Biosynthetic Human Proinsulin Research Articles

Biosynthetic human insulin and proinsulin have additive but not synergistic effects on total body glucose disposal.

Recent studies from our laboratory have demonstrated that human biosynthetic proinsulin has a much slower MCR than insulin as well as a greater effect on hepatic than on peripheral tissues. Since both of these features make proinsulin potentially useful as an adjunct to insulin, the present study was undertaken to characterize the biological effects of combined infusions of insulin and proinsulin. As an initial study, multiple euglycemic clamp studies were performed in six normal subjects to construct dose-response curves for insulin- and proinsulin-mediated glucose disposal. Insulin infusion rates of 0.63, 1.67, 5, and 10 micrograms/M2 X min were compared to proinsulin infusion rates of 2.75, 7.5, 22.5, and 45 micrograms/M2 X min. Analysis of these dose-response curves indicated that proinsulin-mediated glucose disposal was approximately 7% that of insulin. Each subject also received a combined infusion of insulin (0.63 micrograms/M2 X min) and proinsulin (2.75 micrograms/M2 X min). The mean (+/- SE) glucose disposal rate obtained during the combination study (319 +/- 21 mg/M2 X min) was similar to that predicted by adding the results from the individual hormone infusions (307 +/- 21 mg/M2 X min). Analysis of the dose-response curves for insulin and proinsulin was also useful in predicting the glucose disposal rate during the combination study (290 +/- 19 mg/M2 X min). Thus, using either approach to predict the glucose disposal rates, insulin and proinsulin appeared to have additive effects on total body glucose disposal.

In vitro characterization of biosynthetic human proinsulin.

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 degrees C and 37 degrees 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 microM). 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 degrees C. When chloroquine-treated (200 microM) cells were incubated with 125I-proinsulin at 37 degrees C, a 1.5-fold increase in the amount of intracellular proinsulin was observed at 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro characterization of biosynthetic human proinsulin

In vitro characterization of biosynthetic human proinsulin