Chapter One - Assessment of Insulin Proteolysis in Rat Liver Endosomes Its Relationship to Intracellular Insulin Signaling.pdf.pdf

CHAPTER ONE
Assessment of Insulin Proteolysis
in Rat Liver Endosomes: Its
Relationship to Intracellular
Insulin Signaling
François Authier*,1, Bernard Desbuquois†
*Service information scientifique et technique (IST) de l’Inserm, De´le´gation re´gionale Inserm Paris V, Paris, France
†Inserm U567, Institut Cochin, CNRS UMR 8104, Universite´ Paris-Descartes, Paris, France
1Corresponding author: e-mail address: francois.******@
Contents
1. Introduction 4
2. Assays for Insulin Degradation 5
Trichloroacetic acid precipitation assay 5
Reverse-phase HPLC assay using radiolabeled insulin 6
Reverse-phase HPLC assay using native insulin 6
3. Assay for Endosomal Proteolysis of Insulin 7
In vivo studies 7
Cell-free endosome studies 8
In vitro studies 9
4. Sites of Cleavage and Degradation Products of Insulin within Endosomes 12
5. Endosomal Insulinases 14
Cathepsin D 14
Neutral Arg aminopeptidase 14
Insulin-degrading enzyme 15
6. Effects of Insulin and Slowly Processed Insulin Analogs on Insulin Signaling in
Liver Endosomes 15
Endosomal IR tyrosine phosphorylation 15
Translocation of insulin-signaling proteins to endosomes 18
7. Involvement of IR Endocytosis in Positive Regulation of Insulin Signaling 20
References 21
Abstract
Insulin binding to insulin receptor (IR) at the cell surface results in the activation of IR
kinase and initiates the translocation of insulin–plexes to clathrin-coated pits
and to early endosomes containing internalized but still active receptors. In liver paren-
chyma, several mechanisms are involved in the regulation of endosomal IR tyrosine
kinase activity. Two of these regulatory mechanisms are at the level of intraendosomal
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Methods in Enzymology, Volume 535 2014 Elsevier Inc. 3
ISSN 0076-6879 All rights reserved.
http://dx./-0-12-397925--8
4 François Authier and Bernard Desbuquois
ligand. First, a progressive decrease