Peptide intermediates in the cellular metabolism of insulin

We have studied the metabolism of insulin to peptide intermediates which arise during the early stages of cellular hormone processing. [125I]Iodoinsulin (prepared by direct radioiodination of the hormone) and [(125I)iodotyrosylB1]insulin (prepared by semisynthetic procedures and indirect radioiodination) were used as specific probes for the metabolic fates of the A and B chain domains of the hormone, respectively. Incubation of the A chain-labeled probe with isolated rat hepatocytes and subsequent analysis of the cell-associated products revealed low amounts of several insulin-derived peptides. Mapping of these insulin fragments by chemical and enzymatic modification, electrophoresis, and autoradiography showed that they result from specific cleavages in both chains of the hormone. Two A chain-labeled fragments containing residues A14 to A20-21 in disulfide linkage to COOH-terminal portions of the insulin B chain were identified among the cell-associated metabolites. Other products contained intact A chains yet their elution positions during gel filtration were distinct from the position of [125I]iodoinsulin. Incubation of [(125I)iodotyrosylB1]insulin with hepatocytes resulted in cell-associated products containing a set of B chain-labeled fragments complementary to those described above. Structural analysis showed that these metabolites result from sequential cleavage of the insulin molecule at three sites between the interchain disulfide bonds of the B chain domain. Taken together, these A and B chain-labeled peptides form a series of cell-associated and structurally related metabolites. They likely represent the intermediate products which arise during the cellular processing of insulin.