Revisiting the trafficking of insulin and its receptor in rat liver: in vivo and cell-free studies

Endosomes are the main locus where, upon endocytosis in liver cells, insulin and the activated insulin receptor accumulate and insulin is degraded. In this study, ligand and receptor pathways in rat liver have been revisited using analytical subcellular fractionation. Density gradient analysis of microsomal and light mitochondrial fractions confirmed that, upon in vivo uptake into liver, [ ¹²⁵ I]-insulin was rapidly translocated from plasma membranes to endosomes. Internalized [ ¹²⁵ I]-labeled proinsulin and biotinylated insulin, which are less degraded than insulin in endosomes, were in part transferred to lysosomes. Following injection of native insulin, receptor and ligand were also both translocated from the plasma membrane to endosomes, with a maximum at 15-30 min. Receptor translocation was reversed by 2-3 hours and neither endocytosis nor recycling were affected by in vivo cycloheximide treatment. Fractionation of endosomes of insulin-treated rats on density gradients showed that the insulin receptor was recovered at low density, whereas insulin progressively migrated from low to high densities towards the position of acid phosphatase. On SDS polyacrylamide gels, insulin receptor α and β subunits were identified in plasma membrane, endosomal and lysosomal fractions by affinity crosslinking and Western immunoblotting, respectively. Following insulin treatment, insulin receptor expression rapidly decreased in plasma membranes while increasing in endosomes. In a cell-free system, [ ¹²⁵ I]-insulin was in part transferred from endosomes to lysosomes, as was, with organelle content mixing, [ ¹²⁵ I]-biotinylated insulin. In summary: 1) the low lysosomal association of [ ¹²⁵ I]-insulin in vivo is linked to its endosomal degradation; 2) upon ligand-induced endocytosis, the insulin receptor progressively segregates from insulin and is recycled; 3) neither endocytosis no recycling of the receptor requires protein synthesis.