Modulation of 125I-insulin degradation by receptors in liver plasma membranes

doi:10.1016/0006-291X(77)90338-2

Biochemical and Biophysical Research Communications

Volume 74, Issue 2, 24 January 1977, Pages 545-552

https://doi.org/10.1016/0006-291X(77)90338-2 Get rights and content

Abstract

This study investigated the interactions of insulin with receptors in a purified mouse liver plasma membrane. Two processes were found when insulin was incubated with the liver plasma membranes at 30°C; 1) the binding of insulin to these membranes and 2) the degradation of insulin by one or more associated insulinases or proteases. These two activities were physically separated, analyzed, and it was found that bound insulin is a substrate for insulin degradation. In addition, the rate of degradation of free insulin in solution was found to be 1.1% degraded insulin/available insulin/min in a 30 minute period. This rate is compared to 25.3% degraded insulin/available insulin/min under similar conditions when insulin is bound to the plasma membranes. Thus, insulin binding was shown to aid in its degradation. These results suggest that a specific binding by at least one of the receptors for insulin orients the molecule such that its degradation is facilitated.

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Glucagon receptor binding, dissociation and degradation in rat liver plasma membranes studied by a microperifusion method
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The association process of glucagon receptor binding in purified rat liver plasma membranes and prolonged incubation of the hormone-receptor complex at 30°C did not result in degradation of bound labelled glucagon. In contrast, up to 95% of the non-membrane-bound labelled glucagon was degraded. The rate of spontaneous dissociation of the glucagon-receptor complex was slow, and amounted to about 0.1% per min of that bound. GTP greatly enhanced the rate of dissociation. Half the maximal dissociation of the complex was effected by 10⁻⁵ mol/l of GTP under equilibrium binding conditions. At maximally effective concentrations of GTP, 80% of the glucagon-receptor complex was dissociated within 2 min. A microperifusion system for the perifusion of isolated plasma membranes was devised and used for the separation of labelled glucagon from the plasma membranes subsequent to a GTP-induced dissociation of the hormone-receptor complex. Rebinding of the dissociated peptide to fresh membranes showed that maximum binding ability was retained. The glucagon molecule was protected against degradation while bound to the receptor, indicating that the glucagon effector system is completely separate from the inactivating system(s) in isolated plasma membranes. Thus, the hormonal effect of glucagon could be exerted through the sequential interaction of each glucagon molecule with several receptors.
Insulin degradation by insulin target cells
1981, Metabolism
Recent findings illustrate the complexities associated with the interaction between insulin and its target cells. These results suggest that the processes involved in insulin action and those involved in insulin degradation may have certain steps in common. Both apparently begin when insulin binds to the insulin receptor. The next step is unknown but it ultimately leads to the internalization of the hormone before insulin dissociates from the cell surface. Furthermore, internalization appears to be a requirement for efficient degradation of insulin since the vast majority (perhaps all in certain cells) of the degrading activity is intracellular. Internalization may not be required to produce certain actions of the hormone, however, and the two processes may diverge at this point.¹⁶²
It is not clear how insulin enters the target cell other than the process appears to be receptor-mediated. Also, further work is needed to more fully characterize the vesicles that contain internalized insulin. Finally, the actual location of insulin degradation and the enzyme(s) involved need further study, especially to clarify the relative contributions of lysosomes, cytosolic protease, and GIT to physiological insulin destruction. An understanding of the overall process of insulin degradation is required for a complete description of the physiologic disposition of the hormone at the target cell. Moreover, this system has subtle control mechanisms that may have important implications for the management of diabetes and other endocrine and metabolic disorders.
Bacitracin: An inhibitor of the insulin degrading activity of glutathione-insulin transhydrogenase
1981, Biochemical and Biophysical Research Communications
The antibiotic bacitracin, a known inhibitor of insulin degradation by both isolated cells and subcellular organelles, inhibited the ability of purified glutathione-insulin transhydrogenase to split insulin into its constituent A and B chains. This inhibition was demonstrated by measuring the formation of insulin degradative products that were both soluble in 5% trichloroacetic acid and chromatographed as the separate chains of insulin on Sephadex G-50. At concentrations of 90 and 300 μM, bacitracin inhibited 50 and 90%, respectively, of the degrading activity of the purified enzyme. Similarly, degradation by crude liver lysates was inhibited 50 and 90% by 70 and 250 μM bacitracin, respectively. Kinetic studies indicated that this inhibition was by a complex mechanism that decreased both the Vmax and affinity of the enzyme for insulin. These data raise the possibility that the inhibition of glutathione-insulin transhydrogenase by bacitracin could account for part or all of the effects of this antibiotic on inhibition of insulin degradation by target cells.
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1981, International Review of Cytology
This chapter discusses the insulin binding and glucose transport. The insulin binding is explained with the help of recent data on cellular localization, cooperativity, regulation of receptors and receptors in normal and transformed cells. The action of insulin starts with the binding of the hormone to specific receptors on the plasma membrane of target cells. Studies of insulin receptors require a satisfactory reagent probe, one with the same biological properties as the native insulin. The monoiodinated insulin has a biological activity similar to native insulin, based on stimulation of glucose oxidation by fat cells. For glucose transport, the chapter reviews various regulatory aspects, such as cell growth, adaptation, and hormonal influences in normal and neoplastic cells. The chapter focuses on the mechanism and regulation of glucose transport in cells where glucose is transported by facilitated diffusion. This process is mediated by a specific glucose carrier but does not require energy; therefore, the direction of glucose flux is in accordance with its concentration gradient. The most common experimental design for measuring rates of substrate transport is the zero-trans influx procedure.
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Insulin degradation XXIII. Distribution of glutathione-insulin transhydrogenase in isolated rat hepatocytes as studied by immuno-ferritin and electron microscopy
1978, BBA - General Subjects
The distribution of glutathione-insulin transhydrogenase (glutathione: protein-disulphide oxidoreductase, EC 1.8.4.2) in isolated rat hepatocytes that had been first treated with rabbit antiserum against purified rat liver transhydrogenase and then with ferritin-conjugated goat anti-rabbit γ-globulin was examined by electron microscopy. In cells with antact plasma membrane, the immunoferritin labeling of glutathione-insulin transhydrogenase was observed on a few external microvillous projections at the outside of the cell. In cells with breaks in the plasma membrane, the immunoferritin labeling appeared extensively on smooth vesicles just inside the plasma membrane and on smooth endoplasmic reticulum extending to and including the outer nuclear membrane, in addition to the external microvillous projections. There was some immunoferritin labeling on rough endoplasmic reticulum and on the inner surface of the plasma membrane. The mitochondria and the outer surface of the plasma membrane of the cell did not show the ferritin labeling. Control parallel samples in which the antiserum was substituted with normal (i.e. non-immune) serum or with neutralized antiserum (prepared by absorption with the transhydrogenase) showed little or no immunoferritin labeling. These results are consistent with the idea that gluthalione-insulin transhydrogenase probably synthesized in the endoplasmic reticulum and that the transhydrogenase accessible to cell surface (or found in the isolated plasma membrane preparations) probably represents a functional continuity between the endoplasmic reticulum and the plasma membrane.

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Modulation of 125I-insulin degradation by receptors in liver plasma membranes

Abstract

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Comm

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Comm

Modulation of ¹²⁵I-insulin degradation by receptors in liver plasma membranes