Regulation of mitochondrial glycerol-phosphate dehydrogenase by Ca2+ within electropermeabilized insulin-secreting...

(1) A new insulin-secreting cell line (INS-1; Asfari et al. (1992) Endocrinology 130, 167-178) has been used to study the regulation by Ca2+ of mitochondrial FAD-linked glycerol-phosphate dehydrogenase (FAD-GPDH) in situ. (2) Enzyme activity was examined on-line in electropermeabilized cells by a new, sensitive, assay. This involved the reduction of the artificial electron acceptor, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), monitored by the quenching of the fluorescence of rhodamine-18. Using this approach, similar total levels of FAD-GPDH activity (nmol/min per 10(6) cells) were measured in INS-1 cells (1.35 +/- 0.22) and isolated rat islet cells (1.63 +/- 0.02) (3) Ca2+ ions markedly activated the enzyme, lowering the apparent Km-value for added DL-glycerophosphate from 8.8 +/- 1.4 mM to 1.0 +/- 0.1 mM. Ca2+ had no effect on the apparent Vmax. The enzyme displayed cooperative kinetics with respect to DL-glycerophosphate (Hill coefficient of 2.0 +/- 0.2 and 1.6 +/- 0.2 in the absence and presence respectively of Ca2+). Half-maximal effects of Ca2+ were observed in the range 30-130 nM, depending on the concentration of glycerol phosphate. (4) Enzyme activity was weakly (30%) inhibited by diazoxide, but not by the diabetogenic drug, streptozotocin. (5) The data indicate that INS-1 cells represent an excellent model for studying the rôle of FAD-GPDH in the control of insulin secretion.