Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes.
There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism* / Q000528:radiation effects / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D014466:Ultraviolet Rays
D007328:Insulin; C008147:insulin, iodo-; D011972:Receptor, Insulin