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Cell Itinerary and Metabolic Fate of Proinsulin in Rat Liver: In Vivo and in Vitro Studies

Institut National de la Santé et de la Recherche Médicale U567 and Centre National de la Recherche Scientifique Unite Mixte de Recherche 8104 (B.D.), Département d’Endocrinologie, Institut Cochin, 75014 Paris, France; Institut National de la Santé et de la Recherche Médicale U530 (G.C.), Université René Descartes, Unité de Formation et de Recherche Biomédicale des Saints-Pères, 75007 Paris, France; Laboratoire de Spectrométrie de Masse (M.K.), Faculté de Médecine, 59000 Lille, France; and Institut National de la Santé et de la Recherche Médicale U510 (F.A.), Faculté de Pharmacie Paris XI, 92296 Châtenay-Malabry, France

Address all correspondence and requests for reprints to: Bernard Desbuquois, Département d’Endocrinologie, Institut Cochin, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France. E-mail: desbuquois{at}cochin.inserm.fr.

Proinsulin, the insulin precursor in pancreatic ß-cells, displays a slower hepatic clearance than insulin and exerts a more prolonged metabolic effect on liver in vivo. To elucidate the mechanisms underlying these differences, the cellular itinerary and processing of proinsulin and insulin in rat liver have been comparatively studied using cell fractionation. As [¹²⁵I]-insulin, [¹²⁵I]-proinsulin taken up into liver in vivo was internalized and accumulated in endosomes, in which it underwent dissociation from the insulin receptor and degradation in a pH- and ATP-dependent manner. However, relative to [¹²⁵I]-insulin, [¹²⁵I]-proinsulin showed a delayed and prolonged in vivo association with endosomes, a slower in vivo and cell-free endosomal processing, and a higher cell-free endosome-lysosome transfer. Endosomal extracts degraded to a lesser extent proinsulin than insulin at acidic pH; so did, and even proportionally less, at neutral pH, plasma membrane and cytosolic fractions. Proinsulin degradation products generated by soluble endosomal extracts were isolated by HPLC and characterized by mass spectrometry. Under conditions resulting in multiple cleavages in insulin, proinsulin was cleaved at eight bonds in the C peptide but only at the Phe²⁴-Phe²⁵ bond in the insulin moiety. As native insulin, native proinsulin induced a dose- and time-dependent endocytosis and tyrosine phosphorylation of the insulin receptor; but at an inframaximal dose, proinsulin effects on these processes were of longer duration. We conclude that a reduced proteolysis of proinsulin in endosomes, and probably also at the plasma membrane, accounts for its slower hepatic clearance and prolonged effects on insulin receptor endocytosis and tyrosine phosphorylation.

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