Receptor-mediated endocytosis of insulin by cultured endothelial cells

Label-fracture immunochemistry and pre-embedding indirect immunocytochemistry were applied to investigate insulin uptake by endothelial cells. Freeze fracture replicas showed that a small percentage of native insulin receptors are associated with non-coated pits (4%) and coated pits (2%). After warming, receptor bound insulin became increasingly associated with such endocytotic vesicles. After 2 min the percentage of detectable insulin associated with non-coated and coated pits increased to 16% and 8%, respectively. Pre-embedding immunocytochemical localization of insulin gave results consistent with those obtained from the label-fracture studies. Both non-coated and coated vesicles appeared labelled after 5 min of warming. Non-coated vesicles contained 25% of the cell associated insulin while 9% was associated with coated pits and vesicles. After 10 min of warming, 9% of label was located in non-coated vesicles and 7% in coated vesicles. A large proportion (29%) of the label was found in tubular-vesicular endosomes at this time. After 15 min of warming, 30% of the remaining cell-associated gold label was found in multivesicular bodies. These experiments demonstrate that insulin uptake by endothelium is mediated by both coated and non-coated vesicles and that, once internalized, insulin is routed through endosomal pathways that primarily result in transcytosis.