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1.4 Glucose Transport Koester, Anna Magdalena (2021) The spatial dynamics of insulin-regulated GLUT4 dispersal. PhD thesis. http://theses.gla.ac.uk/82077/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] The spatial dynamics of insulin- regulated GLUT4 dispersal Anna Magdalena Koester MSci, MRes Thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Institute of Molecular, Cell and Systems Biology College of Medical, Veterinary and Life Sciences University of Glasgow January 2021 ii Abstract Insulin regulates glucose homeostasis by stimulation of glucose transport into adipose and muscle tissues through the regulated trafficking of glucose transporter 4 (GLUT4). In response to insulin GLUT4 rapidly translocates from intracellular storage sites to the plasma membrane where it facilitates glucose uptake. Significant impairments in glucose transport and GLUT4 trafficking are a major hallmark of diabetes mellitus type II. Recent advances in light microscopy techniques enabled the study of GLUT4 dynamics in the plasma membrane and it was reported that the transporter was clustered in the basal state and insulin stimulation resulted in GLUT4 dispersal. The main aim of this study was to develop a microscopy-based assay to study and quantify insulin-stimulated GLUT4 dispersal dynamics in the plasma membrane. Insulin- stimulated GLUT4 dispersal has only been observed in adipocytes and therefore we have chosen this model as a starting point to investigate the molecular mechanisms behind GLUT4 clustering and dispersal. We explored a range of cluster analysis methods to find the most suitable way to quantify GLUT4 clustering dynamics. Furthermore, this project aimed to optimise super resolution imaging in a variety of cell culture models to determine whether insulin-stimulated GLUT4 dispersal operates in skeletal and cardiac muscle and whether this process is affected by disease. Using a range of approaches we showed that insulin results in GLUT4 translocation and dispersal within the plasma membrane of 3T3-L1 adipocytes. We found that AMPK activation attenuated insulin-stimulated glucose uptake in 3T3-L1 adipocytes and also GLUT4 dispersal. It was observed that cholesterol depletion resulted in increased glucose uptake rates and GLUT4 clustering. Knock down of the membrane-localised protein EFR3 that has previously been shown to be involved in glucose uptake resulted in disruption of GLUT4 dispersal in adipocytes. We also found that HeLa cells show similar insulin-stimulated GLUT4 dispersal as adipocytes and suggest that HeLa cells are a suitable experimental model for initial studies of GLUT4 trafficking and dispersal. Chronic insulin treatment was observed to induce a state of cellular insulin resistance in 3T3-L1 adipocytes and resulted in reduced GLUT4 translocation and a more clustered GLUT4 configuration for both basal and insulin-stimulated cells. This indicates that insulin resistance affects intracellular GLUT4 trafficking pathways as well as the organization of the transporter within the plasma membrane in adipocytes. Moreover, we found a negative correlation between adipocyte cell area and insulin-stimulated GLUT4 translocation. iii We also report that insulin did not stimulate the reorganisation of the transferrin receptor in the plasma membrane of HeLa cells suggesting that insulin-stimulated GLUT4 dispersal did not originate from endosomal compartments in HeLa cells and that this observed effect may be specific for GLUT4. Finally, we observed that insulin did not affect GLUT4 distribution in the membrane of a commercially available model of skeletal muscle from healthy and diabetic donors. Sortilin is a sorting receptor involved in the formation of GLUT4 containing vesicles and levels of this protein were found to be reduced in skeletal muscle myotubes derived from a diabetic donor. Finally, we discovered that insulin stimulated GLUT4 dispersal also operates in stem cell-derived cardiomyocytes and have investigated GLUT4 dispersal in a variety of in vitro models of cardiac muscle tissue. Taken together, this thesis has detailed several novel findings regarding the regulation of GLUT4 clustering in adipose and muscle tissues. A robust assay to measure GLUT4 dispersal has been established and molecular mechanisms behind the observed GLUT4 clustering dynamics have been described in adipocytes. Furthermore GLUT4 clustering was characterised in several cell culture models of skeletal and cardiac muscle for the first time. iv Table of contents Abstract ............................................................................................... ii Table of contents ................................................................................... iv List of tables ...................................................................................... xi List of figures .................................................................................... xii Acknowledgements ............................................................................. xvii Author’s declaration ........................................................................... xix Definitions & abbreviations .................................................................... xx 1. Introduction ....................................................................................... 1 1.1 Glucose homeostasis ......................................................................... 1 1.2 Diabetes ....................................................................................... 3 1.2.1 Diabetes is a growing global healthcare burden.................................... 3 1.2.2 Complications ........................................................................... 3 1.2.3 Causes .................................................................................... 5 1.2.4 Treatment ............................................................................... 5 1.3 Diabetic cardiomyopathy ................................................................... 6 1.3.1 Recognition of diabetic cardiomyopathy ............................................ 6 1.3.2 Pathophysiology of diabetic cardiomyopathy ....................................... 6 1.3.3 Impaired insulin signalling in diabetic cardiomyopathy ........................... 7 1.4 Glucose transport ............................................................................ 8 1.4.1 Glucose transporters ................................................................... 8 1.4.2 Glucose transporter 4 .................................................................. 9 1.4.3 GLUT4 intracellular trafficking overview ........................................... 11 1.4.4 GLUT4 trafficking in muscle tissues ................................................. 14 1.4.5 GLUT4 endocytosis ..................................................................... 15 1.5 Insulin signalling ............................................................................ 15 1.5.1 Mitogen-activated protein (MAP) kinase signalling pathway ..................... 16 1.5.2 Insulin receptor substrate (IRS) signalling pathway ............................... 16 1.5.3 Adaptor protein with pleckstrin homology and Src homology ................... 17 domains (APS) signalling pathway .......................................................... 17 v 1.5.4 Insulin signalling in muscle tissues .................................................. 18 1.5.5 Insulin signalling defects in T2DM ................................................... 19 1.6 GLUT4 in the plasma membrane .......................................................... 20 1.6.1 Total internal fluorescence microscopy reveals plasma membrane GLUT4 dynamics ....................................................................................... 21 1.6.2 The effect of insulin action on plasma membrane GLUT4 dynamics ........... 23 1.7 Aims and hypothesis ........................................................................ 26 2. Materials and Methods .......................................................................... 29 2.1 Materials ..................................................................................... 29 2.1.1 Commercially derived cells ........................................................... 29 2.1.2 Primary cells ............................................................................ 30 2.1.3 Plasmids ................................................................................. 30 2.1.4 General materials and reagents ..................................................... 31 2.1.5 Solutions ................................................................................ 36 2.1.6 Primary Antibodies and Nanobodies ................................................. 38 2.1.7 Secondary Antibodies and Nanobodies .............................................. 39 2.2 Cell Culture Methods ......................................................................
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