Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 398
Filtrar
Mais filtros










Filtros aplicados
Base de dados
Intervalo de ano de publicação
1.
Am J Physiol Cell Physiol ; 317(6): C1324-C1329, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618075

RESUMO

Fatty acid stress can have divergent effects in various cancers. We explored how metabolic and redox flexibility in HepG2 hepatocarcinoma cells mediates protection from palmitoylcarnitine. HepG2 cells, along with HCT 116 and HT29 colorectal cancer cells were incubated with 100 µM palmitoylcarnitine for up to 48 h. Mitochondrial H2O2 emission, glutathione, and cell survival were assessed in HT29 and HepG2 cells. 100 µM palmitoylcarnitine promoted early growth in HepG2 cells by ~8% after 48 h versus decreased cell survival observed in HT29 and HCT 116 cells. Palmitoylcarnitine increased mitochondrial respiration at physiological and maximal concentrations of ADP, while lowering cellular lactate content in HepG2 cells, suggesting a switch to mitochondrial metabolism. HepG2 cell growth was associated with an early increase in H2O2 emission by 10 min, followed by a decrease in H2O2 at 24 h that corresponded with increased glutathione content, suggesting a redox-based compensatory mechanism. In contrast, abrogation of HT29 cell proliferation was related to decreased mitochondrial respiration (likely due to cell death) and decreased glutathione. Concurrent glutathione depletion with BSO prevented palmitoylcarnitine-induced growth in HepG2 cells, indicating that glutathione was critical for promoting growth following palmitoylcarnitine. Inhibiting UCP2 with genipin sensitized HepG2 cells to palmitoylcarnitine, suggesting that activation of UCP2 may be a 2nd redox-based mechanism conferring protection. These findings suggest that HepG2 cells possess inherent metabolic and redox flexibility relative to HT29 cells that confers protection from palmitoylcarnitine-induced stress via adaptive increases in mitochondrial respiratory control, glutathione buffering, and induction of UCP2.


Assuntos
Butionina Sulfoximina/farmacologia , Glutationa/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/efeitos dos fármacos , Palmitoilcarnitina/farmacologia , Proteína Desacopladora 2/genética , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Células HCT116 , Células HT29 , Células Hep G2 , Humanos , Mitocôndrias/metabolismo , Especificidade de Órgãos , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo , Proteína Desacopladora 2/agonistas , Proteína Desacopladora 2/metabolismo
2.
Am J Physiol Cell Physiol ; 317(6): C1278-C1288, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31483701

RESUMO

Previous evidence suggests that palmitoylcarnitine incubations trigger mitochondrial-mediated apoptosis in HT29 colorectal adenocarcinoma cells, yet nontransformed cells appear insensitive. The mechanism by which palmitoylcarnitine induces cancer cell death is unclear. The purpose of this investigation was to examine the relationship between mitochondrial kinetics and glutathione buffering in determining the effect of palmitoylcarnitine on cell survival. HT29 and HCT 116 colorectal adenocarcinoma cells, CCD 841 nontransformed colon cells, and MCF7 breast adenocarcinoma cells were exposed to 0 µM, 50 µM, and 100 µM palmitoylcarnitine for 24-48 h. HCT 116 and HT29 cells showed decreased cell survival following palmitoylcarnitine compared with CCD 841 cells. Palmitoylcarnitine stimulated H2O2 emission in HT29 and CCD 841 cells but increased it to a greater level in HT29 cells due largely to a higher basal H2O2 emission. This greater H2O2 emission was associated with lower glutathione buffering capacity and caspase-3 activation in HT29 cells. The glutathione-depleting agent buthionine sulfoximine sensitized CCD 841 cells and further sensitized HT29 cells to palmitoylcarnitine-induced decreases in cell survival. MCF7 cells did not produce H2O2 when exposed to palmitoylcarnitine and were able to maintain glutathione levels. Furthermore, HT29 cells demonstrated the lowest mitochondrial oxidative kinetics vs. CCD 841 and MCF7 cells. The results demonstrate that colorectal cancer is sensitive to palmitoylcarnitine due in part to an inability to prevent oxidative stress through glutathione-redox coupling, thereby rendering the cells sensitive to elevations in H2O2. These findings suggest that the relationship between inherent metabolic capacities and redox regulation is altered early in response to palmitoylcarnitine.


Assuntos
Antineoplásicos/farmacologia , Butionina Sulfoximina/farmacologia , Células Epiteliais/efeitos dos fármacos , Glutationa/antagonistas & inibidores , Peróxido de Hidrogênio/agonistas , Palmitoilcarnitina/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Células HCT116 , Células HT29 , Humanos , Peróxido de Hidrogênio/metabolismo , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Especificidade de Órgãos , Oxirredução , Estresse Oxidativo , Cultura Primária de Células
3.
J Mol Neurosci ; 69(1): 39-48, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31321646

RESUMO

Neurosyphilis is a chronic central nervous system infectious disease caused by Treponema pallidum. Our aim was to study the metabolic profiling in the cerebrospinal fluid of neurosyphilis patients and identify specific potential biomarkers. Fifteen cerebrospinal fluid samples from neurosyphilis patients and 14 non-neurosyphilis samples were analyzed by liquid chromatography-mass spectrometer (LC-MS). The LC-MS data were preprocessed by supervised pattern recognition to obtain diagnostic models. Both orthogonal projections to a latent structures discriminant analysis (OPLS-DA) and a t test were used to obtain specific metabolites for neurosyphilis. LC-MS data showed that the metabolites in cerebrospinal fluid (CSF) from neurosyphilis are different from the non-neurosyphilis group. The OPLS-DA model parameters R2Y and Q2Y are both more than 0.7 and indicated a satisfactory diagnostic performance. Bilirubin, L-histidine, prostaglandin E2, alpha-kamlolenic acid, and butyryl-L-carnitine and palmitoyl-L-carnitine were identified as novel potential biomarkers for neurosyphilis. The metabolic study of CSF may provide a new way to explore the pathogenesis of neurosyphilis.


Assuntos
Metaboloma , Neurossífilis/líquido cefalorraquidiano , Adulto , Bilirrubina/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Carnitina/análogos & derivados , Carnitina/líquido cefalorraquidiano , Dinoprostona/líquido cefalorraquidiano , Ácidos Graxos Insaturados/líquido cefalorraquidiano , Feminino , Histidina/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-Idade , Palmitoilcarnitina/líquido cefalorraquidiano
5.
Nat Cell Biol ; 20(9): 1043-1051, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30154550

RESUMO

The target of rapamycin complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation following increased PM tension involves redistribution of the Slm1 and 2 paralogues from PM invaginations known as eisosomes into membrane compartments containing TORC2. How Slm1/2 relocalization is triggered, and if/how this plays a role in TORC2 inactivation with decreased PM tension, is unknown. Using osmotic shocks and palmitoylcarnitine as orthogonal tools to manipulate PM tension, we demonstrate that decreased PM tension triggers spontaneous, energy-independent reorganization of pre-existing phosphatidylinositol-4,5-bisphosphate into discrete invaginated membrane domains, which cluster and inactivate TORC2. These results demonstrate that increased and decreased membrane tension are sensed through different mechanisms, highlighting a role for membrane lipid phase separation in mechanotransduction.


Assuntos
Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mecanotransdução Celular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas do Citoesqueleto , Ativação Enzimática , Proteínas Fúngicas/genética , Cinética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Mecanotransdução Celular/efeitos dos fármacos , Pressão Osmótica , Palmitoilcarnitina/farmacologia , Transporte Proteico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos
6.
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
7.
J Orthop Sci ; 23(6): 878-883, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30075996

RESUMO

BACKGROUND: Metabolomics is one of the "omics" technologies, and is a comprehensive analysis of small molecule metabolites which include amino acid, nucleotides, carbohydrates and fatty acid. The purpose of the present study was to compare the differences of metabolite profiling between patients with ossification of the posterior longitudinal ligament (OPLL) and control subjects. METHODS: We analyzed plasma metabolites in patients with cervical OPLL (n = 10) and in control subjects (n = 10). Ionic metabolites were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and lipophilic metabolites were analyzed using liquid chromatograph time-of-flight mass spectrometry (LC-TOFMS). RESULTS: A total of 259 metabolites (144 metabolites in CE-TOFMS and 115 metabolites in LC-TOFMS) were detected. Among the 259 metabolites, six metabolites, namely acylcarnitine (AC) (14:0), palmitoylcarnitine, AC (18:2), fatty acid (FA) (24:2), thyroxine, thiaproline were significantly larger in OPLL group, even in analyzes excluding patients with diabetes mellitus and hyperlipidemia. CONCLUSIONS: We examined the metabolite profiling in patients with OPLL for the first time and detected six metabolites showing suggestive association with disease. These results of the present study could lead to new insights into clarifying the molecular pathomechanisms of OPLL.


Assuntos
Vértebras Cervicais , Metaboloma , Ossificação do Ligamento Longitudinal Posterior/sangue , Idoso , Carnitina/análogos & derivados , Carnitina/sangue , Estudos de Casos e Controles , Ácidos Graxos/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Palmitoilcarnitina/sangue , Tiazolidinas/sangue , Tiroxina/sangue
8.
PLoS One ; 13(8): e0201591, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30133480

RESUMO

BACKGROUND: Patients on dialysis are in a chronic carnitine-deficient state. This condition may be associated with abnormalities of the fatty acid and organic acid metabolisms. Carnitine is required for ß-oxidation of the long-chain fatty acids; therefore, carnitine deficiency decreases the efficiency of ATP synthesis and may incur death. However, the details of this association remain unknown. We examined the relationship between ß-oxidation efficiency represented by the carnitine profile and 4-year all-cause mortality in hemodialysis patients. METHODS: The carnitine profiles of 122 hemodialysis patients were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The associations between the 4-year all-cause mortality and carnitine profile as well as the clinical backgrounds of the patients were investigated. A survival analysis was conducted by the Kaplan-Meier survival method and multivariable Cox proportional hazard analysis. The bootstrap method was performed to confirm the stability and robustness of our model. RESULTS: Of the 122 subjects analyzed, 111 were selected and 24 died during the observation period. Stepwise multivariable Cox regression demonstrated that diabetes state [Hazard ratio (95% confidence interval), 4.981 (2.107-11.77)], age [HR (95% CI), 1.052 (1.014-1.091)], and the acetylcarnitine/(palmitoylcarnitine+octadecenoylcarnitine) [C2/(C16+C18:1)] ratio [HR (95% CI), 0.937 (0.904-0.971)] were independent significant factors of 4-year all-cause mortality. The bootstrap method confirmed the significance of these three factors. CONCLUSION: The 4-year all-cause mortality negatively correlated with the C2/(C16+C18:1) ratio. Improvement of the impaired ß-oxidation state after L-carnitine administration may ameliorate prognosis.


Assuntos
Carnitina/análogos & derivados , Carnitina/análise , Diálise Renal/mortalidade , Acetilcarnitina/análise , Idoso , Causas de Morte , Cromatografia Líquida , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Palmitoilcarnitina/análise , Modelos de Riscos Proporcionais , Análise de Sobrevida , Espectrometria de Massas em Tandem
9.
Can J Diabetes ; 42(4): 382-388.e1, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29129455

RESUMO

OBJECTIVES: Enhanced mitochondrial fatty acid utilization is known to increase radical oxidative stress and induce insulin resistance. An increased level of plasma acylcarnitine (AC) has been proposed to indicate mitochondrial energy substrate overload, a possible mechanism leading to insulin resistance. The aim of our study was to determine fasting and postprandial plasma acetyl-carnitine (AC2:0), palmitoyl-carnitine (AC16:0), oleoyl-carnitine (AC18:1) and linoleoyl-carnitine (AC18:2) levels and their relationships with plasma nonesterified fatty acid appearance and oxidation rates and insulin sensitivity in participants with type 2 diabetes and normoglycemic offspring of 2 parents with type 2 diabetes (FH+) compared to healthy participants without family histories of type 2 diabetes (FH-). METHODS: All participants underwent 3 metabolic protocols: 1) a euglycemic hyperinsulinemic clamp at fasting; 2) a 6-hour steady-state oral standard liquid meal and 3) an identical 6-hour steady-state meal intake study with a euglycemic hyperinsulinemic clamp. AC levels were measured by liquid chromatography with tandem mass spectrometry, and fatty acid oxidation (FAO) rates were measured by stable isotopic tracer techniques with indirect respiratory calorimetry. RESULTS: During the insulin clamp at fasting, AC16:0 was significantly higher in the group with type 2 diabetes vs. FH- (p<0.05). In the postprandial state, AC2:0, AC16:0 and AC18:1 decreased significantly, but this reduction was blunted in type 2 diabetes, even during normalization of postprandial glucose levels during the insulin clamp. Fasting AC16:0 correlated with FAO (ρ=+0.604; p=0.0002); triacylglycerol (ρ=+0.427; p<0.02) and waist circumference (ρ=+0.416; p=0.02). CONCLUSIONS: Spillover of AC occurs in type 2 diabetes but is not fully established in FH+. AC16:0 can be a useful biomarker of excessive FAO.


Assuntos
Biomarcadores/sangue , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Metabolismo dos Lipídeos , Palmitoilcarnitina/sangue , Adulto , Carnitina/análogos & derivados , Carnitina/farmacologia , Jejum/sangue , Feminino , Humanos , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Oxirredução/efeitos dos fármacos , Período Pós-Prandial , Adulto Jovem
10.
Mol Med Rep ; 16(5): 6828-6836, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28901489

RESUMO

Hypoxic preconditioning (HPC) is well­known to exert a protective effect against hypoxic injury; however, the underlying molecular mechanism remains unclear. The present study utilized a serum metabolomics approach to detect the alterations associated with HPC. In the present study, an animal model of HPC was established by exposing adult BALB/c mice to acute repetitive hypoxia four times. The serum samples were collected by orbital blood sampling. Metabolite profiling was performed using ultra­performance liquid chromatography­quadrupole time­of­flight mass spectrometry (UPLC­QTOFMS), in conjunction with univariate and multivariate statistical analyses. The results of the present study confirmed that the HPC mouse model was established and refined, suggesting significant differences between the control and HPC groups at the molecular levels. HPC caused significant metabolic alterations, as represented by the significant upregulation of valine, methionine, tyrosine, isoleucine, phenylalanine, lysophosphatidylcholine (LysoPC; 16:1), LysoPC (22:6), linoelaidylcarnitine, palmitoylcarnitine, octadecenoylcarnitine, taurine, arachidonic acid, linoleic acid, oleic acid and palmitic acid, and the downregulation of acetylcarnitine, malate, citrate and succinate. Using MetaboAnalyst 3.0, a number of key metabolic pathways were observed to be acutely perturbed, including valine, leucine and isoleucine biosynthesis, in addition to taurine, hypotaurine, phenylalanine, linoleic acid and arachidonic acid metabolism. The results of the present study provided novel insights into the mechanisms involved in the acclimatization of organisms to hypoxia, and demonstrated the protective mechanism of HPC.


Assuntos
Cromatografia Líquida de Alta Pressão , Hipóxia , Espectrometria de Massas por Ionização por Electrospray , Aminoácidos/sangue , Animais , Análise Discriminante , Modelos Animais de Doenças , Precondicionamento Isquêmico , Análise dos Mínimos Quadrados , Lisofosfatidilcolinas/sangue , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos BALB C , Palmitoilcarnitina/sangue , Análise de Componente Principal
11.
Biofactors ; 43(5): 718-730, 2017 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-28759135

RESUMO

Acylcarnitine accumulation has been linked to perturbations in energy metabolism pathways. In this study, we demonstrate that long-chain (LC) acylcarnitines are active metabolites involved in the regulation of glucose metabolism in vivo. Single-dose administration of palmitoylcarnitine (PC) in fed mice induced marked insulin insensitivity, decreased glucose uptake in muscles, and elevated blood glucose levels. Increase in the content of LC acylcarnitine induced insulin resistance by impairing Akt phosphorylation at Ser473. The long-term administration of PC using slow-release osmotic minipumps induced marked hyperinsulinemia, insulin resistance, and glucose intolerance, suggesting that the permanent accumulation of LC acylcarnitines can accelerate the progression of insulin resistance. The decrease of acylcarnitine content significantly improved glucose tolerance in a mouse model of diet-induced glucose intolerance. In conclusion, we show that the physiological increase in content of acylcarnitines ensures the transition from a fed to fasted state in order to limit glucose metabolism in the fasted state. In the fed state, the inability of insulin to inhibit LC acylcarnitine production induces disturbances in glucose uptake and metabolism. The reduction of acylcarnitine content could be an effective strategy to improve insulin sensitivity. © 2017 BioFactors, 43(5):718-730, 2017.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Resistência à Insulina/genética , Músculo Esquelético/metabolismo , Palmitoilcarnitina/administração & dosagem , Animais , Glicemia/efeitos dos fármacos , Metabolismo dos Carboidratos/efeitos dos fármacos , Carnitina/análogos & derivados , Carnitina/metabolismo , Gorduras na Dieta , Glucose/metabolismo , Humanos , Insulina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Músculo Esquelético/patologia
12.
Mol Genet Metab ; 122(3): 67-75, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28801073

RESUMO

BACKGROUND: Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder (FAOD). However, newborn screening (NBS) for this potentially fatal disease has not been established partly because reliable indices are not available. METHODS: We diagnosed CPT II deficiency in a 7-month-old boy presenting with hypoglycemic encephalopathy, which apparently had been missed in the NBS using C16 and C18:1 concentrations as indices. By referring to his acylcarnitine profile from the NBS, we adopted the (C16+C18:1)/C2 ratio (cutoff 0.62) and C16 concentration (cutoff 3.0nmol/mL) as alternative indices for CPT II deficiency such that an analysis of a dried blood specimen collected at postnatal day five retroactively yielded the correct diagnosis. Thereafter, positive cases were assessed by measuring (1) the fatty acid oxidation ability of intact lymphocytes and/or (2) CPT II activity in the lysates of lymphocytes. The diagnoses were then further confirmed by genetic analysis. RESULTS: The disease was diagnosed in seven of 21 newborns suspected of having CPT II deficiency based on NBS. We also analyzed the false-negative patient and five symptomatic patients for comparison. Values for the NBS indices of the false-negative, symptomatic patient were lower than those of the seven affected newborns. Although it was difficult to differentiate the false-negative patient from heterozygous carriers and false-positive subjects, the fatty acid oxidation ability of the lymphocytes and CPT II activity clearly confirmed the diagnosis. Among several other indices proposed previously, C14/C3 completely differentiated the seven NBS-positive patients and the false-negative patient from the heterozygous carriers and the false-positive subjects. Genetic analysis revealed 16 kinds of variant alleles. The most prevalent, detected in ten alleles in nine patients from eight families, was c.1148T>A (p.F383Y), a finding in line with those of several previous reports on Japanese patients. CONCLUSIONS: These findings suggested that CPT II deficiency can be screened by using (C16+C18:1)/C2 and C16 as indices. An appropriate cutoff level is required to achieve adequate sensitivity albeit at the cost of a considerable increase in the false-positive rate, which might be reduced by using additional indices such as C14/C3.


Assuntos
Carnitina O-Palmitoiltransferase/análise , Carnitina O-Palmitoiltransferase/deficiência , Erros Inatos do Metabolismo/diagnóstico , Triagem Neonatal , Palmitoilcarnitina/análise , Alelos , Carnitina O-Palmitoiltransferase/genética , Teste em Amostras de Sangue Seco/métodos , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Humanos , Hipoglicemia/complicações , Lactente , Recém-Nascido , Masculino , Erros Inatos do Metabolismo/genética , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem
13.
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
14.
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
15.
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
16.
Clin Nutr ; 36(5): 1310-1319, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-27624997

RESUMO

BACKGROUND: Circulating acyl-carnitines (acyl-CNTs) are associated with insulin resistance (IR) and type 2 diabetes (T2D) in both rodents and humans. However, the mechanisms whereby circulating acyl-CNTs are increased in these conditions and their role in whole-body metabolism remains unknown. The purpose of this study was to determine if, in humans, blood cells contribute in production of circulating acyl-CNTs and associate with whole-body fat metabolism. METHODS AND RESULTS: Eight non-diabetic healthy women (age: 47 ± 19 y; BMI: 26 ± 1 kg·m-2) underwent stable isotope tracer infusion and hyperinsulinemic-euglycemic clamp study to determine in vivo whole-body fatty acid flux and insulin sensitivity. Blood samples collected at baseline (0 min) and after 3 h of clamp were used to determine the synthesis rate of palmitoyl-carnitine (palmitoyl-CNT) in vitro. The fractional synthesis rate of palmitoyl-CNT was significantly higher during hyperinsulinemia (0.788 ± 0.084 vs. 0.318 ± 0.012%·hr-1, p = 0.001); however, the absolute synthesis rate (ASR) did not differ between the periods (p = 0.809) due to ∼30% decrease in blood palmitoyl-CNT concentration (p = 0.189) during hyperinsulinemia. The ASR of palmitoyl-CNT significantly correlated with the concentration of acyl-CNTs in basal (r = 0.992, p < 0.001) and insulin (r = 0.919, p = 0.001) periods; and the basal ASR significantly correlated with plasma palmitate oxidation (r = 0.764, p = 0.027). CONCLUSION: In women, blood cells contribute to plasma acyl-CNT levels and the acyl-CNT production is linked to plasma palmitate oxidation, a marker of whole-body fat metabolism. Future studies are needed to confirm the role of blood cells in acyl-CNT and lipid metabolism under different physiological (i.e., in response to meal) and pathological (i.e., hyperlipidemia, IR and T2D) conditions.


Assuntos
Células Sanguíneas/metabolismo , Carnitina/análogos & derivados , Sobrepeso/sangue , Palmitoilcarnitina/biossíntese , Adulto , Idoso , Glicemia/metabolismo , Índice de Massa Corporal , Carnitina/sangue , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Hiperinsulinismo/sangue , Insulina/sangue , Resistência à Insulina , Metabolismo dos Lipídeos , Pessoa de Meia-Idade , Oxirredução , Palmitatos/sangue , Palmitoilcarnitina/sangue
17.
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
18.
Int J Epidemiol ; 45(5): 1507-1516, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27694567

RESUMO

BACKGROUND: Metabolomics studies in Caucasians have identified a number of novel metabolites in association with the risk of type 2 diabetes (T2D). However, few prospective metabolomic studies are available in Chinese populations. In the present study, we sought to identify novel metabolites consistently associated with incident T2D in two independent cohorts of Chinese adults. METHODS: We performed targeted metabolomics (52 metabolites) of fasting plasma samples by liquid chromatography-mass spectrometry in two prospective case-control studies nested within the Dongfeng-Tongji (DFTJ) cohort and Jiangsu Non-communicable Disease (JSNCD) cohort. After following for 4.61 ± 0.15 and 7.57 ± 1.13 years, respectively, 1039 and 520 eligible participants developed incident T2D in these two cohorts, and controls were 1:1 matched with cases by age (± 5 years) and sex. Multivariate conditional logistic regression models were constructed to identify metabolites associated with future T2D risk in both cohorts. RESULTS: We identified four metabolites consistently associated with an increased risk of developing T2D in the two cohorts, including alanine, phenylalanine, tyrosine and palmitoylcarnitine. In the meta-analysis of two cohorts, the odds ratios (95% confidence intervals, CIs) comparing extreme quartiles were 1.79 (1.32-2.42) for alanine, 1.91 (1.41-2.60) for phenylalanine, 1.85 (1.37-2.48) for tyrosine and 1.63 (1.21-2.20) for palmitoylcarnitine (all Ptrend ≤ 0.01). CONCLUSIONS: We confirmed the association of alanine, phenylalanine and tyrosine with future T2D risk and further identified palmitoylcarnitine as a novel metabolic marker of incident T2D in two prospective cohorts of Chinese adults. Our findings might provide new aetiological insight into the development of T2D.


Assuntos
Aminoácidos/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/epidemiologia , Metabolômica/métodos , Palmitoilcarnitina/sangue , Adulto , Idoso , Biomarcadores/sangue , Glicemia/metabolismo , Estudos de Casos e Controles , China/epidemiologia , Cromatografia Líquida , Feminino , Humanos , Modelos Logísticos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Estudos Prospectivos , Fatores de Risco
19.
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
20.
Eur J Vasc Endovasc Surg ; 52(1): 5-10, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27231199

RESUMO

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/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA