Divergence of the in vitro biological activity and receptor binding affinity of a synthetic insulin analogue, [21-asparaginamide-A]insulin.

The [21-asparaginamide-A]insulin ([Asn-(NH2)21-A]insulin) was synthesized by the procedures developed in this laboratory to investigate the contribution of the C-terminal residue, asparagine, of the A chain to the biological activity and receptor binding affinity of insulin. Its secondary structure was investigated by circular dichroism studies. The biological behavior of this analogue was compared with that of insulin in in vitro and in vivo tests and in receptor binding assays. In contrast to other naturally occurring insulins and to all insulin analogues synthesized thus far, [Asn-(NH2)21-A]insulin displays a disparity between receptor binding affinity and in vitro biological potency. In stimulating glucose oxidation and lipogenesis the analogue exhibited potencies of 12 and 14.8%, respectively, compared to insulin. In insulin receptor binding assays, however, this analogue was found to possess a relative potency at least fourfold higher than the in vitro biological activities. In rat liver membranes and in isolated fat cells the analogue exhibited affinities of ca. 63.9 and 51.4%, respectively, compared to the natural insulin. Both the synthetic analogue and the natural hormone have the same maximal activity in the in vitro assays and their dose-response curves are parallel. When assayed in vivo by the mouse convulsion test, [Asn(NH2)21-A]insulin displays a potency of ca. 72% that of the native insulin. This might indicate partial amidolysis of the analogue in vivo, resulting in conversion to the natural hormone. The implications of these observations are considered with regard to insulin-receptor interactions and the generation of the physiological response to the hormone.