Regulation of insulin synthesis in an insulin-producing cell line (RINm5F): long-term experiments.

The insulin-producing cell line RINm5F, has been used in short-term experiments to evaluate insulin secretion. We sought to maintain the responsiveness of these cells to stimuli for up to 2 days. We examined the course of new insulin synthesis over this period by measuring at intervals immunoreactive insulin (IRI) in two parts: IRI in the medium (M) and IRI extracted from the cells (C). Control cells were incubated in RPMI 1640/2.8 mM glucose/10% fetal bovine serum/200 micrograms/ml bacitracin (to prevent insulin degradation). The addition of dibutyryl cAMP 10 mM to the experimental dishes significantly increased total (M + C) IRI at 48 hr to 37% above the insulin content of the control dishes (p less than 0.01). Theophylline 10 mM increased total (M + C) IRI by 24% over control (p less than 0.05) after 24 hrs. Glucose, glyceraldehyde, leucine, arginine, glucagon and tolbutamide, other stimulants of insulin production, had no effect. Under the experimental conditions reported here, including the use of bacitracin, IRI synthesis can be studied for up to 48 hr.