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


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
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.
Kouach
M
M
Faculté de Pharmacie, Centre Universitaire de Mesures et d'Analyses, Lille, France.
Desbuquois
B
B
Authier
F
F
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001120:Arginine; D015153:Blotting, Western; D011992:Endosomes / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008297:Male; D019000:Phosphotyrosine / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C099655:GRB14 protein, human; D007328:Insulin; D019000:Phosphotyrosine; D001120:Arginine; D048049:Extracellular Signal-Regulated MAP Kinases