Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts.
The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-[3H]glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of [3H]thymidine was stimulated approximately 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor beta-subunit in cells prelabeled with [32P]phosphate was increased 10- to 20-fold within 5 min of adding insulin, with a half-maximal effect at approximately 3 nM insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only approximately 30% that of insulin. Two antibodies (25-49 and 83-14) showed a similar concentration dependence to insulin in their metabolic effects and in the inhibition of 125I-labeled insulin binding to cells. The other two antibodies (83-7 and 18-44) were somewhat less potent and did not inhibit insulin binding. None of the antibodies significantly increased insulin receptor phosphorylation at concentrations up to 100 nM, which at least in the case of 25-49 and 83-14 was sufficient for full receptor occupancy. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway.
Soos
M A
MA
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, United Kingdom.
O'Brien
R M
RM
Brindle
N P
NP
Stigter
J M
JM
Okamoto
A K
AK
Whittaker
J
J
Siddle
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004261:DNA Replication; D003847:Deoxyglucose / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D007328:Insulin; D003847:Deoxyglucose; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin