Insulin increases the cell surface concentration of alpha 2-macroglobulin receptors in 3T3-L1 adipocytes. Altered transit of the receptor among intracellular endocytic compartments.
The present study shows that insulin causes an increase in the binding of alpha 2-macroglobulin (alpha 2M) to 3T3-L1 adipocytes. Scatchard analysis of the binding at 4 degrees C indicated an approximate 2-fold increase in the number of alpha 2M binding sites, with no change in the apparent affinity of the receptor. In addition, a 2-3-fold increase in the binding of monoclonal antibody 2C6, which recognizes a component of the alpha 2M receptor, was found in cells treated at 37 degrees C with insulin and then KCN to inhibit receptor endocytosis. An increased cellular accumulation of alpha 2M was also observed in response to insulin. Interestingly, the increase in the rate of accumulation of alpha 2M was significantly smaller than the increase in the number of alpha 2M receptors on the cell surface, suggesting that the rate of ligand internalization or subsequent processing is altered in response to insulin. Ultrastructural analysis of the internalization pathway of the alpha 2M receptor was performed using colloidal gold-coupled 2C6 monoclonal antibody. Control cells incubated for 20 min at 37 degrees C with the gold-conjugated antibody displayed 40% of cellular gold particles on the cell surface and 60% within intracellular structures. In insulin-treated cells this proportion was reversed, with 64% of the particles being found on the cell surface, and only 36% within intracellular structures. Significant differences in the distribution of gold particles among intracellular structures were detected between control and insulin-treated cells. Whereas in control cells, 18% of the total cellular gold particles internalized into tubulovesicles and multivesicular bodies, in insulin-treated cells only 3% of the gold particles were found within these structures. These data indicate that the movement of this receptor between endocytic compartments is altered in response to insulin, and suggest that the effect of insulin to increase the cell surface concentration of alpha 2M receptors and the accumulation of alpha 2M is due, at least in part, to alterations in the endocytic portion of the receptor recycling pathway.
Corvera
S
S
Department of Pathology, University of Pennsylvania Medical School, Philadelphia 19104-6082.
Graver
D F
DF
Smith
R M
RM
D000273:Adipose Tissue / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology / Q000502:physiology / Q000648:ultrastructure; D002478:Cells, Cultured; D003034:Coated Pits, Cell-Membrane / Q000276:immunology / Q000502:physiology; D004705:Endocytosis; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D051379:Mice; D008854:Microscopy, Electron; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007328:Insulin; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins