Degradation of endocytosed insulin in rat liver is mediated by low-density vesicles

Pease, Richard J.; Smith, Geoffrey D.; Peters, Timothy J.

doi:10.1042/bj2280137

Cited by 53 publications

(32 citation statements)

References 37 publications

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“…On chromatography, most of the degraded insulin eluted as monoiodotyrosine, one major expected product of A chain degradation with the radioiodinated insulin used. These findings extend previous observations (6,121 and suggest that insulin degradation in isolated endosomes may generate similar products to those identified itz vivo [ 111.…”

Section: Discussionsupporting

confidence: 80%

“…Upon incubation at 30°C in buffered isotonic sucrose or KCl, a further loss of insulin integrity occurred, in accordance with the studies of Pease et al [6,121. At pH 5.5, this process followed first-order kinetics until about 40% of the 12sI-insulin present initially had been degraded (Fig.…”

Section: Characteristics Of the Degradation Of Insulin Associated Witsupporting

confidence: 78%

“…When endosomes containing internalized 1251-insulin were incubated at 30'C in isotonic KCl, a rapid degradation of insulin occurred, with a maximum at pH [5][6]. As with a number of insulin degrading systems [2,31, the loss of insulin integrity was more rapid when assessed by antibody-or receptor-binding ability than by trichloroacetic acid precipitation or Sephadex G-50 chromatography.…”

Section: Discussionmentioning

confidence: 99%

“…This assay was carried out both on intact endosomes and on endosomes permeabilized by Brij 35 (0.1 %, mass/vol.) as described by Pease et al [6,121. It was assumed that, in intact endosomes, both receptor-bound and free intraluminal insulin were poly (-ethyleneglyco1)-precipitable, whereas in permeabilized endosomes, only receptor-bound insulin was poly(ethyleneglyco1)-precipitable.…”

Section: Assay For Insulin Dissociation F R O M Liver Endosomesmentioning

confidence: 99%

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Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

Desbuquois¹,

Janicot²,

Dupuis³

1990

European Journal of Biochemistry

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The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 1251-insulin in isotonic KCl at 30°C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5 -6 ( t 1 , 2 : 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglyco1). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5 -8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (dpH about 0.8 -0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of CI-, other anions being less effective (Br-> gluconate = SO:-> NO; = sucrose = mannitol) and/or inhibitory (NO;). Na', K + and Li' supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5 -9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulinreceptor complex, is required for optimum degradation of insulin within liver endosomes.Biochemical and morphological studies on intact liver and isolated hepatocytes have shown that, following binding to its receptor at the cell surface, insulin undergoes rapid endocytosis along with its receptor (reviewed in [l -31). Subsequently, the receptor is recycled in part to the cell surface, while most of the insulin is degraded within the cell. Several lines of evidence indicate that, although initially thought to occur in lysosomes, degradation of internalized insulin occurs mainly in endosomes. First, little association of insulin with authentical lysosomes is demonstr...

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Section: Discussionsupporting

confidence: 80%

Section: Characteristics Of the Degradation Of Insulin Associated Witsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Assay For Insulin Dissociation F R O M Liver Endosomesmentioning

confidence: 99%

See 2 more Smart Citations

Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

Desbuquois¹,

Janicot²,

Dupuis³

1990

European Journal of Biochemistry

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“…Cell-free studies with intact endosomes by Diment and Stahl [1] and Pease et al [2] provided indication that endosomes might be proteolytically active against internalized mannosylated bovine serum albumin (mannose-BSA) and insulin (Fig. 1).…”

Section: Early Observationsmentioning

confidence: 99%

Endosomal proteolysis of internalized proteins

1996

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Endosomal proteases have been implicated in the degradation of internalized regulatory peptides involved in the control of metabolic pathways and in the processing of intracellular antigens for cytolytic immune responses. Processing in the endocytic vesicles is regulated by changes in endosomal acidity due to the presence of an ATP-dependent proton pump which modulates protease activity, protein unfolding and receptor-ligand interactions. A limited number of proteases appear to reside in endosomes which do not contain the full complement of active proteases capable of completely degrading all internalized polypeptides. Retention of some acid hydrolases in endosomes is apparently related to their association with undefined endosomal membrane receptors. The limited number of proteases and the pH gradient from neutral to acidic (pH 7 to 5) within endosomes make possible a selective and controlled processing environment in comparison to lysosomes. The full set of endo-and exopeptidases that break down proteins to amino acids are active later in the pathway in lysosomes.Key words: Endosomes; Endocytosis; Major histocompatibility complex class II; Antigen processing; Endopeptidase; Cathepsin (insulin, glucagon, epidermal growth factor (EGF), invariant chain (Ii), antibodies and vitellogenin) and activation (lysosomal hydrolases, parathyroid hormone (PTH), protein antigens and toxins) of internalized proteins. The intraendosomal cleavage of proteins following endocytosis has been studied in a large number of cells, especially hepatocytes, macrophages, B-lymphocytes, fibroblasts and oocytes. These studies have raised interesting questions regarding the extent of endosomal proteolysis in different cell types, the nature and specificity of endosomal proteases, the points in the endocytic pathway where acid hydrolases are delivered and the mechanisms by which proteases are permanently retained within endosomes. Furthermore, recent studies have demonstrated that the cysteine protease cathepsins B and H, and the aspartic protease cathepsin D play a particularly important role in endosomal proteolysis. The present review focuses on the endosomal pathways involved in the processing of polypeptide hormones and internalized antigenic proteins. Proteolytic modifications of internalized proteins in endosomes

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Liver Receptors for Regulatory Peptides

Rosselin

1989

Comprehensive Physiology

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Degradation of endocytosed insulin in rat liver is mediated by low-density vesicles

Cited by 53 publications

References 37 publications

Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

Endosomal proteolysis of internalized proteins

Liver Receptors for Regulatory Peptides

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