High performance liquid chromatographic analysis of insulin degradation products from a cultured kidney cell line.
The kidney is a major site for insulin removal and degradation, but the subcellular processes and enzymes involved have not been established. We have examined this process by analyzing insulin degradation products by HPLC. Monoiodoinsulin specifically labeled on either the A14 or B26 tyrosine residue was incubated with a cultured kidney epithelial cell line, and both intracellular and extracellular products were examined on HPLC. The products were then compared with products of known structure generated by hepatocytes and the enzyme insulin protease. Intracellular and extracellular products were different, suggesting two different degradative pathways, as previously shown in liver. The extracellular degradation products eluted from HPLC both before and after sulfitolysis similarly with hepatocyte products and products generated by insulin protease. The intracellular products also eluted identically with hepatocyte products. Based on comparisons with identified products, the kidney cell generates two fragments from the A chain of intact insulin, one with a cleavage at A13-A14 and the other at A14-A15. The B chain of intact insulin is cleaved in a number of different sites, resulting in peptides that elute identically with B chain peptides cleaved at B9-B10, B13-B14, B16-B17, B24-B25, and B25-B26. These similarities with hepatocytes and insulin protease suggest that liver and kidney have similar mechanisms for insulin degradation and that insulin protease or a very similar enzyme is involved in both tissues.
Duckworth
W C
WC
Veterans Administration Medical Center, Omaha, Nebraska.
Hamel
F G
FG
Liepnieks
J
J
Frank
B H
BH
Yagil
C
C
Rabkin
R
R
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004848:Epithelium / Q000378:metabolism; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D007339:Insulysin / Q000378:metabolism; D007457:Iodine Radioisotopes; D007668:Kidney / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D009893:Opossums / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism
D007328:Insulin; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; C008147:insulin, iodo-; D007339:Insulysin