INSULIN UPTAKE AND PROCESSING BY CULTURED MOUSE GLOMERULAR ENDOTHELIAL-CELLS

Endothelial cells isolated from a variety of vascular beds bind and tr ansport 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 insul in by glomerular endothelial cells (GEC). When exposed to 2 x 10(-10) M I-125-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 I-125-insulin, as measured by solubility in trichloroace tic 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 plas ma 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 I-125-insulin was examined by incubating acid-washed cells at 37-degr ees-C for 60 min. Over this time 18% of the radioactivity was released as TCA insoluble- and 72% as TCA-soluble radioactivity. Release was i ncreased by insulin (10(-6) M) but not by unrelated peptide hormones. In the presence of chloroquine, I-125-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 th at endothelial cells possess insulin-specific receptors that mediate h ormone 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 t o 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 activi ty, in GEC the degradative pathway predominates.