The role of insulin dissociation from its endosomal receptor in insulin degradation.

Mechanisms that terminate signals from activated receptors have potential to influence the magnitude and nature of cellular responses to insulin. The aims of this study were to determine in rat liver endosomes (the subcellular site of insulin signal termination) whether dissociation of insulin from its receptor was a pre-requisite for ligand degradation and whether the state of receptor phosphorylation influenced the dissociation and hence endosomal degradation of insulin and/or receptor recycling. Following in vivo administration of 125I-[A14]-insulin or analogues (native, X10 or H2, relative binding affinities 1:7:67) livers were removed and endosomes prepared. In the endosomal preparations a significantly greater percentage of both analogues were receptor-bound than native insulin with concomitantly less ligand degradation. When rats were injected with protein-tyrosine phosphatase inhibitors (peroxovanadium compounds bpV(phen) or bpV(pic)) before insulin, endosomal insulin receptor phosphotyrosine content, assessed by Western blotting, was increased as was receptor-bound 125I-[A14]-insulin, whilst insulin degradation was decreased. Peroxovanadiums also completely inhibited recycling of insulin receptors from endosomes. However, treatment of freshly isolated endosomes with acid phosphatase which completely dephosphorylated the insulin receptor, did not return the rate of insulin dissociation and degradation to control values, suggesting that peroxovanadium compounds elicit their effect on binding and degradation via a mechanism other than as protein-tyrosine phosphatase inhibitors. We conclude that promotion of sustained receptor binding decreases endosomal insulin degradation and extends the half-life of the activated endosomal receptor, which in turn would be expected to potentiate insulin signalling from this intracellular compartment.

The characterization of endosomal insulin degradation intermediates and their sequence of production.

Insulin degradation within isolated rat liver endosomes was studied in vitro with the aid of three 125I-insulin isomers specifically labelled at tyrosine (A14, B16 and B26). Chloroquine and 1,10-phenanthroline were used to minimize insulin proteolysis during endosome preparation, whereas the manipulation of endosomal processing of insulin in vitro by Co2+ ions (to activate) and 1,10-phenanthroline (to inhibit) permitted the study of degradation intermediates and their time-dependent production. Structural and kinetic analysis of intermediates isolated from both intra- and extra-endosomal compartments allowed the determination of major cleavage sites and the probable sequence of proteolytic events. It was found that 125I-tyrosine is the ultimate labelled degradation product of all iodo-insulin isomers, suggesting that endosomal proteases are able to degrade insulin to the level of its constituent amino acids. 125I-tyrosine was also the only radiolabelled product able to cross the endosomal membrane. Intra-endosomal insulin degradation proceeds via two inter-related cleavage routes after metalloendoprotease cleavage of the B-chain. One pathway results from an initial cleavage in the centre region of the B-chain (B7-19), probably at B14-15, whereas the major route results from a cleavage at B24-25. B24-25 cleavage removes the B-chain C-terminal hexapeptide (B25-30), which is subsequently cleaved by an aminopeptidase activity to produce first the pentapeptide B26-30 and then 125I-tyrosine. The isolation of intact radiolabelled A-chain from the degradation of 125I-[A14]-insulin suggests that further degradation of proteolytic intermediates containing cleaved B-chain proceeds via interchain disulphide reduction. The A-chain is then processed by several cleavages, one of which occurs at A13-14.

Chloroquine extends the lifetime of the activated insulin receptor complex in endosomes.

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 insulin-stimulated 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 receptor-bound 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.