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

Abstract

A proteolysis study of human monoarginyl-insulin ([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. 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. 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. 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.