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Biologia Computacional/métodos , Processamento de Imagem Assistida por Computador/métodos , Metadados , Algoritmos , Animais , Congressos como Assunto , Microscopia Crioeletrônica/métodos , Mineração de Dados/métodos , Bases de Dados Factuais , Diagnóstico por Imagem/métodos , Humanos , Microscopia , SoftwareRESUMO
CONTEXT: Mupirocin (BactrobanR) is widely prescribed for intra-nasal decolonisation of MRSA for in-patients awaiting surgery or self-medicated for out-patients although adherence for the latter is not monitored. Non-adherence is a widespread pharmaceutical problem but could encourage selection of antibiotic resistance. Mupirocin is only a topical antibiotic because it decomposes in stomach acidity to monic acid A, but this has not previously been exploited as a biomarker for clinical intra-nasal medication. MATERIALS AND METHODS: Urine from three catheterised patients in two London hospitals during and after mupirocin medication, was passed through Waters Oasis cartridges to isolate organic acids. Sensitive LC-MS-MS analysis for monic acid A in methanolic eluate has been developed to identify â¼10 pg. RESULTS: Monic acid A was quantified in all samples from one patient, translating into 6-46 ng from 12 mg mupirocin, assuming 1 L daily urine output. However, no urinary monic acid A was detected for two other patients. DISCUSSION AND CONCLUSIONS: Consistent occurrence of monic acid A in urine of one mupirocin patient shows for the first time that antibiotic distribution across nasal mucous membranes had generally been maintained during medication. In contrast, consistent absence in the two other patients requires wider study in hospital.
Assuntos
Biomarcadores/urina , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/urina , Cromatografia Líquida , Feminino , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Mupirocina/administração & dosagem , Piranos/urina , Infecções Estafilocócicas/tratamento farmacológico , Espectrometria de Massas em TandemRESUMO
Drug-induced phospholipidosis (DIPL) is characterized by an increase in the phospholipid content of the cell and the accumulation of drugs and lipids inside the lysosomes of affected tissues, including in the liver. Although of uncertain pathological significance for patients, the condition remains a major impediment for the clinical development of new drugs. Human Sandhoff disease (SD) is caused by inherited defects of the ß subunit of lysosomal ß-hexosaminidases (Hex) A and B, leading to a large array of symptoms, including neurodegeneration and ultimately death by the age of 4 in its most common form. The substrates of Hex A and B, gangliosides GM2 and GA2, accumulate inside the lysosomes of the CNS and in peripheral organs. Given that both DIPL and SD are associated with lysosomes and lipid metabolism in general, we measured the hepatic lipid profiles in rodent models of these two conditions using untargeted LC/MS to examine potential commonalities. Both model systems shared a number of perturbed lipid pathways, notably those involving metabolism of cholesteryl esters, lysophosphatidylcholines, bis(monoacylglycero)phosphates, and ceramides. We report here profound alterations in lipid metabolism in the SD liver. In addition, DIPL induced a wide range of lipid changes not previously observed in the liver, highlighting similarities with those detected in the model of SD and raising concerns that these lipid changes may be associated with underlying pathology associated with lysosomal storage disorders.
Assuntos
Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/metabolismo , Fosfolipídeos/metabolismo , Doença de Sandhoff/induzido quimicamente , Doença de Sandhoff/metabolismo , Animais , Modelos Animais de Doenças , Fígado/patologia , Lisossomos/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Doença de Sandhoff/patologiaRESUMO
Metabolic traits are molecular phenotypes that can drive clinical phenotypes and may predict disease progression. Here, we report results from a metabolome- and genome-wide association study on (1)H-NMR urine metabolic profiles. The study was conducted within an untargeted approach, employing a novel method for compound identification. From our discovery cohort of 835 Caucasian individuals who participated in the CoLaus study, we identified 139 suggestively significant (P<5×10(-8)) and independent associations between single nucleotide polymorphisms (SNP) and metabolome features. Fifty-six of these associations replicated in the TasteSensomics cohort, comprising 601 individuals from São Paulo of vastly diverse ethnic background. They correspond to eleven gene-metabolite associations, six of which had been previously identified in the urine metabolome and three in the serum metabolome. Our key novel findings are the associations of two SNPs with NMR spectral signatures pointing to fucose (rs492602, Pâ=â6.9×10(-44)) and lysine (rs8101881, Pâ=â1.2×10(-33)), respectively. Fine-mapping of the first locus pinpointed the FUT2 gene, which encodes a fucosyltransferase enzyme and has previously been associated with Crohn's disease. This implicates fucose as a potential prognostic disease marker, for which there is already published evidence from a mouse model. The second SNP lies within the SLC7A9 gene, rare mutations of which have been linked to severe kidney damage. The replication of previous associations and our new discoveries demonstrate the potential of untargeted metabolomics GWAS to robustly identify molecular disease markers.
Assuntos
Metaboloma/genética , Metabolômica , Polimorfismo de Nucleotídeo Único/genética , Urina , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Doença de Crohn/genética , Doença de Crohn/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Nefropatias/genética , Nefropatias/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
One aim of systems toxicology is to deliver mechanistic, mathematically rigorous, models integrating biochemical and pharmacological processes that result in toxicity to enhance the assessment of the risk posed to humans by drugs and other xenobiotics. The benefits of such 'in silico' models would be in enabling the rapid and robust prediction of the effects of compounds over a range of exposures, improving in vitro-in vivo correlations and the translation from preclinical species to humans. Systems toxicology models of organ toxicities that result in high attrition rates during drug discovery and development, or post-marketing withdrawals (e.g., drug-induced liver injury (DILI)) should facilitate the discovery of safe new drugs. Here, systems toxicology as applied to the effects of paracetamol (acetaminophen, N-acetyl-para-aminophenol (APAP)) is used to exemplify the potential of the approach.
Assuntos
Acetaminofen/metabolismo , Glutationa/metabolismo , Modelos Biológicos , Acetaminofen/toxicidade , Animais , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Simulação por Computador , Desenho de Fármacos , Descoberta de Drogas/métodos , Humanos , Toxicologia/métodos , Xenobióticos/toxicidadeRESUMO
Sandhoff disease (SD) is a lysosomal disease caused by mutations in the gene coding for the ß subunit of ß-hexosaminidase, leading to deficiency in the enzymes ß-hexosaminidase (HEX) A and B. SD is characterised by an accumulation of gangliosides and related glycolipids, mainly in the central nervous system, and progressive neurodegeneration. The underlying cellular mechanisms leading to neurodegeneration and the contribution of inflammation in SD remain undefined. The aim of the present study was to measure global changes in metabolism over time that might reveal novel molecular pathways of disease. We used liquid chromatography-mass spectrometry and 1H Nuclear Magnetic Resonance spectroscopy to profile intact lipids and aqueous metabolites, respectively. We examined spinal cord and cerebrum from healthy and Hexb -/- mice, a mouse model of SD, at ages one, two, three and four months. We report decreased concentrations in lipids typical of the myelin sheath, galactosylceramides and plasmalogen-phosphatidylethanolamines, suggesting that reduced synthesis of myelin lipids is an early event in the development of disease pathology. Reduction in neuronal density is progressive, as demonstrated by decreased concentrations of N-acetylaspartate and amino acid neurotransmitters. Finally, microglial activation, indicated by increased amounts of myo-inositol correlates closely with the late symptomatic phases of the disease.
RESUMO
The metabolic fate, toxicity, and effects on endogenous metabolism of paracetamol (acetaminophen, APAP) in 22 female Landrace cross large white pigs were evaluated in a model of acute liver failure (ALF). Anesthetized pigs were initially dosed at 250 mg/kg via an oroduodenal tube with APAP serum concentrations maintained above 300 mg/l using maintenance doses of 0.5-4 g/h until ALF. Studies were undertaken to determine both the metabolic fate of APAP and its effects on the endogenous metabolic phenotype of ALF in using 1H NMR spectroscopy. Increased concentrations of citrate combined with pre-ALF increases in circulating lactate, pyruvate, and alanine in plasma suggest mitochondrial dysfunction and a switch in hepatic energy metabolism to glycolysis in response to APAP treatment. A specific liquid chromatography-tandem mass spectrometry assay was used to quantify APAP and metabolites. The major circulating and urinary metabolite of APAP was the phenolic glucuronide (APAP-G), followed by p-aminophenol glucuronide (PAP-G) formed from N-deacetylated APAP. The PAP produced by N-deacetylation was the likely cause of the methemoglobinemia and kidney toxicity observed in this, and previous, studies in the pig. The phenolic sulfate of APAP, and the glutathione-derived metabolites of the drug were only found as minor components (with the cysteinyl conjugate detected but not the mercapturate). Given its low sulfation, combined with significant capacity for N-deacetylation the pig may represent a poor translational model for toxicology studies for compounds undergoing significant metabolism by sulfation, or which contain amide bonds which when hydrolyzed to unmask an aniline lead to toxicity. However, the pig may provide a useful model where extensive amide hydrolysis is seen for drugs or environmental chemicals in humans, but not in, eg, the rat and dog which are the preclinical species normally employed for safety assessment.
Assuntos
Acetaminofen/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Falência Hepática/metabolismo , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Acetaminofen/toxicidade , Animais , Biotransformação , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Cromatografia Líquida , Modelos Animais de Doenças , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Falência Hepática/induzido quimicamente , Falência Hepática/patologia , Metaboloma , Metabolômica , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Espectroscopia de Prótons por Ressonância Magnética , Sus scrofa , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
The 3T3-L1 murine cell line is a robust and widely used model for the study of adipogenesis and processes occurring in mature adipocytes. The fibroblastic like cells can be induced by hormones to differentiate into mature adipocytes. In this study, the metabolic phenotype associated with differentiation of the 3T3-L1 cell line has been studied using gas chromatography-mass spectrometry, (1)H nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry, direct infusion-mass spectrometry, and 13C substrate labeling in conjunction with multivariate statistics. The changes in metabolite concentrations at distinct periods during differentiation have been defined including alterations in the TCA cycle, glycolysis, the production of odd chain fatty acids by alpha-oxidation, fatty acid synthesis, fatty acid desaturation, polyamine biosynthesis, and trans-esterification to produce complex lipids. The metabolic changes induced during differentiation of the 3T3-L1 cell line were then compared with the metabolic differences between pre- and postdifferentiation primary adipocytes. These metabolic alterations reflect the changing role of the 3T3-L1 cells during differentiation, as well as possibly providing metabolic triggers to stimulate the processes which occur during differentiation.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Modelos Biológicos , Células 3T3-L1 , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos , Citoplasma/metabolismo , Análise Discriminante , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Análise dos Mínimos Quadrados , Metabolismo dos Lipídeos , Espectroscopia de Ressonância Magnética , Camundongos , Fenótipo , Coloração e Rotulagem , Triglicerídeos/metabolismoAssuntos
Metabolismo Energético , Medicina/métodos , Fenótipo , Animais , Genoma Humano , Humanos , Estilo de Vida , Ratos , Resultado do TratamentoRESUMO
With the rise of systems biology, a number of approaches have been developed to globally profile a tier of organization in a cell, tissue or organism. Metabolomics is an approach that attempts to profile all the metabolites in a biological matrix. One of the major challenges of this approach, as with other 'omic' technologies, is that the metabolome is context-dependent and will vary with pathology, developmental stage and environmental factors. Thus, the possibility of globally profiling the metabolome of an organism is a genuine analytical challenge, as by definition this must also take into consideration all relevant factors that influence metabolism. Despite these challenges, the approach has already been applied to understand the metabolism in a range of animal models, and has more recently started to be projected into the clinical situation. In this review, the technologies currently being used in metabolomics will be assessed prior to examining their use to study diseases related to the metabolic syndrome, including Type II diabetes, obesity, cardiovascular disease and fatty liver disease.
Assuntos
Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Dislipidemias/etiologia , Dislipidemias/genética , Genômica/tendências , Metabolismo dos Lipídeos/genética , Metabolismo/genética , Obesidade/genética , Obesidade/metabolismo , Animais , Doenças Cardiovasculares/genética , Ensaios Clínicos como Assunto , Diabetes Mellitus Tipo 2/genética , Fígado Gorduroso/genética , Humanos , Síndrome Metabólica/genética , Obesidade/complicações , Valores de Referência , Projetos de PesquisaRESUMO
This article contains mass spectrometry (MS) data investigating small molecule changes as an effect of a triple peroxisome proliferator-activated receptor (PPAR-pan) agonist GW625019 in the liver as described in the manuscript (Ament et al., 2016) [1]. Samples were measured using gas chromatography-mass spectrometry (GC-MS) for total fatty acid content, and liquid chromatography-mass spectrometry (LC-MS) to measure intact lipids, carnitines and selected aqueous metabolites and eicosanoids. Data files comprise of Excel (Microsoft, WA, USA) spreadsheets of identified metabolites and their area ratio values for total fatty acids, carnitines, aqueous metabolites, and eicosanoids where the intensity of the analytes were normalised to the intensity of the internal standard. In the case of open profiling intact lipid data, the Excel file contains area ratio values of retention time and mass to charge ratio pairs; again, the area ratio values were calculated by normalising to the intensity of the internal standard. It should be noted that several metabolic changes are potentially indirect (secondary, tertiary and ensuing changes).
RESUMO
The peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear receptors that regulate cellular homoeostasis and metabolism. PPARs control the expression of genes involved in fatty-acid and lipid metabolism. Despite evidence showing beneficial effects of their activation in the treatment of metabolic diseases, particularly dyslipidaemias and type 2 diabetes, PPAR agonists have also been associated with a variety of side effects and adverse pathological changes. Agonists have been developed that simultaneously activate the three PPAR receptors (PPARα, γ and δ) in the hope that the beneficial effects can be harnessed while avoiding some of the negative side effects. In this study, the hepatic effects of a discontinued PPAR-pan agonist (a triple agonist of PPAR-α, -γ, and -δ), was investigated after dietary treatment of male Sprague-Dawley (SD) rats. The agonist induced liver enlargement in conjunction with metabolomic and lipidomic remodelling. Increased concentrations of several metabolites related to processes of oxidation, such as oxo-methionine, methyl-cytosine and adenosyl-methionine indicated increased stress and immune status. These changes are reflected in lipidomic changes, and increased energy demands as determined by free fatty acid (decreased 18:3 n-3, 20:5 n-3 and increased ratios of n-6/n-3 fatty acids) triacylglycerol, phospholipid (decreased and increased bulk changes respectively) and eicosanoid content (increases in PGB2 and 15-deoxy PGJ2). We conclude that the investigated PPAR agonist, GW625019, induces liver enlargement, accompanied by lipidomic remodelling, oxidative stress and increases in several pro-inflammatory eicosanoids. This suggests that such pathways should be monitored in the drug development process and also outline how PPAR agonists induce liver proliferation.
Assuntos
Fígado/efeitos dos fármacos , Estresse Oxidativo/genética , PPAR alfa/genética , PPAR gama/genética , PPAR beta/genética , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Ácidos Graxos não Esterificados/metabolismo , Metabolismo dos Lipídeos/genética , Lipídeos/biossíntese , Lipídeos/genética , Fígado/metabolismo , Fígado/patologia , PPAR alfa/agonistas , PPAR gama/agonistas , PPAR beta/agonistas , Ratos , Ratos Sprague-DawleyRESUMO
Phenotyping of 1,200 'healthy' adults from the UK has been performed through the investigation of diverse classes of hydrophilic and lipophilic metabolites present in serum by applying a series of chromatography-mass spectrometry platforms. These data were made robust to instrumental drift by numerical correction; this was prerequisite to allow detection of subtle metabolic differences. The variation in observed metabolite relative concentrations between the 1,200 subjects ranged from less than 5 % to more than 200 %. Variations in metabolites could be related to differences in gender, age, BMI, blood pressure, and smoking. Investigations suggest that a sample size of 600 subjects is both necessary and sufficient for robust analysis of these data. Overall, this is a large scale and non-targeted chromatographic MS-based metabolomics study, using samples from over 1,000 individuals, to provide a comprehensive measurement of their serum metabolomes. This work provides an important baseline or reference dataset for understanding the 'normal' relative concentrations and variation in the human serum metabolome. These may be related to our increasing knowledge of the human metabolic network map. Information on the Husermet study is available at http://www.husermet.org/. Importantly, all of the data are made freely available at MetaboLights (http://www.ebi.ac.uk/metabolights/).
RESUMO
Pattern recognition techniques are effective tools for reducing the information contained in large spectral data sets to a much smaller number of significant features which can then be used to make interpretations about the chemical or biochemical system under study. Often the effectiveness of such approaches is impeded by experimental and instrument induced variations in the position, phase, and line width of the spectral peaks. Although characterizing the cause and magnitude of these fluctuations could be important in its own right (pH-induced NMR chemical shift changes, for example) in general they obscure the process of pattern discovery. One major area of application is the use of large databases of (1)H NMR spectra of biofluids such as urine for investigating perturbations in metabolic profiles caused by drugs or disease, a process now termed metabonomics. Frequency shifts of individual peaks are the dominant source of such unwanted variations in this type of data. In this paper, an automatic procedure for aligning the individual peaks in the data set is described and evaluated. The proposed method will be vital for the efficient and automatic analysis of large metabonomic data sets and should also be applicable to other types of data.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Reconhecimento Automatizado de Padrão , Processamento de Sinais Assistido por Computador , Urina/química , Animais , Carcinógenos/toxicidade , Hidrazinas/toxicidade , Ratos , Ratos WistarRESUMO
UNLABELLED: Metformin, a biguanide derivate, has pleiotropic effects beyond glucose reduction, including improvement of lipid profiles and lowering microvascular and macrovascular complications associated with type 2 diabetes mellitus (T2DM). These effects have been ascribed to adenosine monophosphate-activated protein kinase (AMPK) activation in the liver and skeletal muscle. However, metformin effects are not attenuated when AMPK is knocked out and intravenous metformin is less effective than oral medication, raising the possibility of important gut pharmacology. We hypothesized that the pharmacology of metformin includes alteration of bile acid recirculation and gut microbiota resulting in enhanced enteroendocrine hormone secretion. In this study we evaluated T2DM subjects on and off metformin monotherapy to characterize the gut-based mechanisms of metformin. Subjects were studied at 4 time points: (i) at baseline on metformin, (ii) 7 days after stopping metformin, (iii) when fasting blood glucose (FBG) had risen by 25% after stopping metformin, and (iv) when FBG returned to baseline levels after restarting the metformin. At these timepoints we profiled glucose, insulin, gut hormones (glucagon-like peptide-1 (GLP-1), peptide tyrosine-tyrosine (PYY) and glucose-dependent insulinotropic peptide (GIP) and bile acids in blood, as well as duodenal and faecal bile acids and gut microbiota. We found that metformin withdrawal was associated with a reduction of active and total GLP-1 and elevation of serum bile acids, especially cholic acid and its conjugates. These effects reversed when metformin was restarted. Effects on circulating PYY were more modest, while GIP changes were negligible. Microbiota abundance of the phylum Firmicutes was positively correlated with changes in cholic acid and conjugates, while Bacteroidetes abundance was negatively correlated. Firmicutes and Bacteroidetes representation were also correlated with levels of serum PYY. Our study suggests that metformin has complex effects due to gut-based pharmacology which might provide insights into novel therapeutic approaches to treat T2DM and associated metabolic diseases. TRIAL REGISTRATION: www.ClinicalTrials.gov NCT01357876.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes , Mucosa Intestinal , Intestinos , Metformina , Microbiota/efeitos dos fármacos , Adolescente , Adulto , Idoso , Ácidos e Sais Biliares/metabolismo , Glicemia/metabolismo , Feminino , Peptídeo 1 Semelhante ao Glucagon/sangue , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacocinética , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Masculino , Metformina/administração & dosagem , Metformina/farmacocinética , Pessoa de Meia-Idade , Peptídeo YY/sangueRESUMO
The metabolic investigation of the human population is becoming increasingly important in the study of health and disease. The phenotypic variation can be investigated through the application of metabolomics; to provide a statistically robust investigation, the study of hundreds to thousands of individuals is required. In untargeted and MS-focused metabolomic studies this once provided significant hurdles. However, recent innovations have enabled the application of MS platforms in large-scale, untargeted studies of humans. Herein we describe the importance of experimental design, the separation of the biological study into multiple analytical experiments and the incorporation of QC samples to provide the ability to perform signal correction in order to reduce analytical variation and to quantitatively determine analytical precision. In addition, we describe how to apply this in quality assurance processes. These innovations have opened up the capabilities to perform routine, large-scale, untargeted, MS-focused studies.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Projetos de Pesquisa , Interpretação Estatística de Dados , Humanos , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Overt response to a single 6.25 mg dose of ochratoxin A (OTA) by oral gavage to 15 months male rats was progressive loss of weight during the following four days. Lost weight was restored within one month and animals had a normal life-span without OTA-related terminal disease. Decline in plasma OTA concentration only commenced four days after dosing, while urinary excretion of OTA and ochratoxin alpha was ongoing. During a temporary period of acute polyuria, a linear relationship between urine output and creatinine concentration persisted. Elimination of other common urinary solutes relative to creatinine was generally maintained during the polyuria phase, except that phosphate excretion increased temporarily. (1)H NMR metabolomic analysis of urine revealed a progressive cyclic shift in the group principal components data cluster from before dosing, throughout the acute insult phase, and returning almost completely to normality when tested six months later. Renal insult by OTA was detected by (1)H NMR within a day of dosing, as the most sensitive early indicator. Notable biomarkers were trimethylamine N-oxide and an aromatic urinary profile dominated by phenylacetylglycine. Tolerance of such a large acute insult by OTA, assessed by rat natural lifetime outcomes, adds a new dimension to toxicology of this xenobiotic.
Assuntos
Envelhecimento/urina , Neoplasias Renais/induzido quimicamente , Metabolômica/métodos , Ocratoxinas/farmacocinética , Ocratoxinas/toxicidade , Uremia/induzido quimicamente , Envelhecimento/sangue , Animais , Relação Dose-Resposta a Droga , Testes de Função Renal , Neoplasias Renais/sangue , Neoplasias Renais/patologia , Neoplasias Renais/urina , Espectroscopia de Ressonância Magnética , Masculino , Ocratoxinas/sangue , Ocratoxinas/urina , Análise de Componente Principal , Ratos , Ratos Endogâmicos F344 , Testes de Toxicidade Aguda , Uremia/sangue , Uremia/patologia , Uremia/urina , UrináliseRESUMO
BACKGROUND: The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and peroxisome proliferator-activated receptor δ (PPARδ) play central roles in regulating metabolism in adipose tissue, as well as being targets for the treatment of insulin resistance. While the role of PPARγ in regulating insulin sensitivity has been well defined, research into PPARδ has been limited until recently due to a scarcity of selective PPARδ agonists. RESULTS: The metabolic effects of PPARγ and PPARδ activation have been examined in vivo in white adipose tissue from ob/ob mice and in vitro in cultured 3T3-L1 adipocytes using (1)H nuclear magnetic resonance spectroscopy and mass spectrometry metabolomics to understand the receptors' contrasting roles. These steady state measurements were supplemented with (13)C-stable isotope substrate labeling to assess fluxes, in addition to respirometry and transcriptomic microarray analysis. The metabolic effects of the receptors were readily distinguished, with PPARγ activation characterized by increased fat storage, synthesis and elongation, while PPARδ activation caused increased fatty acid ß-oxidation, tricarboxylic acid cycle rate and oxidation of extracellular branch chain amino acids. Stimulated glycolysis and increased fatty acid desaturation were common pathways for the agonists. CONCLUSIONS: PPARγ and PPARδ restore insulin sensitivity through varying mechanisms. PPARδ activation increases total oxidative metabolism in white adipose tissue, a tissue not traditionally thought of as oxidative. However, the increased metabolism of branch chain amino acids may provide a mechanism for muscle atrophy, which has been linked to activation of this nuclear receptor. PPARδ has a role as an anti-obesity target and as an anti-diabetic, and hence may target both the cause and consequences of dyslipidemia.
Assuntos
Tecido Adiposo Branco/metabolismo , Metabolismo dos Lipídeos , Oxirredução , PPAR delta/metabolismo , PPAR gama/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Resistência à Insulina , Espectroscopia de Ressonância Magnética/métodos , Masculino , Metabolômica/métodos , Camundongos , Camundongos Obesos , PPAR delta/agonistas , PPAR gama/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography-mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography-MS (GC-MS) and ultraperformance liquid chromatography-MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control-based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.
Assuntos
Análise Química do Sangue , Metabolômica/métodos , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Espectrometria de Massas/métodos , Plasma/química , Soro/químicaRESUMO
BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear receptor superfamily. The PPAR family consists of three members: PPARalpha, PPARgamma, and PPARdelta. PPARdelta controls the transcription of genes involved in multiple physiological pathways, including cellular differentiation, lipid metabolism and energy homeostasis. The receptor is expressed almost ubiquitously, with high expression in liver and skeletal muscle. Although the physiological ligands of PPARdelta remain undefined, a number of high affinity synthetic ligands have been developed for the receptor as a therapeutic target for type 2 diabetes mellitus, dyslipidemia and the metabolic syndrome. METHODS: In this study, the metabolic role of PPARdelta activation has been investigated in liver, skeletal muscle, blood serum and white adipose tissue from ob/ob mice using a high affinity synthetic ligand and contrasted with PPARgamma activation. To maximize the analytical coverage of the metabolome, (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) were used to examine metabolites from tissue extracts. RESULTS: Analysis by multivariate statistics demonstrated that PPARdelta activation profoundly affected glycolysis, gluconeogenesis, the TCA cycle and linoleic acid and alpha-linolenic acid essential fatty acid pathways. CONCLUSIONS: Although activation of both PPARdelta and PPARgamma lead to increased insulin sensitivity and glucose tolerance, PPARdelta activation was functionally distinct from PPARgamma activation, and was characterized by increased hepatic and peripheral fatty acid oxidative metabolism, demonstrating the distinctive catabolic role of this receptor compared with PPARgamma.