Energy-Dependent Steps in Insulin-Hepatocyte Interaction*

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Abstract
We have studied the relationship between intracellular ATP levels and insulin binding, internalization, degradation, and dissociation from isolated rat hepatocytes. ATP depletion (40% decrease) was accompanied by significantly diminished insulin binding (5.9 ± 0.2% vs. 9.4 ± 0.5% in controls)and degradation (16.8 ± 0.5% vs. 24.5 ±1.1%).Decreased insulin binding to ATP-depleted hepatocytes was accounted for by reduction in receptor affinity (Kd = 7.25 ± 0.39 × 10-9 vs. 3.03 ± 0.29 × 10-9 M) without a change in receptor number. We have observed a time-dependent diminution of the dissociation rate of insulin from both control and ATP-depleted hepatocytes. The longer the insulin was allowed to associate with the cells, the slower the rate of its dissociation. ATP depletion accelerated the rate of insulin dissociation, confirming the lower affinity of insulin receptors in energy-depleted cells. An accelerated effect of unlabeled insulin on the ligand dissociation (negative cooperativity) was attenuated by ATP depletion. To examine the role of energy in the process of insulin internalization and intracellular degradation, we have incubated liver cells with the lysosomatotropic agent chloroquine (0.2 mM) in the presence and absence of DNP. Chloroquine significantly increased the amount of cell-associated radioactivity (150%). DNP effectively blocked chloroquine-induced increase of cellassociated radioactivity. This suggests that the energy-dependent endocytosis precedes intracellular insulin accumulation. We have demonstrated that an extracellular insulin degradation (5–6%) is energy independent, whereas nearly all intracellular lysosomal and 25% of nonlysosomal insulin degradation are energy dependent. ATP depletion results in decreased insulin receptor affinity, with diminished insulin binding and accelerated insulin dissociation from freshly isolated hepatocytes. The investigation of receptor-mediated insulin degradation reveals that energy-dependent endocytosis of the ligand precedes intracellular insulin degradation. The intracellular lysosomal pathway of insulin degradation appears to be almost totally energy dependent, whereas the nonlysosomal pathway demonstrates only partial energy dependency.