Use of a soluble tetrazolium compound to assay metabolic activation of intact β cells

Abstract

Although assessments of metabolic activation are central to studies of β-cell function, available techniques are tedious, insensitive, and/or require cell disruption. We have investigated the use of a new water-soluble tetrazolium salt, MTS (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H -tetrazolium, inner salt), in the presence of phenazine methosulfate (PMS), an intermediate electron acceptor that amplifies its signal (fluorescence at 490 nm). During static incubations of glucose-responsive (HIT-T15 or INS-1) dispersed β cells with increasing glucose concentrations, there was a progressive increase in MTS reduction, with a maximum signal-to-noise ( S N ) ratio of 24 with HIT-T15 cells and 10 with INS-1 cells. This was associated with, but not attributable to, parallel increases in insulin secretion. Pure mitochondrial fuels (α-ketoisocaproate [KIC], methyl pyruvate [MP], or l-glutamine [GLN] + l-leucine [LEU]) also increased the reduction of MTS in INS-1 cells (6.5-, 4.8-, and 14.4-fold, respectively), but generally less than glucose, suggesting a major role of glycolysis in the signal induced by glucose. Inhibitors of glucose metabolism (mannoheptulose [MH], Iodoacetate [IA], or 2-deoxyglucose [2-DG]) markedly reduced the glucose-stimulated MTS signal. In comparison to another tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), MTS assay provided a better S N ratio with glucose or other nutrient secretagogues. Extant theory holds that activation of mitochondrial dehydrogenases by increments in Ca 2+ influx couples glycolysis to mitochondrial oxidation of glucose-derived fuels. However, reduction of fuel-induced calcium influx (by Ca 2+-free medium or diazoxide [DZX]) or direct stimulation of calcium influx (by 40 mmol/L K +) failed to significantly modulate the signal, arguing against this theory. We conclude that the MTS assay is a facile test that reflects the global metabolic function of insulin-secreting β cells. Furthermore, since this assay does not require disruption of cells to solubilize the formazan product, and therefore also allows concomitant measurement of insulin secretion, it offers considerable advantages over earlier methods.