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1.
Arch Biochem Biophys ; 655: 56-66, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30092229

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 ∼10-20 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 Aminoácidos
2.
Sci Rep ; 7(1): 2786, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28584281

RESUMO

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome.


Assuntos
Fatores Imunológicos/metabolismo , Espectrometria de Massas , Palmitoilcarnitina/metabolismo , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Salmonella typhimurium/imunologia , Animais , Biomarcadores , Feminino , Camundongos , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
J Cell Sci ; 130(11): 1940-1951, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28424233

RESUMO

Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status.


Assuntos
Ciclo do Ácido Cítrico/genética , DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas de Ligação a RNA/metabolismo , Trifosfato de Adenosina/biossíntese , Sistemas CRISPR-Cas , Ciclo do Ácido Cítrico/efeitos dos fármacos , Dano ao DNA , DNA Mitocondrial/metabolismo , Etídio/toxicidade , Deleção de Genes , Células HeLa , Humanos , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/efeitos dos fármacos , Imagem Óptica , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Palmitoilcarnitina/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Ligação a RNA/genética
4.
Brain Dev ; 39(1): 48-57, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27591119

RESUMO

INTRODUCTION: We evaluated the effects of bezafibrate (BEZ) on ß-oxidation in fibroblasts obtained from patients with glutaric acidemia type II (GA2) of various clinical severities using an in vitro probe (IVP) assay. METHODS: Cultured fibroblasts from 12 patients with GA2, including cases of the neonatal-onset type both with and without congenital anomalies (the prenatal- and neonatal-onset forms, respectively), the infantile-onset, and the myopathic forms, were studied. The IVP assay was performed by measuring acylcarnitines (ACs) in the cell culture medium of fibroblasts incubated with palmitic acid for 96h in the presence of 0-800µM BEZ using tandem mass spectrometry. RESULTS: The IVP assay showed that 100µM BEZ markedly reduced the level of palmitoylcarnitine (C16) in the neonatal-onset, infantile-onset, and myopathic forms of GA2, either increasing or maintaining a high level of acetylcarnitine (C2), which serves as an index of energy production via ß-oxidation. In the prenatal-onset form, although a small reduction of C16 was also observed in the presence of 100µM BEZ, the level of C2 remained low. At concentrations higher than 100µM, BEZ further decreased the level of ACs including C16, but a concentration over 400µM decreased the level of C2 in most cases. DISCUSSION: BEZ at 100µM was effective for all GA2 phenotypes except for the prenatal-onset form, as a reduction of C16 without deterioration of C2 is considered to indicate improvement of ß-oxidation. The effects of higher doses BEZ could not be estimated by the IVP assay but might be small or nonexistent.


Assuntos
Bezafibrato/farmacologia , Carnitina/análogos & derivados , Fibroblastos/efeitos dos fármacos , Reguladores do Metabolismo de Lipídeos/farmacologia , Deficiência Múltipla de Acil Coenzima A Desidrogenase/tratamento farmacológico , Adolescente , Adulto , Idade de Início , Carnitina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ativadores de Enzimas/farmacologia , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Deficiência Múltipla de Acil Coenzima A Desidrogenase/metabolismo , Palmitoilcarnitina/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Pele/citologia , Pele/efeitos dos fármacos , Pele/metabolismo
5.
Biochem J ; 474(4): 557-569, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27941154

RESUMO

The obligatory role of carnitine palmitoyltransferase-I (CPT-I) in mediating mitochondrial lipid transport is well established, a process attenuated by malonyl-CoA (M-CoA). However, the necessity of reducing M-CoA concentrations to promote lipid oxidation has recently been challenged, suggesting external regulation on CPT-I. Since previous work in hepatocytes suggests the involvement of the intermediate filament fraction of the cytoskeleton in regulating CPT-I, we investigated in skeletal muscle if CPT-I sensitivity for M-CoA inhibition could be regulated by the intermediate filaments, and whether AMP-activated protein kinase (AMPK) could be involved in this process. Chemical disruption (3,3'-iminodipropionitrile, IDPN) of the intermediate filaments did not alter mitochondrial respiration or sensitivity for numerous substrates (palmitoyl-CoA, ADP, palmitoyl carnitine and pyruvate). In contrast, IDPN reduced CPT-I sensitivity for M-CoA inhibition in permeabilized muscle fibers, identifying M-CoA kinetics as a specific target for intermediate filament regulation. Importantly, exercise mimicked the effect of IDPN on M-CoA sensitivity, suggesting that intermediate filament disruption in vivo is physiologically important for CPT-I regulation. To ascertain a potential mechanism, since AMPK is activated during exercise, AMPK ß1ß2-KO mice were utilized in an attempt to ablate the observed exercise response. Unexpectedly, these mice displayed drastic attenuation in resting M-CoA sensitivity, such that exercise and IDPN could not further alter M-CoA sensitivity. These data suggest that AMPK is not required for the regulation of the intermediate filament interaction with CPT-I. Altogether, these data highlight that M-CoA sensitivity is important for regulating mitochondrial lipid transport. Moreover, M-CoA sensitivity appears to be regulated by intermediate filament interaction with CPT-I, a process that is important when metabolic homeostasis is challenged.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Filamentos Intermediários/metabolismo , Malonil Coenzima A/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Difosfato de Adenosina/metabolismo , Animais , Carnitina O-Palmitoiltransferase/genética , Regulação da Expressão Gênica , Filamentos Intermediários/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Musculares/genética , Músculo Esquelético/efeitos dos fármacos , Nitrilos/farmacologia , Oxirredução , Fosforilação Oxidativa , Palmitoil Coenzima A/metabolismo , Palmitoilcarnitina/metabolismo , Condicionamento Físico Animal , Ácido Pirúvico/metabolismo , Transdução de Sinais , Especificidade por Substrato
6.
Prostate ; 76(14): 1326-37, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27403764

RESUMO

BACKGROUND: Acylcarnitines are intermediates of fatty acid oxidation and accumulate as a consequence of the metabolic dysfunction resulting from the insufficient integration between ß-oxidation and the tricarboxylic acid (TCA) cycle. The aim of this study was to investigate whether acylcarnitines accumulate in prostate cancer tissue, and whether their biological actions could be similar to those of dihydrotestosterone (DHT), a structurally related compound associated with cancer development. METHODS: Levels of palmitoylcarnitine (palcar), a C16:00 acylcarnitine, were measured in prostate tissue using LC-MS/MS. The effect of palcar on inflammatory cytokines and calcium (Ca(2+) ) influx was investigated in in vitro models of prostate cancer. RESULTS: We observed a significantly higher level of palcar in prostate cancerous tissue compared to benign tissue. High levels of palcar have been associated with increased gene expression and secretion of the pro-inflammatory cytokine IL-6 in cancerous PC3 cells, compared to normal PNT1A cells. Furthermore, we found that high levels of palcar induced a rapid Ca(2+) influx in PC3 cells, but not in DU145, BPH-1, or PNT1A cells. This pattern of Ca(2+) influx was also observed in response to DHT. Through the use of whole genome arrays we demonstrated that PNT1A cells exposed to palcar or DHT have a similar biological response. CONCLUSIONS: This study suggests that palcar might act as a potential mediator for prostate cancer progression through its effect on (i) pro-inflammatory pathways, (ii) Ca(2+) influx, and (iii) DHT-like effects. Further studies need to be undertaken to explore whether this class of compounds has different biological functions at physiological and pathological levels. Prostate 76:1326-1337, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.


Assuntos
Cálcio/metabolismo , Mediadores da Inflamação/metabolismo , Palmitoilcarnitina/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Cromatografia Líquida , Humanos , Masculino , Espectrometria de Massas , Palmitoilcarnitina/análise , Próstata/metabolismo , Neoplasias da Próstata/patologia
7.
Br J Pharmacol ; 173(9): 1529-40, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26844527

RESUMO

BACKGROUND AND PURPOSE: Trimetazidine, known as a metabolic modulator, is an anti-anginal drug used for treatment of stable coronary artery disease (CAD). It is proposed to act via modulation of cardiac metabolism, shifting the mitochondrial substrate utilization towards carbohydrates, thus increasing the efficiency of ATP production. This mechanism was recently challenged; however, these studies used indirect approaches and animal models, which made their conclusions questionable. The goal of the current study was to assess the effect of trimetazidine on mitochondrial substrate oxidation directly in left ventricular myocardium from CAD patients. EXPERIMENTAL APPROACH: Mitochondrial fatty acid (palmitoylcarnitine) and carbohydrate (pyruvate) oxidation were measured in permeabilized left ventricular fibres obtained during coronary artery bypass grafting surgery from CAD patients, which either had trimetazidine included in their therapy (TMZ group) or not (Control). KEY RESULTS: There was no difference between the two groups in the oxidation of either palmitoylcarnitine or pyruvate, and in the ratio of carbohydrate to fatty acid oxidation. Activity and expression of pyruvate dehydrogenase, the key regulator of carbohydrate metabolism, were also not different. Lastly, acute in vitro exposure of myocardial tissue to different concentrations of trimetazidine did not affect myocardial oxidation of fatty acid. CONCLUSION AND IMPLICATIONS: Using myocardial tissue from CAD patients, we found that trimetazidine (applied chronically in vivo or acutely in vitro) had no effect on cardiac fatty acid and carbohydrate oxidation, suggesting that the clinical effects of trimetazidine are unlikely to be due to its metabolic effects, but rather to an as yet unidentified intracardiac mechanism.


Assuntos
Doença da Artéria Coronariana/tratamento farmacológico , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Trimetazidina/farmacologia , Idoso , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Feminino , Humanos , Masculino , Oxirredução/efeitos dos fármacos , Palmitoilcarnitina/metabolismo , Ácido Pirúvico/metabolismo , Trimetazidina/administração & dosagem
8.
Am J Physiol Endocrinol Metab ; 310(9): E715-23, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26908505

RESUMO

Oxidation of fatty acids is a major source of energy in the heart, liver, and skeletal muscle. It can be measured accurately using respirometry in isolated mitochondria, intact cells, and permeabilized cells or tissues. This technique directly measures the rate of oxygen consumption or flux at various respiratory states when appropriate substrates, uncouplers, and inhibitors are used. Acylcarnitines such as palmitoylcarnitine or octanoylcarnitine are the commonly used substrates. The ß-oxidation pathway is prone to feedforward inhibition resulting from accumulation of short-chain acyl-CoA and depletion of CoA, but inclusion of malate or carnitine prevents accumulation of these intermediaries and CoA depletion.


Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Acil Coenzima A/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Coenzima A/metabolismo , Eletrodos , Retroalimentação Fisiológica , Humanos , Malatos/metabolismo , Oxirredução , Fosforilação Oxidativa , Palmitoilcarnitina/metabolismo
9.
Am J Physiol Endocrinol Metab ; 309(3): E256-64, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26037250

RESUMO

Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma acylcarnitine profiles have been shown to poorly reflect whole body acylcarnitine metabolism. We aimed to clarify the individual role of different organ compartments in whole body acylcarnitine metabolism in a fasted and postprandial state in a porcine transorgan arteriovenous model. Twelve cross-bred pigs underwent surgery where intravascular catheters were positioned before and after the liver, gut, hindquarter muscle compartment, and kidney. Before and after a mixed meal, we measured acylcarnitine profiles at several time points and calculated net transorgan acylcarnitine fluxes. Fasting plasma acylcarnitine concentrations correlated with net hepatic transorgan fluxes of free and C2- and C16-carnitine. Transorgan acylcarnitine fluxes were small, except for a pronounced net hepatic C2-carnitine production. The peak of the postprandial acylcarnitine fluxes was between 60 and 90 min. Acylcarnitine production or release was seen in the gut and liver and consisted mostly of C2-carnitine. Acylcarnitines were extracted by the kidney. No significant net muscle acylcarnitine flux was observed. We conclude that liver has a key role in acylcarnitine metabolism, with high net fluxes of C2-carnitine both in the fasted and fed state, whereas the contribution of skeletal muscle is minor. These results further clarify the role of different organ compartments in the metabolism of different acylcarnitine species.


Assuntos
Carnitina/análogos & derivados , Metabolismo dos Lipídeos , Fígado/metabolismo , Modelos Biológicos , Acetilcarnitina/sangue , Acetilcarnitina/metabolismo , Animais , Carnitina/biossíntese , Carnitina/sangue , Carnitina/metabolismo , Cateteres de Demora , Cruzamentos Genéticos , Feminino , Mucosa Intestinal/metabolismo , Intestinos/irrigação sanguínea , Rim/irrigação sanguínea , Rim/metabolismo , Fígado/irrigação sanguínea , Azeite de Oliva , Especificidade de Órgãos , Palmitoilcarnitina/sangue , Palmitoilcarnitina/metabolismo , Óleos Vegetais/administração & dosagem , Óleos Vegetais/metabolismo , Período Pós-Prandial , Sus scrofa
10.
Scand J Med Sci Sports ; 25(1): e59-69, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24845952

RESUMO

High-intensity interval training (HIT) is known to increase mitochondrial content in a similar way as endurance training [60-90% of maximal oxygen uptake (VO2peak)]. Whether HIT increases the mitochondria's ability to oxidize lipids is currently debated. We investigated the effect of HIT on mitochondrial fat oxidation in skeletal muscle and adipose tissue. Mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial substrate sensitivity (K(m)(app)), and mitochondrial content were measured in skeletal muscle and adipose tissue in healthy overweight subjects before and after 6 weeks of HIT (three times per week at 298 ± 21 W). HIT significantly increased VO2peak from 2.9 ± 0.2 to 3.1 ± 0.2 L/min. No differences were seen in maximal fat oxidation in either skeletal muscle or adipose tissue. K(m)(app) for octanoyl carnitine or palmitoyl carnitine were similar after training in skeletal muscle and adipose tissue. Maximal OXPHOS capacity with complex I- and II-linked substrates was increased after training in skeletal muscle but not in adipose tissue. In conclusion, 6 weeks of HIT increased VO2peak. Mitochondrial content and mitochondrial OXPHOS capacity were increased in skeletal muscle, but not in adipose tissue. Furthermore, mitochondrial fat oxidation was not improved in either skeletal muscle or adipose tissue.


Assuntos
Carnitina/análogos & derivados , Exercício/fisiologia , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Sobrepeso/metabolismo , Palmitoilcarnitina/metabolismo , Gordura Subcutânea/metabolismo , Adulto , Carnitina/metabolismo , Feminino , Humanos , Masculino , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologia
11.
Biochem Biophys Res Commun ; 448(2): 175-81, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24780397

RESUMO

INTRODUCTION: Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving ß-oxidation of long-chain fatty acids (FAO), which leads to rhabdomyolysis and subsequent acute renal failure. The detailed mechanisms of disease pathogenesis remain unknown; however, the availability of relevant human cell types for investigation, such as skeletal muscle cells, is limited, and the development of novel disease models is required. METHODS: We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a Japanese patient with CPT II deficiency. Mature myocytes were differentiated from the patient-derived hiPSCs by introducing myogenic differentiation 1 (MYOD1), the master transcriptional regulator of myocyte differentiation. Using an in vitro acylcarnitine profiling assay, we investigated the effects of a hypolipidemic drug, bezafibrate, and heat stress on mitochondrial FAO in CPT II-deficient myocytes and controls. RESULTS: CPT II-deficient myocytes accumulated more palmitoylcarnitine (C16) than did control myocytes. Heat stress, induced by incubation at 38°C, leads to a robust increase of C16 in CPT II-deficient myocytes, but not in controls. Bezafibrate reduced the amount of C16 in control and CPT II-deficient myocytes. DISCUSSION: In this study, we induced differentiation of CPT II-deficient hiPSCs into mature myocytes in a highly efficient and reproducible manner and recapitulated some aspects of the disease phenotypes of CPT II deficiency in the myocyte disease models. This approach addresses the challenges of modeling the abnormality of FAO in CPT II deficiency using iPSC technology and has the potential to revolutionize translational research in this field.


Assuntos
Carnitina O-Palmitoiltransferase/deficiência , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Células Musculares/patologia , Células-Tronco Pluripotentes/patologia , Bezafibrato/farmacologia , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Diferenciação Celular , Células Cultivadas , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Masculino , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Palmitoilcarnitina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Adulto Jovem
12.
Mol Cell Biochem ; 393(1-2): 191-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24771065

RESUMO

Arrhythmias have been treated for a long time with drugs that mainly target the ionic pumps and channels. These anti-arrhythmic regimens per se introduce new arrhythmias, which can be detrimental to patients. Advances in development of novel pharmacology without introduction of iatrogenic arrhythmias are thus favorable for an effective treatment of arrhythmias. Electrophysiological stability of the heart has been shown to be closely associated with cardiac metabolism. The present effective anti-arrhythmic drugs such as beta-blockers and amiodarone have profound beneficial effects in regulating myocardial metabolism. Aiming at decreasing production of toxic metabolites or preventing accumulation of arrhythmogenic lipids perhaps is a good strategy to effectively control arrhythmias. Therefore, a better understanding of the pro-arrhythmic profiles of cardiac metabolites helps to explore a new generation of metabolically oriented anti-arrhythmic medications. In this review, we present several lipid metabolites and summarize their arrhythmogenic characteristics.


Assuntos
Arritmias Cardíacas/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Lisofosfatidilcolinas/metabolismo , Palmitoilcarnitina/metabolismo , Antagonistas Adrenérgicos beta/metabolismo , Antagonistas Adrenérgicos beta/uso terapêutico , Antiarrítmicos/metabolismo , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/patologia , Ceramidas/metabolismo , Humanos , Metabolismo dos Lipídeos , Terapia de Alvo Molecular
13.
PLoS One ; 9(1): e87205, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24498043

RESUMO

BACKGROUND: Intralipid® administration at reperfusion elicits protection against myocardial ischemia-reperfusion injury. However, the underlying mechanisms are not fully understood. METHODS: Sprague-Dawley rat hearts were exposed to 15 min of ischemia and 30 min of reperfusion in the absence or presence of Intralipid® 1% administered at the onset of reperfusion. In separate experiments, the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine was added either alone or with Intralipid®. Left ventricular work and activation of Akt, STAT3, and ERK1/2 were used to evaluate cardioprotection. ROS production was assessed by measuring the loss of aconitase activity and the release of hydrogen peroxide using Amplex Red. Electron transport chain complex activities and proton leak were measured by high-resolution respirometry in permeabilized cardiac fibers. Titration experiments using the fatty acid intermediates of Intralipid® palmitoyl-, oleoyl- and linoleoylcarnitine served to determine concentration-dependent inhibition of complex IV activity and mitochondrial ROS release. RESULTS: Intralipid® enhanced postischemic recovery and activated Akt and Erk1/2, effects that were abolished by the ROS scavenger N-(2-mercaptopropionyl)glycine. Palmitoylcarnitine and linoleoylcarnitine, but not oleoylcarnitine concentration-dependently inhibited complex IV. Only palmitoylcarnitine reached high tissue concentrations during early reperfusion and generated significant ROS by complex IV inhibition. Palmitoylcarnitine (1 µM), administered at reperfusion, also fully mimicked Intralipid®-mediated protection in an N-(2-mercaptopropionyl)-glycine -dependent manner. CONCLUSIONS: Our data describe a new mechanism of postconditioning cardioprotection by the clinically available fat emulsion, Intralipid®. Protection is elicited by the fatty acid intermediate palmitoylcarnitine, and involves inhibition of complex IV, an increase in ROS production and activation of the RISK pathway.


Assuntos
Cardiotônicos/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/metabolismo , Palmitoilcarnitina/metabolismo , Fosfolipídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Óleo de Soja/farmacologia , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Emulsões/farmacologia , Coração/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Função Ventricular Esquerda/efeitos dos fármacos
14.
Redox Biol ; 1: 304-12, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24024165

RESUMO

Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/H2O2 production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoylcarnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site IQ), with small contributions from the flavin site in complex I (site IF) and the quinol oxidation site in complex III (site IIIQo). However, with glutamate plus malate as substrate, site IQ made little or no contribution, and production was shared between site IF, site IIIQo and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site IIF) was a major contributor (together with sites IF and IIIQo), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo.


Assuntos
Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Superóxidos/metabolismo , Animais , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Glicerofosfatos/metabolismo , Malatos/metabolismo , Palmitoilcarnitina/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Ácido Succínico/metabolismo
15.
PLoS Comput Biol ; 9(8): e1003186, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23966849

RESUMO

Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid ß-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the ß-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the ß-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD⁺]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of ß-oxidation and mitochondrial respiration.


Assuntos
Ácidos Graxos/metabolismo , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Feminino , Cinética , Fígado/enzimologia , Fígado/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , NAD/metabolismo , Obesidade/metabolismo , Oxirredução , Palmitoil Coenzima A/metabolismo , Palmitoilcarnitina/metabolismo , Ratos , Ratos Wistar , Reprodutibilidade dos Testes
16.
Am J Physiol Endocrinol Metab ; 305(4): E540-8, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23820622

RESUMO

We hypothesized that insulin alters plasma free fatty acid (FFA) trafficking into intramyocellular (im) long-chain acylcarnitines (imLCAC) and triglycerides (imTG). Overnight-fasted adults (n = 41) received intravenous infusions of [U-¹³C]palmitate (0400-0900 h) and [U-¹³C]oleate (0800-1400 h) to label imTG and imLCAC. A euglycemic-hyperinsulinemic (1.0 mU·kg fat-free mass⁻¹·min⁻¹) clamp (0800-1400 h) and two muscle biopsies (0900 h, 1400 h) were performed. The patterns of [U-¹³C]palmitate incorporation into imTG-palmitate and palmitoylcarnitine were similar to those we reported in overnight postabsorptive adults (saline control); the intramyocellular palmitoylcarnitine enrichment was not different from and correlated with imTG-palmitate enrichment for both the morning (r = 0.38, P = 0.02) and afternoon (r = 0.44, P = 0.006) biopsy samples. Plasma FFA concentrations, flux, and the incorporation of plasma oleate into imTG-oleate during hyperinsulinemia were ~1/10th of that observed in the previous saline control studies (P < 0.001). At the time of the second biopsy, the enrichment in oleoylcarnitine was <25% of that in imTG-oleate and was not correlated with imTG-oleate enrichment. The intramyocellular nonesterified fatty acid-palmitate-to-imTG-palmitate enrichment ratio was greater (P < 0.05) in women than men, suggesting that sex differences in intramyocellular palmitate trafficking may occur under hyperinsulinemic conditions. We conclude that plasma FFA trafficking into imTG during hyperinsulinemia is markedly suppressed, and these newly incorporated FFA fatty acids do not readily enter the LCAC preoxidative pools. Hyperinsulinemia does not seem to inhibit the entry of fatty acids from imTG pools that were labeled under fasting conditions, possibly reflecting the presence of two distinct imTG pools that are differentially regulated by insulin.


Assuntos
Regulação para Baixo , Ácidos Graxos não Esterificados/metabolismo , Hiperinsulinismo/metabolismo , Músculo Esquelético/metabolismo , Adulto , Radioisótopos de Carbono , Carnitina/análogos & derivados , Carnitina/metabolismo , Estudos de Coortes , Regulação para Baixo/efeitos dos fármacos , Ácidos Graxos não Esterificados/administração & dosagem , Ácidos Graxos não Esterificados/sangue , Feminino , Técnica Clamp de Glucose , Humanos , Hiperinsulinismo/sangue , Hiperinsulinismo/etiologia , Infusões Intravenosas , Insulina/efeitos adversos , Insulina/sangue , Insulina/metabolismo , Insulina/farmacologia , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Ácido Oleico/administração & dosagem , Ácido Oleico/sangue , Ácido Oleico/metabolismo , Ácido Palmítico/administração & dosagem , Ácido Palmítico/sangue , Ácido Palmítico/metabolismo , Palmitoilcarnitina/metabolismo , Caracteres Sexuais , Triglicerídeos/metabolismo , Adulto Jovem
17.
Endocrinology ; 154(8): 2650-62, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23709089

RESUMO

In obesity, reduced cardiac glucose uptake and mitochondrial abnormalities are putative causes of cardiac dysfunction. However, high-fat diet (HFD) does not consistently induce cardiac insulin resistance and mitochondrial damage, and recent studies suggest HFD may be cardioprotective. To determine cardiac responses to HFD, we investigated cardiac function, glucose uptake, and mitochondrial respiration in young (3-month-old) and middle-aged (MA) (12-month-old) male Ldlr(-/-) mice fed chow or 3 months HFD to induce obesity, systemic insulin resistance, and hyperinsulinemia. In MA Ldlr(-/-) mice, HFD induced accelerated atherosclerosis and nonalcoholic steatohepatitis, common complications of human obesity. Surprisingly, HFD-fed mice demonstrated increased cardiac glucose uptake, which was most prominent in MA mice, in the absence of cardiac contractile dysfunction or hypertrophy. Moreover, hearts of HFD-fed mice had enhanced mitochondrial oxidation of palmitoyl carnitine, glutamate, and succinate and greater basal insulin signaling compared with those of chow-fed mice, suggesting cardiac insulin sensitivity was maintained, despite systemic insulin resistance. Streptozotocin-induced ablation of insulin production markedly reduced cardiac glucose uptake and mitochondrial dysfunction in HFD-fed, but not in chow-fed, mice. Insulin injection reversed these effects, suggesting that insulin may protect cardiac mitochondria during HFD. These results have implications for cardiac metabolism and preservation of mitochondrial function in obesity.


Assuntos
Glucose/farmacocinética , Hiperinsulinismo/fisiopatologia , Resistência à Insulina/fisiologia , Mitocôndrias Cardíacas/fisiologia , Miocárdio/metabolismo , Fatores Etários , Animais , Aterosclerose/etiologia , Aterosclerose/fisiopatologia , Western Blotting , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/etiologia , Fígado Gorduroso/fisiopatologia , Ácido Glutâmico/metabolismo , Hiperinsulinismo/etiologia , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Obesidade/etiologia , Obesidade/fisiopatologia , Oxirredução , Palmitoilcarnitina/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Estreptozocina/farmacologia , Ácido Succínico/metabolismo
18.
Free Radic Biol Med ; 61: 298-309, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23583329

RESUMO

H2O2 production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H2O2 production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H2O2 production was dominated by complex II (44% from site IIF in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site IF). With added carnitine, H2O2 production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site IIIQo) and 25% from site IF. Thus complex II (site IIF in the forward reaction) is a major source of H2O2 production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H2O2 production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site IIF in the reverse reaction). About half the rest was from site IF, but a significant portion, ∼40pmol H2O2·min(-1)·mg protein(-1), was not from complex I, II, or III and was attributed to the proteins of ß-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ∼200pmol H2O2·min(-1)·mg protein(-1) under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H2O2 productionduring fatty acid oxidation under appropriate conditions.


Assuntos
Ácidos Graxos/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Superóxidos/metabolismo , Animais , Complexo II de Transporte de Elétrons/fisiologia , Feminino , Oxirredução , Consumo de Oxigênio , Palmitoilcarnitina/metabolismo , Ratos , Ratos Wistar
19.
Obesity (Silver Spring) ; 21(3): 516-24, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23404793

RESUMO

OBJECTIVE: Estrogen-related receptors (ERRs) are important regulators of energy metabolism. Here we investigated the hypothesis that ERRγ impacts on differentiation and function of brown adipocytes. DESIGN AND METHODS: We characterize the expression of ERRγ in adipose tissues and cell models and investigate the effects of modulating ERRγ activity on UCP1 gene expression and metabolic features of brown and white adipocytes. RESULTS: ERRγ was preferentially expressed in brown compared to white fat depots, and ERRγ was induced during cold-induced browning of subcutaneous white adipose tissue and brown adipogenesis. Overexpression of ERRγ positively regulated uncoupling protein 1 (UCP1) expression levels during brown adipogenesis. This ERRγ-induced augmentation of UCP1 expression was independent of the presence of peroxisome proliferator-activated receptor coactivator-1 (PGC-1α) but was associated with increased rates of fatty acid oxidation in adrenergically stimulated cells. ERRγ did not influence mitochondrial biogenesis, and its reduced expression in white adipocytes could not explain their low expression level of UCP1. CONCLUSIONS: Through its augmenting effect on expression of UCP1, ERRγ may physiologically be involved in increasing the potential for energy expenditure in brown adipocytes, a function that is becoming of therapeutic interest.


Assuntos
Adipócitos Marrons/metabolismo , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos , Proteínas Mitocondriais/metabolismo , Receptores Estrogênicos/metabolismo , Adipócitos Brancos/metabolismo , Adipogenia , Tecido Adiposo Branco/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Metabolismo Energético , Feminino , Canais Iônicos/genética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lipólise/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Oxirredução , Palmitoilcarnitina/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Estrogênicos/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1
20.
J Lipid Res ; 53(11): 2318-30, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22904346

RESUMO

During cocaine-induced hepatotoxicity, lipid accumulation occurs prior to necrotic cell death in the liver. However, the exact influences of cocaine on the homeostasis of lipid metabolism remain largely unknown. In this study, the progression of subacute hepatotoxicity, including centrilobular necrosis in the liver and elevation of transaminase activity in serum, was observed in a three-day cocaine treatment, accompanying the disruption of triacylglycerol (TAG) turnover. Serum TAG level increased on day 1 of cocaine treatment but remained unchanged afterwards. In contrast, hepatic TAG level was elevated continuously during three days of cocaine treatment and was better correlated with the development of hepatotoxicity. Lipidomic analyses of serum and liver samples revealed time-dependent separation of the control and cocaine-treated mice in multivariate models, which was due to the accumulation of long-chain acylcarnitines together with the disturbances of many bioactive phospholipid species in the cocaine-treated mice. An in vitro function assay confirmed the progressive inhibition of mitochondrial fatty acid oxidation after the cocaine treatment. Cotreatment of fenofibrate significantly increased the expression of peroxisome proliferator-activated receptor α (PPARα)-targeted genes and the mitochondrial fatty acid oxidation activity in the cocaine-treated mice, resulting in the inhibition of cocaine-induced acylcarnitine accumulation and other hepatotoxic effects. Overall, the results from this lipidomics-guided study revealed that the inhibition of fatty acid oxidation plays an important role in cocaine-induced liver injury.


Assuntos
Cocaína/toxicidade , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cromatografia Líquida , Ácidos Graxos , Técnicas In Vitro , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , PPAR alfa/metabolismo , Palmitoilcarnitina/metabolismo
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