Endosomal proteolysis of internalised [ArgA0]-human insulin at neutral pH generates the mature insulin peptide in rat liver in vivo

Abstract

Aims/hypothesis A proteolysis study of human monoarginylinsulin ([Arg A0 ]-HI) and diarginyl-insulin ([Arg B31 -Arg B32 ]-HI) within hepatic endosomes was undertaken to determine whether the endosomal compartment represents a physiological site for the removal of Arg residues and conversion of Arg-extended insulins into fully processed human insulin. Methods The metabolic fate of arginyl-insulins has been studied using the in situ rat liver model system following ligand administration to rats and cell-free hepatic endosomes. Results While the kinetics of insulin receptor endocytosis after the administration of arginyl-insulins were similar to those observed using human insulin, a more prolonged concentration of endosomal insulin receptor was observed in response to [Arg A0 ]-HI. [Arg A0 ]-HI induced a marked increase in the phosphotyrosine content of endosomal insulin receptor, coinciding with a more sustained endosomal association of growth factor receptor-bound protein 14 (GRB14), and a higher and prolonged activation of mitogen-activated protein kinase pathways. At acidic pH, the endosomal cathepsin D rapidly degraded insulin peptides with similar binding affinity, and generated comparable intermediates for both arginyl-insulins without affecting amino and carboxyl arginyl-peptide bonds. At neutral pH, hepatic endosomes fully processed [Arg A0 ]-HI into mature human insulin while no conversion was observed with [Arg B31 -Arg B32 ]-HI. The neutral endosomal Arg-convertase was sensitive to bestatin, immunologically distinct from insulin-degrading enzyme, nardilysin or furin, and was potentially related to aminopeptidase-B-type enzyme. Conclusions/interpretation The data describe a unique processing pathway for the endosomal proteolysis of [Arg A0 ]-HI which involves the removal of Arg A0 and subsequent generation of mature human insulin through an uncovered neutral Arg-aminopeptidase activity. The endosomal conversion of [Arg A0 ]-HI into human insulin might extend the insulin receptor signalling at this locus.

The M1 family of vertebrate aminopeptidases: Role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B

“…To date, only two of its physiological substrates are identified: NRD convertase hydrolyses the glucagon into Arg-Arg-miniglucagon that is converted by Ap-B into miniglucagon, a peptide involved in the maintenance of glucose homeostasis [5]; cathepsin L hydrolyzes cholecystokinin (CCK) precursors into CCK9, which is processed by Ap-B into CCK8, a pleiotropic neuropeptide [6]. This enzyme may also be implicated in the in vivo processing of enkephalins [7] and Arg-extended forms of human insulin [8]. More recently, Ap-B was identified as a new LTA4H, leukotriene A4 hydrolase; LTB4, leukotriene B4; PSA, puromycin-sensitive aminopeptidase; TRH-DE, thyrotropin-releasing hormone degrading enzyme.…”