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J Cell Biol. Jan 1, 1990; 110(1): 35–42.
PMCID: PMC2115978

Selective degradation of insulin within rat liver endosomes

Abstract

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p- chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Affholter JA, Fried VA, Roth RA. Human insulin-degrading enzyme shares structural and functional homologies with E. coli protease III. Science. 1988 Dec 9;242(4884):1415–1418. [PubMed]
  • Ajioka RS, Kaplan J. Characterization of endocytic compartments using the horseradish peroxidase-diaminobenzidine density shift technique. J Cell Biol. 1987 Jan;104(1):77–85. [PMC free article] [PubMed]
  • Anderson RG, Orci L. A view of acidic intracellular compartments. J Cell Biol. 1988 Mar;106(3):539–543. [PMC free article] [PubMed]
  • Baudhuin P, Evrard P, Berthet J. Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles. J Cell Biol. 1967 Jan;32(1):181–191. [PMC free article] [PubMed]
  • Bergeron JJ, Sikstrom R, Hand AR, Posner BI. Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study. J Cell Biol. 1979 Feb;80(2):427–443. [PMC free article] [PubMed]
  • Bergeron JJ, Rachubinski R, Searle N, Borts D, Sikstrom R, Posner BI. Polypeptide hormone receptors in vivo: demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography. J Histochem Cytochem. 1980 Aug;28(8):824–835. [PubMed]
  • Bergeron JJ, Rachubinski RA, Sikstrom RA, Posner BI, Paiement J. Galactose transfer to endogenous acceptors within Golgi fractions of rat liver. J Cell Biol. 1982 Jan;92(1):139–146. [PMC free article] [PubMed]
  • Bergeron JJ, Cruz J, Khan MN, Posner BI. Uptake of insulin and other ligands into receptor-rich endocytic components of target cells: the endosomal apparatus. Annu Rev Physiol. 1985;47:383–403. [PubMed]
  • Bergeron JJ, Paiement J, Khan MN, Smith CE. Terminal glycosylation in rat hepatic Golgi fractions: heterogeneous locations for sialic acid and galactose acceptors and their transferases. Biochim Biophys Acta. 1985 Dec 19;821(3):393–403. [PubMed]
  • Bergeron JJ, Searle N, Khan MN, Posner BI. Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin. Biochemistry. 1986 Apr 8;25(7):1756–1764. [PubMed]
  • Blundell TL, Wood SP. Is the evolution of insulin Darwinian or due to selectively neutral mutation? Nature. 1975 Sep 18;257(5523):197–203. [PubMed]
  • Boulay F, Doms RW, Wilson I, Helenius A. The influenza hemagglutinin precursor as an acid-sensitive probe of the biosynthetic pathway. EMBO J. 1987 Sep;6(9):2643–2650. [PMC free article] [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Bretz R, Bretz H, Palade GE. Distribution of terminal glycosyltransferases in hepatic Golgi fractions. J Cell Biol. 1980 Jan;84(1):87–101. [PMC free article] [PubMed]
  • Brown WJ, Goodhouse J, Farquhar MG. Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes. J Cell Biol. 1986 Oct;103(4):1235–1247. [PMC free article] [PubMed]
  • Carpenter G, Cohen S. Epidermal growth factor. Annu Rev Biochem. 1979;48:193–216. [PubMed]
  • Carpenter G, Lembach KJ, Morrison MM, Cohen S. Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts. J Biol Chem. 1975 Jun 10;250(11):4297–4304. [PubMed]
  • Carpentier JL, Gorden P, Freychet P, Le Cam A, Orci L. Lysosomal association of internalized 125I-insulin in isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography. J Clin Invest. 1979 Jun;63(6):1249–1261. [PMC free article] [PubMed]
  • Cuatrecasas P. Isolation of the insulin receptor of liver and fat-cell membranes (detergent-solubilized-( 125 I)insulin-polyethylene glycol precipitation-sephadex). Proc Natl Acad Sci U S A. 1972 Feb;69(2):318–322. [PMC free article] [PubMed]
  • De Meyts P, Van Obberghen E, Roth J. Mapping of the residues responsible for the negative cooperativity of the receptor-binding region of insulin. Nature. 1978 Jun 15;273(5663):504–509. [PubMed]
  • Desbuquois B, Lopez S, Burlet H. Ligand-induced translocation of insulin receptors in intact rat liver. J Biol Chem. 1982 Sep 25;257(18):10852–10860. [PubMed]
  • Diment S, Stahl P. Macrophage endosomes contain proteases which degrade endocytosed protein ligands. J Biol Chem. 1985 Dec 5;260(28):15311–15317. [PubMed]
  • Diment S, Leech MS, Stahl PD. Cathepsin D is membrane-associated in macrophage endosomes. J Biol Chem. 1988 May 15;263(14):6901–6907. [PubMed]
  • Duckworth WC. Insulin degradation: mechanisms, products, and significance. Endocr Rev. 1988 Aug;9(3):319–345. [PubMed]
  • Duckworth WC, Kitabchi AE. Insulin metabolism and degradation. Endocr Rev. 1981 Spring;2(2):210–233. [PubMed]
  • Duckworth WC, Hamel FG, Peavy DE. Hepatic metabolism of insulin. Am J Med. 1988 Nov 28;85(5A):71–76. [PubMed]
  • Duckworth WC, Hamel FG, Liepnieks JJ, Peavy DE, Ryan MP, Hermodson MA, Frank BH. Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes. Biochem Biophys Res Commun. 1987 Sep 15;147(2):615–621. [PubMed]
  • Duckworth WC, Hamel FG, Peavy DE, Liepnieks JJ, Ryan MP, Hermodson MA, Frank BH. Degradation products of insulin generated by hepatocytes and by insulin protease. J Biol Chem. 1988 Feb 5;263(4):1826–1833. [PubMed]
  • Dunn WA, Hubbard AL. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J Cell Biol. 1984 Jun;98(6):2148–2159. [PMC free article] [PubMed]
  • ELGEE NJ, WILLIAMS RH, LEE ND. Distribution and degradation studies with insulin I131. J Clin Invest. 1954 Sep;33(9):1252–1260. [PMC free article] [PubMed]
  • Geuze HJ, Stoorvogel W, Strous GJ, Slot JW, Bleekemolen JE, Mellman I. Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles. J Cell Biol. 1988 Dec;107(6 Pt 2):2491–2501. [PMC free article] [PubMed]
  • Glickman J, Croen K, Kelly S, Al-Awqati Q. Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance. J Cell Biol. 1983 Oct;97(4):1303–1308. [PMC free article] [PubMed]
  • Goldfine ID. The insulin receptor: molecular biology and transmembrane signaling. Endocr Rev. 1987 Aug;8(3):235–255. [PubMed]
  • Hamel FG, Posner BI, Bergeron JJ, Frank BH, Duckworth WC. Isolation of insulin degradation products from endosomes derived from intact rat liver. J Biol Chem. 1988 May 15;263(14):6703–6708. [PubMed]
  • HUNTER WM, GREENWOOD FC. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. [PubMed]
  • Karnovsky MJ. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol. 1967 Oct;35(1):213–236. [PMC free article] [PubMed]
  • Kay DG, Khan MN, Posner BI, Bergeron JJ. In vivo uptake of insulin into hepatic Golgi fractions: application of the diaminobenzidine-shift protocol. Biochem Biophys Res Commun. 1984 Sep 28;123(3):1144–1148. [PubMed]
  • Kay DG, Lai WH, Uchihashi M, Khan MN, Posner BI, Bergeron JJ. Epidermal growth factor receptor kinase translocation and activation in vivo. J Biol Chem. 1986 Jun 25;261(18):8473–8480. [PubMed]
  • Khan RJ, Khan MN, Bergeron JJ, Posner BI. Prolactin uptake into liver endocytic components. Reduced sensitivity to chloroquine. Biochim Biophys Acta. 1985 Jan 28;838(1):77–83. [PubMed]
  • Khan MN, Savoie S, Bergeron JJ, Posner BI. Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures. J Biol Chem. 1986 Jun 25;261(18):8462–8472. [PubMed]
  • Khan MN, Baquiran G, Brule C, Burgess J, Foster B, Bergeron JJ, Posner BI. Internalization and activation of the rat liver insulin receptor kinase in vivo. J Biol Chem. 1989 Aug 5;264(22):12931–12940. [PubMed]
  • Lai WH, Cameron PH, Wada I, Doherty JJ, 2nd, Kay DG, Posner BI, Bergeron JJ. Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo. J Cell Biol. 1989 Dec;109(6 Pt 1):2741–2749. [PMC free article] [PubMed]
  • Matrisian LM, Planck SR, Magun BE. Intracellular processing of epidermal growth factor. I. Acidification of 125I-epidermal growth factor in intracellular organelles. J Biol Chem. 1984 Mar 10;259(5):3047–3052. [PubMed]
  • Mellman I, Fuchs R, Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. [PubMed]
  • Pease RJ, Smith GD, Peters TJ. Characterization of insulin degradation by rat-liver low-density vesicles. Eur J Biochem. 1987 Apr 1;164(1):251–257. [PubMed]
  • Planck SR, Finch JS, Magun BE. Intracellular processing of epidermal growth factor. II. Intracellular cleavage of the COOH-terminal region of 125I-epidermal growth factor. J Biol Chem. 1984 Mar 10;259(5):3053–3057. [PubMed]
  • Posner BI, Josefsberg Z, Bergeron JJ. Intracellular polypeptide hormone receptors. Characterization of insulin binding sites in Golgi fractions from the liver of female rats. J Biol Chem. 1978 Jun 10;253(11):4067–4073. [PubMed]
  • Posner BI, Patel B, Verma AK, Bergeron JJ. Uptake of insulin by plasmalemma and Golgi subcellular fractions of rat liver. J Biol Chem. 1980 Jan 25;255(2):735–741. [PubMed]
  • Posner BI, Patel BA, Khan MN, Bergeron JJ. Effect of chloroquine on the internalization of 125I-insulin into subcellular fractions of rat liver. Evidence for an effect of chloroquine on Golgi elements. J Biol Chem. 1982 May 25;257(10):5789–5799. [PubMed]
  • Roederer M, Bowser R, Murphy RF. Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes. J Cell Physiol. 1987 May;131(2):200–209. [PubMed]
  • Savage CR, Jr, Inagami T, Cohen S. The primary structure of epidermal growth factor. J Biol Chem. 1972 Dec 10;247(23):7612–7621. [PubMed]
  • Savoie S, Rindress D, Posner BI, Bergeron JJ. Tunicamycin sensitivity of prolactin, insulin and epidermal growth factor receptors in rat liver plasmalemma. Mol Cell Endocrinol. 1986 May;45(2-3):241–246. [PubMed]
  • Scharschmidt BF, Van Dyke RW. Proton transport by hepatocyte organelles and isolated membrane vesicles. Annu Rev Physiol. 1987;49:69–85. [PubMed]
  • Schaudies RP, Gorman RM, Savage CR, Jr, Poretz RD. Proteolytic processing of epidermal growth factor within endosomes. Biochem Biophys Res Commun. 1987 Mar 13;143(2):710–715. [PubMed]
  • Shii K, Roth RA. Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4147–4151. [PMC free article] [PubMed]
  • Shii K, Baba S, Yokono K, Roth RA. Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme. J Biol Chem. 1985 Jun 10;260(11):6503–6506. [PubMed]
  • Simionescu N, Simionescu M. Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect. J Cell Biol. 1976 Sep;70(3):608–621. [PMC free article] [PubMed]
  • Smith CE, Paiement J, Bergeron JJ. Subcellular distribution of acid NADPase activity within the parenchymal cells of rat liver. J Histochem Cytochem. 1986 May;34(5):649–658. [PubMed]
  • Wileman T, Harding C, Stahl P. Receptor-mediated endocytosis. Biochem J. 1985 Nov 15;232(1):1–14. [PMC free article] [PubMed]
  • Wiley HS, VanNostrand W, McKinley DN, Cunningham DD. Intracellular processing of epidermal growth factor and its effect on ligand-receptor interactions. J Biol Chem. 1985 May 10;260(9):5290–5295. [PubMed]
  • Yokono K, Roth RA, Baba S. Identification of insulin-degrading enzyme on the surface of cultured human lymphocytes, rat hepatoma cells, and primary cultures of rat hepatocytes. Endocrinology. 1982 Oct;111(4):1102–1108. [PubMed]

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