Translocation of the insulin-regulated aminopeptidase to the cell surface: detection by radioligand binding.
OBJECTIVE
Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes.


METHODS
After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H).


RESULTS
We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface.


CONCLUSIONS
Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.
Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Brussels, Belgium. hdemaegd@vub.ac.be
H
H
Demaegdt
L
L
Smitz
J-P
JP
De Backer
M T
MT
Le
M
M
Bauwens
E
E
Szemenyei
G
G
Tóth
Y
Y
Michotte
P
P
Vanderheyden
G
G
Vauquelin
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D010122:Cystinyl Aminopeptidase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D021381:Protein Transport; D011869:Radioligand Assay / Q000379:methods*; D013997:Time Factors
D051275:Glucose Transporter Type 4; D007328:Insulin; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase