Selective effects of inhibitors of hormone processing on insulin action in isolated hepatocytes

Several compounds known to alter receptor-mediated endocytosis were tested for their effect on insulin action in isolated rat hepatocytes. Dansylcadaverine blocked the inhibitory effect of insulin on hepatic protein degradation, but was without effect on the inhibition produced by high concentrations of amino acids. Dansylcadaverine also was without effect on the stimulatory action of insulin on glucose incorporation into glycogen, but totally blocked the insulin stimulation of alanine uptake. No inhibition of basal or (Bu)2cAMP-stimulated alanine uptake was observed with dansylcadaverine. These results indicate that dansylcadaverine has selective effects on some but not all of insulin's actions. Methylamine also blocked insulin stimulation of alanine transport, although at higher doses, methylamine decreased basal and (Bu)2cAMP-stimulated uptake due to cellular toxicity. Chloroquine, an inhibitor of intracellular insulin processing, had no effect on insulin-stimulated alanine transport. To examine the site of action of dansylcadaverine on insulin processing, the binding (cell-associated radioactivity) and degradation of [125I]iodoinsulin by hepatocytes were examined in the presence and absence of dansylcadaverine. Dansylcadaverine delayed the time required to achieve maximal insulin binding and produced a dose-dependent decrease in cell-mediated insulin degradation. Acid dissociation and proteolytic digestion methods were used to differentiate between [125I]iodoinsulin bound to the cell surface vs. that which had been internalized. Dansylcadaverine did not block internalization as measured by either of these criteria, but did block the increase in cell-associated radioactivity produced by chloroquine, suggesting that dansylcadaverine blocks insulin processing after internalization but before the chloroquine-sensitive step. These data show that some of the hepatic actions of insulin, namely inhibition of protein degradation and stimulation of alanine transport, are blocked by certain inhibitors of cellular hormone processing, suggesting that these actions of insulin may require postreceptor events involving insulin metabolism.