CHLOROQUINE EXTENDS THE LIFETIME OF THE ACTIVATED INSULIN-RECEPTOR COMPLEX IN ENDOSOMES

Insulin signal transduction, initiated by binding of insulin to its re ceptor at the plasma membrane, activates the intrinsic receptor tyrosi ne kinase and leads to internalization of the activated ligand-recepto r complex into endosomes, This study addresses the role played by the activated insulin receptor within hepatic endosomes and provides evide nce for its central role in insulin-stimulated events in vivo. Rats we re treated with chloroquine, an acidotrophic agent that has been shown previously to inhibit endosomal insulin degradation, and then with in sulin. Livers were removed and fractionated by density gradient centri fugation to obtain endosomal and plasma membrane preparations, Chloroq uine treatment increased the amount of receptor bound insulin in endos omes at 2 min after insulin injection by 93% as determined by exclusio n from G-50 columns and by 90% as determined by polyethylene glycol pr ecipitation (p < 0.02), Chloroquine treatment also increased the insul in receptor content of endosomes after insulin injection (integrated o ver 0-45 min) by 31% when compared with controls (p < 0.05), Similarly , chloroquine increased both insulin receptor phosphotyrosine content and its exogenous tyrosine kinase activity after insulin injection (64 %; p < 0.01 and 96% and p < 0.001, respectively), In vivo chloroquine treatment was without any observable effect on insulin binding to plas ma membrane insulin receptors, nor did it augment insulin-stimulated r eceptor autophosphorylation or kinase activity in the plasma membrane. Concomitant with its effects on endosomal insulin receptors, chloroqu ine treatment augmented insulin-stimulated incorporation of glucose in to glycogen in diaphragm (p < 0.001), These observations are consisten t with the hypothesis that chloroquine-dependent inhibition of endosom al insulin receptor dissociation and subsequent degradation prolongs t he half-life of the active endosomal receptor and potentiates insulin signaling from this compartment.