Insulin receptor binding and receptor-mediated insulin degradation in human adipocytes.
125I-insulin binding and receptor-mediated insulin degradation were studied in isolated human fat cells from subcutaneous tissue. A high albumin concentration during cell isolation and incubation protected the fragile human adipocyte from lysis. Binding of tracer was pH dependent with an optimum between 7.4 and 7.6. At 37 degrees C steady state was reached by 45 min and maintained for at least 2 h. The binding of labelled insulin in the presence of 10 mumol/l unlabelled insulin was only 1-4% of the total insulin binding. The half-maximal displacement of tracer iodoinsulin (10 pmol/l) by unlabelled insulin occurred at 0.25 nmol/l. Kinetic studies of the dissociation of labelled iodoinsulin from fat cells showed a slight acceleration in the presence of a high concentration of unlabelled insulin in the washout buffer as compared to a buffer containing no insulin. At steady state binding about 95% of the cell-associated radioactivity was extracted as iodoinsulin as judged by gel filtration. The remaining 5% co-eluted with iodotyrosine. During 60 min about 90% of the cell-associated radioactivity dissociated as iodoinsulin and the rest as iodotyrosine.


CONCLUSIONS
1) A high albumin content of buffers prevents traumatization of the human adipocyte; 2) under these conditions steady state binding of insulin is readily measured at 37 degrees C; 3) the use of a washing procedure makes the non-specific binding negligible; 4) the human adipocyte insulin receptor has a very high affinity; 5) receptor-mediated insulin degradation is minimal.
D000273:Adipose Tissue / Q000378:metabolism*; D000328:Adult; D000368:Aged; D002452:Cell Count; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D011972:Receptor, Insulin / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D007328:Insulin; D011972:Receptor, Insulin