Insulin and α2‐macroglobulin‐methylamine undergo endocytosis by different mechanisms in rat adipocytes: I. Comparison of cell surface events

Abstract

This ultrastructural study compared the endocytosis of a peptide hormone, ferritin‐labeled insulin (Fm‐I) or gold‐labeled insulin (Au‐I), and a non‐hormonal ligand, gold‐labeled alpha‐2‐macroglobulin‐methylamine (Au‐α₂MGMA), by rat adipocytes. Quantitative analysis of the cell surface showed that coated pits occupied 0.4% of the adipocyte surface. This was one fifth to one tenth of that which has been reported on fibroblasts and hepatocytes, cell types in which receptor‐mediated endocytosis has been extensively studied. In contrast, uncoated micropinocytotic invaginations were quite numerous and occupied 13.1% of the adipocyte cell surface. The frequency of microphinocytotic invaginations, 13.8 per μm² of plasma membrane, was 7–12 times greater than has been reported on fibroblasts. Therefore, the ultrastructure of the endocytic apparatus on rat adipocytes was different from more commonly studied cell types. At 4°C, Au‐α₂MGMA concentrated within coated pits to a density that was 52 times greater than that on the uncoated plasma membrane. Au‐α₂MGMA was excluded from micropinocytotic invaginations by more than 93%; this exclusion was unrelated to the size of the Au‐α₂MGMA particle. In contrast, at 4°C, Fm‐I did not concentrate within coated pits and occupied micropinocytotic invaginations in a random manner. At 37°C, coated pits accounted for all of the endocytosis of Au‐α₂MGMA, proving that these structures were functional despite their atypically low density. In contrast, greater than 99% of the endocytosis of Fm‐I or Au‐I occurred through micropinocytotic invaginations. These results demonstrated for the first time by a comparative, quantitative, ultrastructural method that insulin and Au‐α₂MGMA undergo endocytosis by dissimilar mechanisms on rat adipocytes. Dissimilarities in the endocytosis of insulin and Au‐α₂MGMA may be related to the different biological roles of these two molecules.