Insulin uptake and processing by cultured mouse glomerular endothelial cells

Endothelial cells isolated from a variety of vascular beds bind and transport insulin but exhibit relatively low insulin degrading activity. Because endothelial cells exhibit heterogeneity and since kidney is a major site of insulin degradation, we studied the processing of insulin by glomerular endothelial cells (GEC). When exposed to 2 x 10^-10 M ¹²⁵I- labeled insulin, GEC associated with the hormone in a specific manner. This interaction was inhibited by insulin but not by a number of unrelated peptide hormones. Over a 90-min period, GEC degraded 42 ± 3% of the ¹²⁵I-insulin, as measured by solubility in trichloroacetic acid (TCA). Degradation was inhibited 90% by an excess of insulin or adrenocorticotropic hormone (10^-6 M) and 57% by glucagon, whereas growth hormone and calcitonin were without effect. Separation of plasma membrane bound from internalized insulin was achieved by decreasing extracellular pH. In the steady state, 43% of cell- associated insulin was membrane bound and 57% internalized. The fate of the internalized ¹²⁵I-insulin was examined by incubating acid-washed cells at 37°C for 60 min. Over this time 18% of the radioactivity was released as TCA insoluble- and 72% as TCA-soluble radioactivity. Release was increased by insulin (10^-6 M) but not by unrelated peptide hormones. In the presence of chloroquine, ¹²⁵I-insulin release increased by one third while degradation fell. High-performance liquid chromatography revealed that GEC released both intact insulin and large intermediates and that chloroquine inhibited intermediate formation. We conclude that endothelial cells possess insulin- specific receptors that mediate hormone internalization. Internalized insulin is then processed either through a major chloroquine-sensitive degradative pathway or a lesser exocytosis pathway that returns intact and partially cleaved insulin to the extracellular compartment. Thus GEC share the same two pathways common to endothelial cells from other vascular beds. However, unlike these other endothelial cells that have minor insulin-degrading activity, in GEC the degradative pathway predominates.