Liver cells binding with high insulin doses at 37 C.

Insulin binding and receptor mediated insulin degradation were studied in isolated rat hepatocytes under physiological conditions (37 C, 100% oxygen, Krebs improved Ringer III with glutamate, pyruvate and fumarate, 150 mg% glucose, 1% bovine albumin). 10(6) rat hepatocytes/tube were incubated with various doses of insulin. Steady state binding with low insulin doses (0.05, 0.5 and 66 ng/tube) was reached in 15 minutes, that state being kept for the rest of the experimental time (75 min). Receptor mediated degradation (Kap) at 15 minutes was 0.0479 min-1, including doses of 5 000 and 50 000 ng/tube. Direct correlation was found between degradation and low doses of insulin, being the slope value equal to Kap. Intracellular accumulation of insulin was found at pharmacological concentrations of insulin (5 000 and 50 000 ng/tube) from the first 15 minutes. That accumulation was dose and time dependent. At 75 minutes, with a 0.2 microM insulin concentration, at least 53% of insulin was estimated as insulin accumulated in the cell, since it was not filtrable with acid medium on Sephadex G 50 superfine. When Triton or dodecyl sulphate were used to solubilize the cells, insulin recovery was complete after binding. Intracellular accumulation, however, was not demonstrated at the first two minutes. Binding studies with 16.67 microM insulin in the presence of degradation inhibitors, such as 2 mM N-ethylmaleimide and 5 mM tetracaine hydrochloride, demonstrated that intracellular accumulation of the hormone occurs when degradation is blocked. On the contrary, after trypsin digestion of receptors, degradation was not observed, while increases in binding were abolished, resembling non-specific binding. Under the experimental conditions reported here, neither intracellular accumulation of insulin nor extracellular release of insulin degradation products can be demonstrated at 2 minutes; insulin accumulation is dose dependent, and it is suggested by the fact that the velocity of insulin internalization exceeds its velocity of degradation.