RESUMO
The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.
Assuntos
Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Interferência de RNA/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Dióxido de Carbono/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Ciclo do Ácido Cítrico/fisiologia , Embrião de Mamíferos/citologia , Metabolismo Energético/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Fibroblastos/metabolismo , Fumarato Hidratase/deficiência , Fumarato Hidratase/genética , Deleção de Genes , Glicólise/fisiologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Ácido Láctico/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas de Transporte de Nucleotídeos/genética , Oligomicinas/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , RNA Interferente Pequeno/genéticaRESUMO
A fumarase-deficient patient expressed a novel phenotype of congenital cerebral ventricular dilatation and periventricular cysts. The patient was a compound heterozygote for two mutations that are the only ones among the 12 published mutations that have been found in multiple, unrelated, fumarase-deficient patients.