Substrate-dependence of reduction of MTT: a tetrazolium dye differs in cultured astroglia and neurons

Abstract

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is widely used to evaluate cell proliferation and viability. MTT reduction is interpreted to be indicative of cellular metabolic activity, and the site of reduction includes both mitochondrial and cytosolic redox reactions. Astrocytes are believed to rely mainly on glycolysis for ATP generation, whereas neurons are considered to depend more on oxidative metabolism. The present study, therefore, tested the substrate-preference of glucose and its metabolites for MTT reduction in cultures of rat type 1 astroglia and neurons.MTT specific activity of astroglia was much higher than that of neurons. Astroglial MTT reducing activity in glucose-free medium or 2mM glucose with iodoacetate (5mM) was completely blocked. In glucose-depleted medium, 2mM lactate, pyruvate, malate, or acetate elicited minimal increases in MTT reduction by astroglia. In contrast, MTT reducing activity in neurons was enhanced two-fold by pyruvate and the reducing activity of lactate was equivalent to that of glucose, while malate had a small and acetate had no effect on MTT reduction. These results indicate that these two cell types differ markedly in their substrate-preferences for MTT reduction. In astroglia, MTT reduction reflects mainly cytosolic redox activity and is dependent on glyceraldehyde-3-phosphate dehydrogenase. In neurons, pyruvate dehydrogenase supports MTT reduction more effectively than glucose or lactate, even though both of these substrates can produce NADH and pyruvate.