Insulin degradation by Madin-Darby canine kidney cells expressing the insulin receptor.
Prior studies have shown that Madin-Darby canine kidney cells (MDCK) overexpressing the human insulin receptor bind and respond normally to insulin (T.C. Yeh, R.A. Roth, Diabetes 43 (1994) 1297-1303). Moreover, the insulin receptor preferentially localizes to the basolateral membrane of these cells. In the present studies, insulin was added to either the apical or the basolateral side of these cells and the extent of degradation of the insulin was assessed. Radioactive insulin added to either side was bound to its receptor and the radioactivity which reached the other side of the cell was to a large extent degraded fragments. Insulin added to the apical side was degraded to a larger extent (83%) than when added to the basolateral side (49%) although the basolateral side has much more insulin receptors than the apical side. This degradation process was not inhibitors of either lysosomal enzymes, the proteasome complex or cathepsins. The degradation process could however, be potently inhibited by the sulfhydryl alkylating agent N-ethylmaleimide. Further, cell surface biotinylation study showed that the insulin degrading enzyme was preferentially localized on the apical membranes. These results suggest that insulin added on the apical side of MDCK cells are more closely linked to the degradation process than that added on the basolateral side.
Takeuchi
F
F
Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305, USA. fumito@kanazawa-med.ac.jp
Seta
K A
KA
Omura
S
S
Roth
R A
RA
D000818:Animals; D001711:Biotransformation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007668:Kidney; D007700:Kinetics; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D007457:Iodine Radioisotopes; C008147:insulin, iodo-; D011972:Receptor, Insulin