PROINSULIN AND ITS CONVERSION INTERMEDIATES IN HUMAN PANCREAS AND ISOLATED ISLET TISSUE - KINETICS AND STEADY-STATE ANALYSIS

In non-insulin-dependent diabetes, circulating insulin-related immunor eactivity (IRI) is often composed of a higher fraction of the incomple tely converted forms proinsulin and des-31,32 proinsulin. The present study describes an immunoadsorption method for measuring the proportio ns of proinsulin, its two split products, and insulin in human pancrea tic tissue and for determining their rates of formation in human isola ted islets. The method uses two junction-specific monoclonal proinsuli n antibodies in a protein G fractionation; it is validated by greater than or equal to 90% specificity and recovery. The peptide contents me asured in tissue extracts were comparable to those determined in a pre viously developed immunoradiometric assay. In the nine tissue extracts from nondiabetic donor organs, 97% of IRI corresponded to insulin, 1% to proinsulin, 2% to the des-31,32 proinsulin conversion product, and 0.1% to des-64,65 proinsulin. Two samples from non-insulin-dependent diabetics under sulfonylurea treatment contained a fourfold lower cont ent of IRI but the peptide distribution was comparable except for a lo w percentage (0.3) of proinsulin in one case. In pulse-chase experimen ts on three preparations of human islets isolated from nondiabetic don ors, proinsulin represented the major (> 90%) IRI that was synthesized at the end of the 30-min pulse; a subsequent 90-min chase at either 2 .5 or 10 mM glucose resulted in conversion of 75% of proinsulin to des -31,32 (20%) and des-64,65 (2%) intermediates and to insulin (50%); af ter a 180-min chase, 88% of proinsulin was converted to insulin, but 1 0% remained present as proinsulin. In a pulse-chase experiment on isle ts isolated from tissue with a high proportion of des-31,32 intermedia te (5% instead of 2%), the conversion process was slower (45% after 90 min and 70% after 180 min) and resulted in a higher fraction of des-3 1,32 intermediate, suggesting that the elevated tissue content in this intermediate is caused by a reduced PC2 converting activity. These da ta confirm that des-31,32 proinsulin represents the major conversion i ntermediate in normal human islets and indicate the existence of slow converters, possibly as a result of decreased enzymatic processing of the prohormone's AC junction.