Internalization and catabolism of insulin by an established renal cell line

Proximal renal tubules are a key site of insulin metabolism. To explore the kinetics and metabolic requirements of insulin internalization and catabolism, we used the opossum kidney cell line, which has proximal tubular-like features and possesses insulin-specific receptors. Internalization was determined by separating membrane-bound insulin from intracellular insulin by exposure to an acidified medium. Internalization of membrane-bound insulin was rapid, and half-maximal internalization occurred within 2.5 min. Degradation products did not accumulate in the cell but appeared in the medium after a delay of 5 min from the onset of internalization. In other experiments, addition of KCN (2 mM) or omission of glucose did not alter degradation, but KCN, combined with the omission of glucose, inhibited degradation by 64%. This was associated with a 240% increase in membrane-bound insulin and an 81% decrease in intracellular insulin. Accordingly, it appears that under these circumstances impaired degradation was a consequence of impaired internalization. In contrast, although 0.1 mM chloroquine, an endosomal-lysosomal inhibitor, also depressed degradation (by 57%), intracellular insulin increased fourfold, indicating failure of intracellular processing. We conclude that these cultured kidney cells rapidly internalize and degrade insulin and that internalization, a prerequisite for degradation, is dependent on energy that can be derived from anaerobic glycolysis or oxidative metabolism. Furthermore, the intracellular degradative processing of insulin by these cells involves a chloroquine-sensitive pathway.