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-alpha 2MGMA), 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 micropinocytotic invaginations, 13.8 per micron 2 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 degrees C, Au-alpha 2MGMA concentrated within coated pits to a density that was 52 times greater than that on the uncoated plasma membrane. Au-alpha 2MGMA was excluded from micropinocytotic invaginations by more than 93%; this exclusion was unrelated to the size of the Au-alpha 2MGMA particle. In contrast, at 4 degrees C, Fm-I did not concentrate within coated pits and occupied micropinocytotic invaginations in a random manner. At 37 degrees C, coated pits accounted for all of the endocytosis of Au-alpha 2MGMA, 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-alpha 2MGMA undergo endocytosis by dissimilar mechanisms on rat adipocytes. Dissimilarities in the endocytosis of insulin and Au-alpha 2MGMA may be related to the different biological roles of these two molecules.

A previous ultrastructural study showed that gold-labeled insulin (Au-I) and the non-hormonal ligand gold-labeled alpha-2-macroglobulin-methylamine (Au-alpha 2MGMA) underwent endocytosis by dissimilar cell surface structures on rat adipocytes. The present ultrastructural study compared the intracellular routes taken by these two ligands in adipocytes. Intracellular Au-alpha 2MGMA was initially found within apparent coated vesicles but Au-I was not, consistent with the previous demonstration that Au-alpha 2MGMA underwent endocytosis by coated pits whereas Au-I was internalized by uncoated micropinocytotic invaginations. Early in the endocytic pathway, the two ligands were segregated within separate small vesicles and tubulovesicles. Au-alpha 2MGMA was concentrated in a small number of these structures whereas Au-I was sparsely distributed among a relatively large number. Subsequently, the two endocytic pathways converged as the ligands intermingled within pale multivesicular bodies and lysosome-like structures. Au-I was less efficiently transferred to lysosomes than Au-alpha 2MGMA since a greater proportion of intracellular Au-I remained associated with small vesicles and tubulovesicles. This study indicates that early intracellular events in the endocytic pathways of insulin and alpha 2MGMA are distinct. These findings are discussed in light of the fundamentally dissimilar biological roles of these two molecules and the possible involvement of the endocytic pathway in the insulin signaling mechanism.

We studied the binding and biological activities of gold-insulin complexes to develop a complex with properties identical to native insulin. Stabilizing amounts ofinsulin absorbed to 5-, 10-, or 15-nm gold partides resulted in complexes with 40-327 insulin molecules per gold particle and 4-1 1 1 times the biological activity ofunlabeled insulin, based on the molan concentration ofgold complex. These data suggested that these complexes behaved as multivalent ligands. Gold-insulin complexes were prepared with 5% ofthe stabilizing insulin concentration and were stabilized with bovine serum albumm. This resulted in a complex with 5-7 insulin molecules per 10-nm gold partide, which stimulated glucose oxidation in rat adipocytes and competed with ['251]-insulin for binding to the insulin receptor identically to unlabeled in-SMITH, GOLDBERG, JARETT from 150 to seven and with binding and biological activities vintually indistinguishable from unlabeled insulin. This monovalentbehaving product with native ligand properties should be useful for studying insulin and insulin receptor processing by cells. In addition, the technique for producing a monovalent-behaving product may be useful with other ligands. Materials and Methods Reagents and Supplies. Bovine serum albumin (BSA) free of insulinlike activity was purchased from United States Biochemical Corp.

No abstract