Stoichiometric binding and regulation of insulin receptors on human diploid fibroblasts using physiologic insulin levels.

Cultured diploid human fibroblasts bind insulin to specific receptors which can be regulated with physiologic levels of insulin. Fibroblast monolayers incubated with insulin in concentrations of 5 to 100 rig/ml at 37”C, suspended, washed, and assayed for insulin binding at 15”C, showed a decrease in receptor number with no significant change in affinity. The insulin-mediated regulation of insulin receptors increased with incubation temperatures between 6” and 37°C and reached a maximum decrease in specific binding (40 to 65%) by 4.0 h at 37°C. A stoichiometric relationship between insulin binding and down regulation in the physiologic insulin range was indicated by the similar values obtained for the high affinity insulin dissociation constant (0.61 nM) and the insulin concentration required for half-maximal down regulation (0.58 11~). The order of effectiveness of insulin analogues for binding and down regulation was identical: porcine insulin > guinea pig insulin > proinsulin. Twenty per cent fetal calf serum had no significant effect on insulin binding to control cells (not incubated with insulin) or on the extent of down regulation with 50 rig/ml of insulin. The identity of the insulin-binding site with the down regulation site and its distinction from a mitogen site was demonstrated because: (a) similar values were obtained with insulin for half-maximal down regulation and the high affinity dissociation constant for insulin binding, (b) the order of analogue effectiveness for binding and down regulation were the same, and (c) there was no significant serum effect on insulin binding or down regulation. The in vitro demonstration of down regulation in fibroblasts using physiologic insulin concentrations will permit investigation of mechanisms for insulin regulation of cellular metabolism.