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 after subcutaneous administration. Profiles of circulating insulin-like immunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Both techniques indicated the presence of circulating immunoreactivity having retention characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention characteristics of des(64,65)-human proinsulin. The peaks of immunoreactive material were isolated and their structure determined using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two circulating metabolites were identified. These metabolites co-eluted by reversed-phase high-performance liquid chromatography with human insulin and diarginyl(B31,32)-human insulin and had mass spectra identical to the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at the carboxy side of ArgB32, with the formation of human insulin by the subsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dipeptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin may be mediated in part by circulating insulin-like metabolites.
Department of Drug Metabolism and Disposition, Lilly Research Laboratories, Division of Eli Lilly and Company, Indianapolis, Indiana.
V J
VJ
Wroblewski
M
M
Masnyk
R E
RE
Kaiser
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000502:physiology*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007328:Insulin / Q000097:blood / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D011916:Rats, Inbred F344
D007328:Insulin; D011384:Proinsulin; D001120:Arginine; D010450:Endopeptidases