Effect of Insulin on the Conversion of Glucose-C-14 to C-14-02 by Normal and Diabetic Fibroblasts in Culture

Abstract

In an attempt to elucidate the basic genetic defect (s) in diabetes, the acute effect of insulin on the conversion of glucose-C-14 to C-14-02 in cultured human diploid fibroblasts was measured under various conditions. Freshly trypsinized cells did not respond to insulin, but after more than sixteen hours in growth medium, a small but significant stimulation, requiring high concentrations of insulin (0.26 U. per ml.) was observed. Stimulation by insulin in various normal strains ranged from 5 to 45 per cent with a mean of 18 per cent (p < .001). C-14-O2 production was approximately linear with time after an initial lag phase and proportional to the number of cells inoculated. Increasing the concentration of glucose in the medium increased C-14-O2 production, with stimulation by insulin apparent at only very low concentrations and at 250 mg. per 100 ml., the highest concentration studied. Experiments comparing the oxidation of glucose-l-C-14 versus glucose-6-C-14 showed that substantial C-14-O2 production occurred via the hexose monophosphate shunt, but that most of the increase in C-14-O2 production in the presence of insulin occurred via the Krebs cycle. A comparison of basal and insulin-stimulated C-14-O2 production in normal and diabetic fibroblasts revealed no significant differences. Possible explanations for the failure to distinguish between the two groups are discussed.