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

Abstract

Insulin signal transduction, initiated by binding of insulin to its receptor at the plasma membrane, activates the intrinsic receptor tyrosine kinase and leads to internalization of the activated ligand-receptor complex into endosomes. This study addresses the role played by the activated insulin receptor within hepatic endosomes and provides evidence for its central role in insulinstimulated events in vivo. Rats were treated with chloroquine, an acidotrophic agent that has been shown previously to inhibit endosomal insulin degradation, and then with insulin. Livers were removed and fractionated by density gradient centrifugation to obtain endosomal and plasma membrane preparations. Chloroquine treatment increased the amount of receptorbound insulin in endosomes at 2 min after insulin injection by 93% as determined by exclusion from G-50 columns and by 90% as determined by polyethylene glycol precipitation (p < 0.02). Chloroquine treatment also increased the insulin receptor content of endosomes after insulin injection (integrated over 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 plasma membrane insulin receptors, nor did it augment insulin-stimulated receptor autophosphorylation or kinase activity in the plasma membrane. Concomitant with its effects on endosomal insulin receptors, chloroquine treatment augmented insulin-stimulated incorporation of glucose into glycogen in diaphragm (p < 0.001). These observations are consistent with the hypothesis that chloroquine-dependent inhibition of endosomal insulin receptor dissociation and subsequent degradation prolongs the half-life of the active endosomal receptor and potentiates insulin signaling from this compartment.Insulin signal transduction is initiated by binding of insulin to its receptor at the plasma membrane, which in turn leads to the rapid autophosphorylation of multiple tyrosine residues on the intracellular portion of the ␤-subunit and the activation of the receptor tyrosine kinase toward exogenous substrates (1, 2). Following autophosphorylation, the activated ligand-receptor complex is internalized into endosomes in liver (3-6) and low density membranes in adipocytes (7,8) and muscle (9). Endocytosis of activated receptors has the twin effects of concentrating receptors within endosomes and allowing the insulin receptor tyrosine kinase to phosphorylate substrates that are spatio-temporally distinct from the plasma membrane (Ref. 10; reviewed in Ref. 11). Subsequent termination of signal transduction is achieved by endosomal insulin degradation (12-16) following dissociation of insulin from its receptor (14, 17) as the intralumenal environment of the endosome acidifies (18). This loss of the ligand-receptor complex attenuates any further ligand-driven recept...

An arginine to cysteine252 mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events

“…In addition, similarly to what is occurring in cells expressing IR R252C , IRS 1 phosphorylation could be achieved by IR C860S localised mainly on microvilli supporting the idea that some IR biological signals can be elicited from these surface domains. However, endosome could also be a site of IR signal transduction and biological responsiveness, as suggested by studies of the kinetics of IR kinase activation and of the subcellular localisation of this kinase domain [18,19,20,42].…”

Effect of Inhibiting Vacuolar Acidification on Insulin Signaling in Hepatocytes

“…In previous work we showed that inhibition of endosomal acidification with chloroquine inhibited both insulin degradation and dissociation of insulin-IRK complexes within endosomes (18), with the consequent augmentation of IRK activity in this compartment (19). Because a critical role for endosomal IRK in insulin signaling has been clearly demonstrated (5), it was postulated that the inhibition of vacuolar acidification might potentiate insulin signaling from endosomes (19).…”

“…This suggests that inhibitors of vacuolar acidification would potentiate insulin signaling from the endosomal compartment, and there are studies consistent with this view. Thus, in rats, chloroquine treatment augmented and prolonged IRK activity in hepatic endosomes (19). Further, the administration of chloroquine to patients with type 2 diabetes mellitus decreased fasting plasma glucose levels, improved glucose tolerance, and increased plasma insulin levels (20 -22).…”

Increase in epidermal growth factor receptor protein induced in osteoblastic cells after exposure to flow of culture media

“…4), because all the three inhibitors are thought to work inside the cells. Chloroquine is thought to inhibit proteolysis at the late stage of endosomes (5,20,34), probably due to inhibition of acidification of endosomes. MG-132 is an inhibitor of proteasomes (20,32) that is thought to degrade the ubiquitinated EGF receptor downstream of the endosomes.…”

“…Therefore, we examined the participation of this pathway by interfering with it. The cells were pretreated with MDC, an inhibitor of clathrin-dependent endocytosis (13), chloroquine, an inhibitor of endosomal proteolysis (5,20,34), MG-132, a proteasome inhibitor (20,32), or herbimycin A, a tyrosine kinase inhibitor, in media containing EGF (10 ng/ml) but no serum. In untreated cells, which were not treated with any of the inhibitors, the relative intensity of the EGF receptor increased 1.2-fold on average at 2 min after shaking for 5 min.…”