitoilcarnitina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação a Ácido Graxo/química , Heme/química , Cavalos , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Mioglobina/química , Oxigênio/química , Ácido Palmítico/química , Palmitoilcarnitina/química , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVE: Stroke is a major cause of death and disability. That three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, the utility of comprehensive metabolic phenotyping is assessed to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples. METHOD: Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n = 5), and from asymptomatic patients (n = 5). Two adjacent biological replicates were obtained from each tissue. Organic and aqueous metabolite extracts were obtained separately and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were used for statistical analysis. RESULTS: The two study groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p = 10(-4)-10(-5)). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine, and palmitoylcarnitine), intermediates of the ß-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected. CONCLUSIONS: Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and ß-oxidation) were implicated in differentiating symptomatic from asymptomatic patients and will be further investigated. These results indicate that metabolic phenotyping should be further explored to investigate the chemistry of the unstable plaque, in the pursuit of candidate biomarkers for risk-stratification and targets for pharmacotherapeutic intervention.
Assuntos
Estenose das Carótidas/metabolismo , Acidente Vascular Cerebral/etiologia , Idoso , Idoso de 80 Anos ou mais , Ácido Araquidônico/análise , Ácido Araquidônico/metabolismo , Biomarcadores/química , Carnitina/análogos & derivados , Carnitina/química , Estenose das Carótidas/complicações , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Palmitoilcarnitina/química , Fenótipo , Placa Aterosclerótica/química , Fatores de Risco , Acidente Vascular Cerebral/metabolismoRESUMO
The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane α-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [(3)H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.
Assuntos
Carnitina Aciltransferases/metabolismo , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Mitocôndrias/enzimologia , Mutagênese Sítio-Dirigida/métodos , Acilação/efeitos dos fármacos , Animais , Sítios de Ligação , Carnitina/análogos & derivados , Carnitina/farmacologia , Carnitina Aciltransferases/antagonistas & inibidores , Carnitina Aciltransferases/química , Carnitina Aciltransferases/genética , Biologia Computacional , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Mitocôndrias/efeitos dos fármacos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Palmitoilcarnitina/química , Palmitoilcarnitina/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Fatores de TempoRESUMO
Bacteria growing in biofilms are often in metabolic and physiological states that do not respond well to antibiotics, and thus, are major contributors to chronic diseases. Biofilm inhibitors, therefore, have the potential to be used alone or as adjuvants to conventional antibiotic therapies. Here, we screened a chemically diverse collection of protein kinase inhibitors for molecules that perturb biofilm development. Among the inhibitory molecules identified, palmitoyl-DL-carnitine (pDLC) impaired Pseudomonas aeruginosa and Escherichia coli biofilm formation in a dose-dependent manner. The pDLC affected multiple pathways implicated in P. aeruginosa biofilm development; it stimulated motility, inhibited activity of the Las quorum sensing system, and overrode the biofilm-promoting effects of subminimal inhibitory concentrations of aminoglycosides and high levels of the second messenger, cyclic-di-GMP. Palmitic acid, but not carnitine, inhibited biofilm formation but did not stimulate motility, suggesting that pDLC works through unique mechanisms. The ability to target multiple pathways involved in biofilm formation is desirable in an inhibitor, which makes pDLC an interesting lead for antibiofilm therapies.
Assuntos
Biofilmes , Palmitoilcarnitina/farmacologia , Pseudomonas aeruginosa/metabolismo , Palmitoilcarnitina/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The analysis of amino acids (AA) and acylcarnitines (AC) by tandem mass spectrometry (MS/MS) is performed in newborn screening laboratories worldwide. While butyl esterification assays are routine, it is possible to detect AAs and ACs as their native free acids (underivatized). The Centers for Disease Control and Prevention's Newborn Screening Quality Assurance Program provides dried blood spot (DBS) quality control (QC) and proficiency testing (PT) programs for numerous MS/MS analytes. We describe empirical differences between derivatization and non-derivatization techniques for selected AAs and ACs. METHODS: DBS materials were prepared at levels near, above and below mean domestic laboratory cut-offs, and distributed to program participants for MS/MS analysis. Laboratories reported quantitative and qualitative results. QC DBS materials were assayed in-house following established protocols. RESULT: Minor differences (<15%) between quantitative values resulting from butyl esters and free acid techniques were observed for the majority of the analytes. Mass spectrometric response from underivatized dicarboxylic acid acylcarnitines was less intense than their butyl esters. CONCLUSIONS: The use of underivatized techniques may also result in the inability to differentiate isobaric acylcarnitines. Laboratories should establish their own protocols by focusing on the decisions that identify test results requiring additional follow-up testing versus those that do not.
Assuntos
Aminoácidos/análise , Carnitina/análogos & derivados , Triagem Neonatal/métodos , Espectrometria de Massas em Tandem/métodos , Aminoácidos/sangue , Aminoácidos/química , Butanóis/química , Carnitina/análise , Carnitina/sangue , Carnitina/química , Humanos , Recém-Nascido , Leucina/análise , Leucina/sangue , Leucina/química , Doenças Metabólicas/diagnóstico , Metionina/análise , Metionina/sangue , Metionina/química , Palmitoilcarnitina/análise , Palmitoilcarnitina/sangue , Palmitoilcarnitina/química , Fenilalanina/análise , Fenilalanina/sangue , Fenilalanina/química , Controle de QualidadeRESUMO
A robust bioanalytical method capable of measuring acetyl and palmitoyl carnitines was developed and validated. Application of hydrophilic interaction chromatography (HILIC) enabled retention of these highly polar and difficult to analyze compounds on a silica HPLC column. The chromatography was conducted with a high percentage of an organic component in the mobile phase, allowing high sensitivity for the pre-existing positively charged quaternary ammonium ions by electrospray ionization mass spectrometry. Successful application of the method to reliably quantify naturally occurring acyl carnitines in mouse plasma depended on the use of corresponding deuterated analogues. The specificity of the method, achieved through the use of stable isotope labeled compounds in combination with a mass spectral multiple reaction monitoring technique, permitted a non-invasive assessment of the overall change in the levels of these acyl carnitines in the plasma of intact animals administered peroxisome proliferator activated receptor (PPAR) agents. These acyl carnitines, as carriers of the corresponding long-chain fatty acids for transport into mitochondria, can be employed as potential biomarkers for significant alteration in the beta-oxidation process in an intact animal.
Assuntos
Carnitina/análogos & derivados , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Palmitoilcarnitina/sangue , Animais , Biomarcadores/sangue , Biomarcadores/química , Calibragem , Carnitina/sangue , Marcação por Isótopo , Masculino , Camundongos , Camundongos Endogâmicos , Estrutura Molecular , Oxirredução , Palmitoilcarnitina/química , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND PURPOSE: The P2X(7) receptor exhibits a high degree of plasticity with agonist potency increasing after prolonged receptor activation. In this study we investigated the ability of lipids to modulate agonist potency at P2X(7) receptors. EXPERIMENTAL APPROACH: A variety of lipids, including lysophosphatidylcholine, sphingosylphosphorylcholine and hexadecylphosphorylcholine were studied for their effect on P2X(7) receptor-stimulated ethidium bromide accumulation in cells expressing human recombinant P2X(7) receptors and on P2X(7) receptor-stimulated interleukin-1 beta (IL1 beta) release from THP-1 cells. The effects of the lipids were also assessed in radioligand binding studies on human P2X(7) receptors. KEY RESULTS: At concentrations (3-30 microM) below the threshold to cause cell lysis, the lipids increased agonist potency and/or maximal effects at P2X(7) receptors in both ethidium accumulation and IL1 beta release studies. There was little structure activity relationship (SAR) for this effect and sub-lytic concentrations of Triton X-100 partially mimicked the effects of the lipids. The lipids caused cell lysis and increased intracellular calcium at higher concentrations (30-100 microM) which complicated interpretation of their effects in functional studies. However, the lipids (3-100 microM) also increased agonist potency 30-100 fold in radioligand binding studies. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that a diverse range of lipids increase agonist potency at the P2X(7) receptor in functional and binding studies. The broad SAR, including the effect of Triton X-100, suggests this may reflect changes in membrane properties rather than a direct effect on the P2X(7) receptor. Since many of the lipids studied accumulate in disease states they may enhance P2X(7) receptor function under pathophysiological conditions.
Assuntos
Lipídeos/farmacologia , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Etídio/metabolismo , Fluorometria , Humanos , Interleucina-1beta/metabolismo , Lipídeos/química , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/farmacologia , Camundongos , Octoxinol/farmacologia , Palmitoilcarnitina/química , Palmitoilcarnitina/farmacologia , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologia , Ensaio Radioligante , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X7 , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/farmacologia , Tensoativos/química , Tensoativos/farmacologiaRESUMO
We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.
Assuntos
Carnitina/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Isótopos de Carbono/análise , Carnitina/análise , Carnitina/química , Deutério/análise , Fluorocarbonos/química , Masculino , Métodos , Músculo Esquelético/química , Ácido Palmítico/sangue , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Palmitoilcarnitina/análise , Palmitoilcarnitina/química , Palmitoilcarnitina/normas , Coelhos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
A strategy for detection of carnitine and acylcarnitines is introduced. This versatile system has four components: (1) isolation by protein precipitation/desalting and cation-exchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase chromatography using a single non-end-capped C8 column, and (4) detection of carnitine and acylcarnitine pentafluorophenacyl esters using an ion trap mass spectrometer. Recovery of carnitine and acylcarnitines from the isolation procedure is 77-85%. Derivatization is rapid and complete with no evidence of acylcarnitine hydrolysis. Sequential ion-exchange/reversed-phase HPLC results in separation of reagent byproducts from derivatized carnitine and acylcarnitines, followed by reversed-phase separation of carnitine and acylcarnitine pentafluorophenacyl esters. Detection by MS/MS is highly selective, with carnitine pentafluorophenacyl ester yielding a strong product ion at m/z 311 and acylcarnitine pentafluorophenacyl ester fragmentation yielding two product ions: (1) loss of m/z 59 and (2) generation of an ion at m/z 293. To demonstrate this analytical strategy, phosphate buffered serum albumin was spiked with carnitine and 15 acylcarnitines and analyzed using the described protein precipitation/desalting and cation-exchange solid-phase extraction isolation, derivatization with pentafluorophenacyl trifluoromethanesulfonate, chromatography using the sequential ion-exchange/reversed-phase chromatography HPLC system, and detection by MS and MS/MS. Successful application of this strategy to the quantification of carnitine and acetylcarnitine in rat liver is shown.
Assuntos
Carnitina/análogos & derivados , Carnitina/análise , Acetonitrilas/química , Acetofenonas/química , Acetilcarnitina/análise , Acetilcarnitina/química , Animais , Análise Química do Sangue/métodos , Carnitina/química , Carnitina/isolamento & purificação , Precipitação Química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Humanos , Fígado/química , Masculino , Espectrometria de Massas , Metanol/química , Estrutura Molecular , Palmitoilcarnitina/análise , Palmitoilcarnitina/química , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Albumina Sérica/química , Sílica Gel , Dióxido de Silício/química , Extração em Fase Sólida/métodosRESUMO
The syntheses of (R)- and (S)-norcarnitine ethyl esters are described starting with an optimized, chiral chemical reduction of ethyl 4-chloroacetoacetate followed by azide substitution, reduction, and dimethylation. The reaction of (R)- and (S)-norcarnitine ethyl esters with 1-bromoheptadecan-2-one gives (+)- and (-)-6-[(methoxycarbonyl)methyl]-2-pentadecyl-4,4-dimethylmorpholinium bromide, respectively, which hydrolyzes to (+)- and (-)-6-(carboxylatomethyl)-2-pentadecyl-4,4-dimethylmorpholinium (hemipalmitoylcarnitinium, (+)- and (-)-HPC), respectively, upon treatment with a hydroxide resin. (+)- and (-)-HPC are reversible active-site directed inhibitors of hepatic mitochondrial CPTs. Both stereoisomers inhibit CPT I and CPT II in control and streptozotocin diabetic rat to the same extent (Imax=100%). Using intact mitochondria (CPT I), I50values for (-)-HPC and (+)-HPC were 15.5 microM and 47.5 microM, respectively. The I50 values for CPT II were 6.7 microM and 38.5 microM for (-)-HPC and (+)-HPC, respectively. The mode of inhibition was uncompetitive for CPT I with respect to acyl-CoA. The apparent K(i) for (-)-HPC is about 5 microM. These data suggest that (-)-HPC may be useful for further evaluation as an antidiabetic agent.
Assuntos
Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Diabetes Mellitus Experimental/enzimologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Palmitoilcarnitina/análogos & derivados , Animais , Sítios de Ligação , Carnitina O-Palmitoiltransferase/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Masculino , Mitocôndrias Hepáticas/metabolismo , Conformação Molecular , Palmitoilcarnitina/síntese química , Palmitoilcarnitina/química , Palmitoilcarnitina/farmacologia , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.
Assuntos
Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/genética , Cisteína/química , Alanina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Carnitina/química , Catálise , Primers do DNA/química , Humanos , Cinética , Malonil Coenzima A/química , Modelos Químicos , Dados de Sequência Molecular , Mutagênese , Mutação , Miocárdio/metabolismo , Ácido Palmítico , Palmitoilcarnitina/química , Pichia/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina/químicaRESUMO
The carnitine-dependent transport of long-chain fatty acids is essential for fatty acid catabolism. In this system, the fatty acid moiety of acyl-CoA is transferred enzymatically to carnitine, and the resultant product, acylcarnitine, is imported into the mitochondrial matrix through a transporter named carnitine-acylcarnitine translocase (CACT). Here we report a novel mammalian protein homologous to CACT. The protein, designated as CACL (CACT-like), is localized to the mitochondria and has palmitoylcarnitine transporting activity. The tissue distribution of CACL is similar to that of CACT; both are expressed at a higher level in tissues using fatty acids as fuels, except in the brain, where only CACL is expressed. In addition, CACL is induced by partial hepatectomy or fasting. Thus, CACL may play an important role cooperatively with its homologue CACT in a stress-induced change of lipid metabolism, and may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.
Assuntos
Carnitina Aciltransferases/química , Carnitina Aciltransferases/metabolismo , Carnitina Aciltransferases/farmacologia , Carnitina/análogos & derivados , Fígado/fisiologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/farmacologia , Células 3T3 , Sequência de Aminoácidos , Animais , Transporte Biológico , Northern Blotting , Western Blotting , Encéfalo/fisiologia , Carnitina/química , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Privação de Alimentos , Teste de Complementação Genética , Humanos , Imuno-Histoquímica , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Dados de Sequência Molecular , Palmitoilcarnitina/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
We investigated, in skeletal muscle mitochondria isolated from semistarved and refed rats, the relation between the protein expression of uncoupling protein 3 (UCP3) and mitochondrial oxidative capacity, assessed as state 4 and state 3 respiration rates in presence of substrates that are either non-lipids (glutamate, succinate) or lipids (palmitoyl CoA, palmitoylcarnitine). During semistarvation, when whole-body thermogenesis is diminished, state 3 respiration was lower than in fed controls by about 30% independently of substrate types, while state 4 respiration was lower by 20% only during succinate oxidation, but UCP3 was unaltered. After 5 days of refeeding, when thermogenesis is still diminished, neither state 4, state 3 nor UCP3 were lower than in controls. Refeeding on a high-fat diet, which exacerbates the suppression of thermogenesis, resulted in a two-fold elevation in UCP3 but no change in state 4 or state 3 respiration. These results during semistarvation and refeeding, in line with those previously reported for fasting, are not in support of the hypothesis that UCP3 is a mediator of adaptive thermogenesis pertaining to weight regulation, and underscore the need for caution in interpreting parallel changes in UCP3 and mitochondrial oxidative capacity as the reflection of mitochondrial uncoupling by UCP3.
Assuntos
Proteínas de Transporte/metabolismo , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Oxigênio/metabolismo , Ração Animal , Animais , Western Blotting , Peso Corporal , Privação de Alimentos , Ácido Glutâmico/química , Canais Iônicos , Metabolismo dos Lipídeos , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Músculo Esquelético/citologia , Palmitoil Coenzima A/química , Palmitoilcarnitina/química , Ratos , Ratos Sprague-Dawley , Ácido Succínico/química , Proteína Desacopladora 3RESUMO
Accumulation of lipid metabolites, such as palmitoylcarnitine and lysophosphatidylcholine, is thought to be a major contributor to the development of cardiac arrhythmias during myocardial ischemia. This arrhythmogenicity is likely due to the effects of these metabolites on various ion channels. Diabetic hearts have been shown to accumulate much higher concentrations of these lipid metabolites during ischemia, which may be an important factor in the enhanced incidence of arrhythmias in diabetic hearts. However, it is not known whether these metabolites have similar effects on the ion channels of diabetic hearts as in non-diabetic hearts. Previous studies on myocytes from non-diabetic hearts have reported either enhancement or inhibition of L-type calcium current (I(Ca)) by these lipid metabolites. Thus, it is not clear whether the effects of palmitoylcarnitine and/or lysophosphatidlycholine on I(Ca) contribute to the enhanced arrhythmogenicity of diabetic hearts or protect against arrhythmias. We determined the effect of exogenous palmitoylcarnitine and lysophosphatidylcholine on the (I(Ca)) in ventricular myocytes from streptozotocin-diabetic and non-diabetic rat hearts under identical conditions. We found that palmitoylcarnitine and lysophosphatidylcholine exhibited a dose-dependent inhibition of I(Ca), which was virtually identical in diabetic and non-diabetic cardiac myocytes. Thus, we conclude that these arrhythmogenic lipid metabolites have similar actions on calcium channels in diabetic and non-diabetic hearts. Therefore, the greater susceptibility of diabetic hearts to arrhythmias during myocardial ischemia is not due to an altered sensitivity of the L-type calcium channels to lipid metabolites, but may be explained, in large part, by the greater accumulation of these metabolites during ischemia.
Assuntos
Arritmias Cardíacas , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Metabolismo dos Lipídeos , Animais , Relação Dose-Resposta a Droga , Eletrofisiologia , Isquemia/metabolismo , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Masculino , Miocárdio/metabolismo , Palmitoilcarnitina/química , Palmitoilcarnitina/metabolismo , Técnicas de Patch-Clamp , Ratos , Fatores de TempoAssuntos
Acil Coenzima A/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina/síntese química , Carnitina/análogos & derivados , Carnitina O-Palmitoiltransferase/deficiência , Humanos , Oxirredução , Palmitoilcarnitina/análogos & derivados , Palmitoilcarnitina/síntese química , Palmitoilcarnitina/química , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Palmitoylcarnitine is a well-known intermediate in mitochondrial fatty acid oxidation. Less known are its properties as a surfactant, with a capacity to solubilize biological membranes similar to that of many synthetic detergents used in the biochemical laboratory. Some of the physico-chemical properties of palmitoylcarnitine may help to explain the need for coenzyme A-carnitine-coenzyme A acyl exchange during mitochondrial fatty acid import. The amphiphilic character of palmitoylcarnitine may also explain its proposed involvement in the pathogenesis of myocardial ischemia.
Assuntos
Coenzima A/metabolismo , Palmitoilcarnitina/química , Palmitoilcarnitina/fisiologia , Tensoativos , Animais , Carnitina/metabolismo , Ácidos Graxos/metabolismo , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Palmitoilcarnitina/metabolismo , Frações Subcelulares/metabolismoRESUMO
The effect of a series of long-chain acyl-CoAs and acylcarnitines has been tested by differential scanning calorimetry on the gel-fluid transition of saturated phosphatidylcholines and of dietai-doylphosphatidylethanolamine (DEPE), and on the lamellar-Hexagonal transition of DEPE. Both series of acylderivatives have similar effects (the acylcarnitines being more potent): a decrease in the gel-fluid transition enthalpy and an increase in the gel-fluid transition width. Mixtures of dipalmitoylphosphatidylcholine with palmitoyl-CoA or palmitoylcarnitine (i.e. when all three hydrocarbon chains are 16C in length) display a peculiar behaviour, in that the main endotherm remains unchanged until a high proportion of palmitoylderivative is present, then it collapses suddenly. The disappearance of the gel-fluid main endotherm in the presence of palmitoylcarnitine is due to the fragmentation of the bilayer below the cooperative unit size of the phospholipid, while the same effect is caused by palmitoyl-CoA through the interaction of the coenzyme A polar moiety with the lipid-water interface, the overall bilayer structure being maintained. The effect of both series of compounds on the lamellar to inverted hexagonal phase transition of DEPE is also similar: they both stabilize the lamellar phase, increase the transition temperature and smear out the transition endotherm. Their behaviour may be rationalized considering that they are compounds with a bulky polar head, relative to their single hydrophobic chain, that would favour a positive curvature in the monolayer, while the inverted hexagonal phase requires a negative curvature.
Assuntos
Acil Coenzima A/farmacologia , Carnitina/farmacologia , Fosfolipídeos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Varredura Diferencial de Calorimetria , Carnitina/análogos & derivados , Dimiristoilfosfatidilcolina/química , Géis/química , Palmitoilcarnitina/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/químicaRESUMO
The heart is known for its ability to produce energy from fatty acids (FA) because of its important beta-oxidation equipment, but it can also derive energy from several other substrates including glucose, pyruvate, and lactate. The cardiac ATP store is limited and can assure only a few seconds of beating. For this reason the cardiac muscle can adapt quickly to the energy demand and may shift from a 100% FA-derived energy production (after a lipid-rich food intake) or any balanced situation (e.g., diabetes, fasting, exercise). These situations are not similar for the heart in terms of oxygen requirement because ATP production from glucose is less oxygen-consuming than from FA. The regulation pathways for these shifts, which occur in physiologic as well as pathologic conditions (ischemia-reperfusion), are not yet known, although both insulin and pyruvate dehydrogenase activation are clearly involved. It becomes of strategic importance to clarify the pathways that control these shifts to influence the oxygen requirement of the heart. Excess FA oxidation is closely related to myocardial contraction disorders characterized by increased oxygen consumption for cardiac work. Such an increased oxygen cost of cardiac contraction was observed in stunned myocardium when the contribution of FA oxidation to oxygen consumption was increased. In rats, an increase in n-3 polyunsaturated FA in heart phospholipids achieved by a fish-oil diet improved the recovery of pump activity during postischemic reperfusion. This was associated with a moderation of the ischemia-induced decrease in mitochondrial palmitoylcarnitine oxidation. In isolated mitochondria at calcium concentrations close to that reported in ischemic cardiomyocytes, a futile cycle of oxygen wastage was reported, associated with energy wasting (constant AMP production). This occurs with palmitoylcarnitine as substrate but not with pyruvate or citrate. The energy wasting can be abolished by CoA-SH and other compounds, but not the oxygen wasting. Again, the calcium-induced decrease in mitochondrial ADP/O ratio was reduced by increasing the n-3 polyunsaturated FA in the mitochondrial phospholipids. These data suggest that in addition to the amount of circulating lipids, the quality of FA intake may contribute to heart energy regulation through the phospholipid composition. On the other hand, other intervention strategies can be considered. Several studies have focused on palmitoylcarnitine transferase I to achieve a reduction in beta-oxidation. In a different context, trimetazidine was suggested to exert its anti-ischemic effect on the heart by interfering with the metabolic shift, either at the pyruvate dehydrogenase level or by reducing the beta-oxidation. Further studies will be required to elucidate the complex system of heart energy regulation and the mechanism of action of potentially efficient molecules.
Assuntos
Cálcio/metabolismo , Ácidos Graxos/metabolismo , Óleos de Peixe/uso terapêutico , Miocárdio/metabolismo , Consumo de Oxigênio/fisiologia , Traumatismo por Reperfusão/dietoterapia , Trifosfato de Adenosina/biossíntese , Animais , Ácidos Graxos/química , Humanos , Ácido Láctico/metabolismo , Mitocôndrias Cardíacas/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/citologia , Oxirredução , Palmitoilcarnitina/química , Palmitoilcarnitina/metabolismo , Ácido Pirúvico/metabolismo , Ratos , Traumatismo por Reperfusão/fisiopatologiaRESUMO
Fatty acyl-coenzyme A's are temporarily converted into fatty acylcarnitines while transferred across the inner mitochondrial membrane, in their catabolic pathway. In search of an explanation for the need of this coenzyme exchange, the present work describes comparatively the abilities of both kinds of fatty acyl derivatives (represented by palmitoyl-coenzyme A and palmitoylcarnitine) in binding to and perturbing the structure of phosphatidylcholine bilayers in the form of large unilamellar vesicles. Both palmitoyl-coenzyme A and palmitoylcarnitine partition preferentially into the bilayer lipids, so that their free concentration in water is in practice negligible. However, palmitoylcarnitine is able to disrupt the membrane barrier to solutes, leading to vesicle leakage, and, at higher concentrations, it produces complete membrane solubilization, while palmitoyl-coenzyme A produces neither leakage nor solubilization. Palmitoylcarnitine has the properties of many commonly used biochemical detergents. The different behavior of both fatty acyl derivatives helps to explain the need for the transitory coenzyme A/carnitine exchange, and provides a pathogenic mechanism for some genetic defects of mitochondrial fatty acid transport. Other pathophysiological processes in which palmitoylcarnitine has been putatively involved are examined in light of the above results.
Assuntos
Membrana Celular/efeitos dos fármacos , Lipossomos/metabolismo , Palmitoil Coenzima A/farmacologia , Palmitoilcarnitina/farmacologia , Membrana Celular/metabolismo , Cinética , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Micelas , Nefelometria e Turbidimetria , Palmitoil Coenzima A/química , Palmitoil Coenzima A/metabolismo , Palmitoilcarnitina/química , Palmitoilcarnitina/metabolismo , Fosfatidilcolinas/metabolismo , Solubilidade , Espectrometria de FluorescênciaRESUMO
Mixtures of di-(perdeuteropalmitoyl)-sn-glycero-3 choline ([2H62]Pam2GroPCho) with palmitoylcarnitine or palmitoyl-CoA in aqueous suspension have been examined by Fourier-transform infrared spectroscopy. The C-H (or C-D) region of the spectrum shows that an order-disorder transition exists in pure aqueous palmitoylcarnitine at 45 degrees C; palmitoylcarnitine mixes with [2H62]Pam2GroPCho without perturbing the gel-fluid transition of the phospholipid even at [2H62]Pam2GroPCho/palmitoylcarnitine 1:2 molar ratios; and palmitoyl-CoA, however, at similar proportions, smears out the [2H62]Pam2GroPCho transition as detected from C-D stretching vibrations. Relevant data from the carbonyl region include; the high-frequency (non-hydrogen bound) carbonyl subpopulation, but not the low frequency one, detects the gel-to-fluid transition of the phospholipid; the carbonyl region detects the thermotropic transition over a wider temperature range than the methylene stretching region, i.e. detects changes starting well below and ending several degrees above the methylene transition temperature, and a significant interaction may occur between some coenzyme A group and the carbonyl groups of the phospholipid. The latter interaction may contribute to explain the coenzyme A/carnitine exchange during mitochondrial fatty acid import.