UPTAKE AND METABOLIC-FATE OF [HIS(A8),HIS(B4),GLU(B10),HIS(B27)]INSULIN IN RAT-LIVER IN-VIVO

Receptor-mediated endocytosis and subsequent endosomal proteolysis of [I-125]Tyr(A14)- [His(A8), His(B4), Glu(B10), His(B27)]insulin ([I-125 ]Tyr(A14)-H2 analogue), an insulin analogue exhibiting a high affinity for the insulin receptor, has been studied in liver parenchymal cells by quantitative subcellular fractionation and compared with that of w ild-type [I-125]Tyr(A14)-insulin. Whereas the kinetics of uptake of th e H2 analogue by liver was not different from that of insulin, the H2 analogue radioactivity after the 2 min peak declined significantly mor e slowly. A significant retention of the H2 analogue compared with ins ulin in both plasma membrane and endosomal fractions was observed and corresponded to decreased processing and dissociation of the H2 analog ue. Cell-free endosomes preloaded in vivo with radiolabelled ligands a nd incubated in vitro processed insulin and extraluminally released in sulin intermediates at a 2-3-fold higher rate than the H2 analogue. In vitro proteolysis of both non-radiolabelled and monoiodinated molecul es by endosomal lysates showed a decreased response to the endosomal p roteolytic machinery for the H2 analogue. However, in cross-linking an d competition studies the H2 analogue exhibited an affinity for insuli n-degrading enzyme identical with that of wild-type insulin. Brij-35-p ermeabilized endosomes revealed a 2-fold higher rate of dissociation o f insulin from internalized receptors compared with the H2 analogue. A fter the administration of a saturating dose of both ligands, a rapid and reversible ligand-induced translocation of insulin receptor was ob served, but without receptor loss. The H2 analogue induced a higher re ceptor concentration and tyrosine autophosphorylation of the receptor beta subunit in endosomes. Moreover, a prolonged temporal interaction of the in vivo injected H2 analogue with receptor was observed by dire ct binding assays performed on freshly prepared subcellular fractions. These results indicate that endosomal proteolysis for the H2 analogue is slowed as a result of an increased residence time of the analogue on the insulin receptor and a low affinity of endosomal acidic insulin ase for the dissociated H2 molecule.