Chloroquine augments the binding of insulin to its receptor

Abstract

The effect of chloroquine on the interaction of insulin with its receptor has been investigated under both equilibrium and non-equilibrium conditions. Chloroquine was found to augment insulin binding in a pH-dependent manner between pH 6.0 and pH 8.5, with the maximum occurring at approximately pH 7.0. Analysis of the equilibrium binding data in terms of independent binding sites gave equivocal results but suggested an increase in the high-affinity component. Analysis using the negative co-operativity binding model of De Meyts, Bianco and Roth [J. Biol. Chem. (1976) 251, 1877-1888] suggested that the affinity at both high and low occupancy was increased equally. The kinetics of association of insulin with the plasma-membrane receptor indicated that, although the net rate of association increased in the presence of chloroquine, this was due to a reduction in the dissociation rate rather than an increase in the association rate. This was confirmed by direct measurement of the rates of dissociation. Dissociation was found to be distinctly biphasic, with fast and slow components. Curve fitting suggested that the decrease in dissociation rate in the presence of chloroquine was not due to a decrease in either of the two dissociation rate constants, but rather to an increase in the amount of insulin dissociating by the slow component. It was also found that the increase in dissociation rate in the presence of excess insulin, ascribed to negative co-operativity, could be accounted for by an increase in the amount of insulin dissociating by the faster pathway, rather than by an increase in the dissociation rate constant. Thus chloroquine appears to have the opposite effect to excess insulin, and evidence was found for the induction of positive co-operativity in the insulin-receptor interaction at high chloroquine concentrations. Evidence was also found for the presence of low-affinity chloroquine binding sites with binding parameters similar to the concentration dependence of the chloroquine-induced augmentation of insulin binding.

Chloroquine Extends the Lifetime of the Activated Insulin Receptor Complex in Endosomes

“…1-4). These findings are in agreement with previous in vitro studies, where it was observed that chloroquine augmented the binding of insulin to its receptor in purified rat liver plasma membrane preparations (28) and inhibited endosomal insulin degradation in isolated vesicles (12).…”

“…Together, these findings have prompted the suggestion that endosomes play a pivotal role in signal transduction, giving the receptor tyrosine kinase access to substrates distal to and topographically distinct from the plasma membrane (11). In this study, we have demonstrated that in vivo administration of chloroquine, a compound that inhibits both dissociation of insulin from its receptor (28) and subsequent endosomal degradation of insulin (12), leads to a prolongation of the activated insulin-receptor complex and augments insulin-stimulated responses. A key observation is that the in vivo locus for both the inhibition of insulin dissociation and degradation by chloroquine is restricted almost entirely to the endosomal compartment (Figs.…”

“…Thus, the effect of chloroquine on insulin dissociation and degradation in vivo is restricted to endosomes. The findings that in vivo chloroquine treatment inhibited insulin dissociation from its receptor and reduced insulin degradation led us to investigate whether in vivo chloroquine treatment could augment the insulin signaling cascade from this compartment as hypothesized previously (28). The flux of insulin receptors through the endosomal compartment of the liver following insulin administration was significantly increased in amount and duration in the presence of chloroquine (Fig.…”

“…A previous study (28) had shown chloroquine capable of enhancing the association of insulin to its receptor. The effect of chloroquine in vitro was examined on insulin-receptor dissociation and degradation.…”

“…Additionally, chloroquine has been shown to increase insulin binding in cultured hepatoma cells (25,26) and IM9 lymphocytes (27). Modeling of insulin binding to purified plasma membranes in the presence of chloroquine demonstrated that the augmentation of binding was due to a decrease in the rate constant for insulin dissociation (28). Clinical manifestations of chloroquine action have also been observed.…”