In Vitro Characterization of Biosynthetic Human Proinsulin

Abstract

The binding and biologic properties of human proinsulin produced by recombinant DNA technology have been determined. The biosynthetic human proinsulin was iodinated using lactoperoxidase and subsequently purified by HPLC to yield the [(125I)TyrA14]-proinsulin isomer. Using isolated rat adipocytes, biosynthetic human proinsulin was shown to have approximately 11% of the binding potency of native insulin. At 16°C and 37°C, the ED50 values of biosynthetic human proinsulin were 3.7 nM and 15 nM, respectively, which was significantly different from the insulin values of 0.4 nM and 1.7 nM, respectively. Kinetic analysis suggested that the decreased affinity of biosynthetic human proinsulin was due primarily to a decreased association rate rather than an increased dissociation rate. Similar to insulin, biosynthetic human proinsulin exhibited a decreased half-time of dissociation in the presence of insulin (16.7 nM) or proinsulin (111 nM); however, this negative cooperative effect was lost in the presence of high concentrations of proinsulin (11 μM). Biologic potency, assessed by measuring glucose transport in rat adipocytes, showed that biosynthetic human proinsulin had 10% of the biologic activity of insulin, suggesting close coupling between binding to receptors and membrane generated cellular response. By extracting cell surface bound proinsulin with acidic buffer, the amount of 125I-proinsulin that internalized following binding to surface receptors was measured. At equilibrium, 55% of the cell-associated radioactivity was internalized at 37°C. When chloroquine-treated (200 μM) cells were incubated with 125I-proinsulin at 37°C, a 1.5-fold increase in the amount of intra-cellular proinsulin was observed at 1 h. This suggests that like insulin, chloroquine treatment augments the intracellular accumulation of proinsulin by inhibiting intracellular processing of internalized proinsulin. In conclusion, (1) biosynthetic human proinsulin exhibits ∼ 10% of the binding and biologic potency of native insulin in adipocytes, (2) despite its decreased affinity for surface receptors, proinsulin is readily internalized by adipocytes, where it is degraded by a chloroquine-sensitive pathway.