The effect of insulin concentration on retroendocytosis in isolated rat adipocytes

After internalization by isolated rat adipocytes, insulin can be degraded or released intact from the cell, a process termed retroendocytosis. To determine whether the amount of ligand entering the cell could modulate its ultimate intracellular disposition, adipocytes were incubated with 0.4-25 ng/ml radiolabeled insulin for 20 min at 37 C to reach steady state binding and internalization. After this, surface bound insulin was removed by acid extraction and the cells were reincubated in insulin-free 37 C buffer. The fractional rate of release of internalized cell associated radioactivity was similar at all insulin concentrations. However, insulin enhanced the appearance of trichloroacetic acid (TCA)-precipitable material in a dose-dependent manner reaching a maximum at an insulin concentration of 10 ng/ml. At 0.4 ng/ml insulin, 18 +/- 2% of the released radioactivity was TCA precipitable whereas 36 +/- 3% was precipitable at 25 ng/ml. Sephadex G-50 gel chromatography and reverse phase HPLC analysis of the reincubation medium confirmed TCA-precipitable material was intact insulin. To further investigate the dual pathways of intracellular insulin processing, adipocytes were incubated with 0.4 and 25 ng/ml insulin for 20 min, acid extracted to remove surface receptor insulin, and solubilized. Sephadex G-50 and HPLC analysis revealed that proportionately less insulin intermediates and low molecular weight degradation products are found in cells incubated at the higher insulin concentrations. In conclusion, as adipocytes internalize more insulin, less is converted into insulin intermediates and low molecular weight degradation products and more is diverted to retroendocytosis.