Selective Effects of Inhibitors of Hormone Processing on Insulin Action in Isolated Hepatocytes*

Abstract

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.

Insulin-like Growth Factor-I Receptor Internalization Regulates Signaling via the Shc/Mitogen-activated Protein Kinase Pathway, but Not the Insulin Receptor Substrate-1 Pathway

“…In the CHO cells investigated in this study, we have demonstrated a similar marked inhibition of ligand-induced IGF-I receptor internalization at 15°C. Previous studies have shown that dansylcadaverine appears to inhibit a step in endocytotic vesicle formation that involves the pinching off of vesicles from the plasma membrane (33,37). Based on these sites of inhibition, our findings are consistent with a model in which the tyrosine phosphorylation of Shc occurs predominantly by activated IGF-I receptors in endosomes, or at least by receptors in vesicles that have moved past the point in the endocytotic pathway inhibited by dansylcadaverine.…”

Multiple metabolic abnormalities in normal glucose tolerant relatives of NIDDM families

“…Inhibition of rat hepatocyte insulin clearance and degradation has been shown to impair the action of insulin on amino acid but not glycogen metabolism [39]. The biological significance of decreased hepatic insulin clearance for hepatic metabolism in the normal glucose tolerant relatives requires further study, although it is interesting to note that hepatic glucose output was found to be normal in a group of comparable relatives of NIDDM families [16].…”

The Sulfonylurea Glimepiride Regulates Intracellular Routing of the Insulin-Receptor Complexes through Their Interaction with Specific Protein Kinase C Isoforms

“…Previous in vivo and in vitro investigations have provided substantial evidence for a relationship between insulin action and intracellular processing of insulin and its receptor (Ferrannini et al, 1982;Flier et al, 1982;Peavy et al, 1984;Veda et al, 1985;Jochen and Berhanu, 1987;Miller, 1988). Furthermore, it has been suggested that abnormalities in insulin receptor recycling and intracellular processing of the insulinreceptor complex might contribute to impair insulin action in patients with type 2 diabetes mellitus (Jochen et al, 1986;Grunberger et al, 1989;Trischitta et al, 1989;Benzi et al, 1990Benzi et al, , 1997Sesti et al, 1996).…”

“…By contrast, most of the internalized receptor is recycled to the cell surface to be reutilized, and only a small fraction is degraded (Marshall, 1985b). Several lines of evidence suggest that internalization and intracellular processing of the insulin-receptor complex may play a role in insulin action (Peavy et al, 1984;Veda et al, 1985;Jochen and Berhanu, 1987;Miller, 1988). Association between impaired in vivo insulin clearance and action have been reported in subjects with insulin resistance (Ferrannini et al, 1982;Flier et al, 1982).…”