METABOLISM OF DES(64,65)-HUMAN PROINSULIN IN THE RAT - EVIDENCE FOR THE PROTEOLYTIC PROCESSING TO INSULIN

The metabolism of des(64,65)-human proinsulin was examined in rats aft er subcutaneous administration. Profiles of circulating insulin-like i mmunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange last protein liquid chromatography a nd reversed-phase high-performace liquid chromatography. Both techniqu es indicated the presence of circulating immunoreactivity having reten tion characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention cha racteristics of des(64,65)-human proinsulin. The peaks of immunoreacti ve material were isolated and their structure determined using reverse d-phase high-performace liquid chromatography and electrospray ionizat ion mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two ci rculating metabolites were identified. These metabolites co-eluted by reversed-phase high-performace liquid chromatography with human insuli n and diarginyl(B31,32)-human insulin and had mass spectra identical t o the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at th e carboxy side of ArgB32, with the formation of human insulin by the s ubsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dip eptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin ma y be mediated in part by circulating insulin-like metabolites.