Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells

In vivo, filtered insulin is absorbed and degraded in proximal tubules after binding to the apical membrane. Peritubular removal also occurs and involves basolateral receptor binding and degradation. Whether basolateral degradation proceeds within the cell or on the cell surface is unknown. Because of the difficulties in addressing this question in vivo, this study was carried out with a cultured opossum kidney epithelium cell line with proximal-like features and insulin receptors. Cells were grown in partitioned wells on polycarbonate filters and, when confluent, the monolayer effectively separated the culture well into apical and basolateral compartments. Apical and basolateral binding, internalization, and degradation were studied separately by incubating monolayers with I-insulin added to either the apical or basal compartment. At 37°C insulin associated with either pole in a time-dependent manner. This interaction was specific, for it was competitively inhibited by cold insulin but not by unrelated peptides. Separation of surface-bound from internalized insulin was achieved by lowering extracellular pH. At 4°C, 92% of the radioactivity added to either side of the monolayer was surface-bound, whereas at 37°C and after 1 h, 57% was surface-bound and 43% internalized. Affinity of apical and basolateral receptors were similar (1-2 nM), but basolateral receptor number was greater, for at high insulin concentrations (5 x 10 M) basolateral membrane binding exceeded apical by fivefold (250 ± 81 vs. 56 ± 11 fm/10 cells). Degradation followed exposure to either pole of the cell. Although an excess of insulin inhibited degradation by both poles, adrenocorticotropic hormone inhibited apical degradation only. Other unrelated hormones were without effect on either side. In the presence of 5 x 10 M I-insulin, apical degradation averaged 6.5 ± 0.3%/h and basolateral degradation 4.2 ± 0.3%/h. As medium exhibited negligible degrading activity and as degradation products appeared in the medium after a delay of ~ 10 min, it appears that internalization is a prerequisite for degradation. Thus we concude that the cultured opossum kidney epithelial cell line possesses insulin-specific receptors, with more located on the basolateral than apical pole. Binding is followed by internalization and degradation. Insulin-specific basolateral internalization with degradation is an important finding, which if operative in vivo, could explain the large postglomerular peritubular component of renal insulin metabolism.