Use of a soluble tetrazolium compound to assay metabolic activation of intact beta cells.
Although assessments of metabolic activation are central to studies of beta-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 beta 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 (alpha-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], lodoacetate [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 Ca2+ influx couples glycolysis to mitochondrial oxidation of glucose-derived fuels. However, reduction of fuel-induced calcium influx (by Ca2+-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 beta 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.
Section of Endocrinology and the Medical Service, William S. Middleton Memorial Veterans Administration Hospital, Madison, WI, USA.
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D003000:Clonidine / Q000494:pharmacology; D006224:Cricetinae; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000037:antagonists & inhibitors / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008773:Methylphenazonium Methosulfate / Q000378:metabolism / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D013004:Somatostatin / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000378:metabolism* / Q000494:pharmacology; D013844:Thiazoles / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007328:Insulin; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D008773:Methylphenazonium Methosulfate; D013004:Somatostatin; D005947:Glucose; D003000:Clonidine; D002118:Calcium