ACUTE REGULATION OF GLUT1 FUNCTION: THE ROLE OF DETERGENT-RESISTANT MEMBRANE DOMAINS by DARRELL RUBIN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Dr. Faramarz Ismail-Beigi Department of Pathology CASE WESTERN RESERVE UNIVERSITY August 2004 Copyright © 2004 by Darrell Casimir Rubin All rights reserved CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication To my family and all the people providing inspiration or motivation along the way. Table of Contents Table of Contents …………………………………………………………………. v List of Tables and Figures ………………………………………………………... vii Acknowledgements ………………………………………………………………... ix List of Abbreviations ……………………………………………………………... x Abstract ……………………………………………………………………………. xi Chapter 1 Introduction Members of the Glucose Transporter Family …………………….. 1 Clone 9 cells and the Regulation of Glucose Transport ………….. 6 Protocols for Subcellular Fractionation …………………………... 8 Glucose Transporter Compartmentalization ……………………… 16 The Activation of Glucose Transporters ………………………….. 20 Membrane Microdomains and Glucose Transport Regulation …… 31 Chapter 2 Distribution of Glut1 in Detergent-Resistant Membranes (DRMs) and non-DRM domains: Effect of Treatment with Azide Introduction ……………………………………………………….. 52 Materials and Methods ……………………………………………. 55 Results …………………………………………………………….. 60 Discussion ………………………………………………………… 65 v Chapter 3 Distribution of Glut1 in Clone 9 cells and to Plasma Membrane DRM and non-DRM Domains: Effect of Azide Introduction ……………………………………………………….. 79 Materials and Methods ……………………………………………. 82 Results …………………………………………………………….. 89 Discussion ………………………………………………………… 94 Chapter 4 The Effects of Methyl-β-Cyclodextrin on Glut1 and Glucose Transport Introduction ……………………………………………………….. 117 Materials and Methods ……………………………………………. 120 Results …………………………………………………………….. 123 Discussion ………………………………………………………… 125 Chapter 5 Summary and Future Studies Summary ………………………………………………………….. 140 Future Studies ……………………………………………………... 141 Works Cited ……………………………………………………………………... 145 vi List of Figures and Tables Table 1.1 The glucose transporter family ………………………………... 37 Figure 1.1 Schematics of SLCA2 glucose transporters …………………... 39 Figure 1.2 Dendrogram SLCA2 family glucose transporters ……………... 41 Figure 1.3 Protocols for subcellular fractionation ………………………… 43 Figure 2.1 Low density DRMs in Triton X-100 cell lysate ………………... 69 Figure 2.2 DRMs in cell lysate produced with other detergents …………... 71 Figure 2.3 Low density DRMs in Triton X-100 cell lysate: Azide effect … 73 Table 2.1 The azide effect on glucose transport and Glut1 in the DRMs ... 75 Figure 2.4 Low density DRMs in the plasma membrane: Azide effect …… 77 Figure 3.1 Electron microscopy of preparative gradient fractions …………. 101 Figure 3.2 Protein distribution in Clone 9 cell post-nuclear homogenate …... 103 Table 3.1 Protein distribution in Clone 9 cell post-nuclear homogenate …. 104 Figure 3.3 Glut1 colocalization with other proteins ………………………… 106 Table 3.2 Glut1 colocalization with other proteins ………………………... 107 Figure 3.4 Caveolin-1 in plasma membrane microdomains ………………... 109 Figure 3.5 Glut1 in plasma membrane microdomains: Azide effect ………. 111 Figure 3.6 Glut1 distribution in plasma membrane microdomains …………. 113 Figure 3.7 Glut1 content in plasma membrane microdomains ……………... 115 Table 3.3 Glut1 in plasma membrane microdomains: Azide effect ……… 116 vii Figure 4.1 mβcd blocks azide – induced glucose transport ………………… 130 Table 4.1 Glucose transport by inhibitors of respiration blocked by mβcd ... 131 Figure 4.2 mβcd-treated Clone 9 cells can recover the azide response ……... 133 Figure 4.3 mβcd blocks glucose transport stimulated by ETC inhibition …... 135 Figure 4.4 mβcd decreases Glut1 association with the DRM ………………. 137 Table 4.2 mβcd decreases Glut1 association with the DRM ………………. 139 viii Acknowledgements I thank Dr. Faramarz Ismail – Beigi for his patient guidance and his generous dedication to my education and progress. The other members of my committee Dr. George Dubyak, Dr. Alan Levine, Dr. Sanjay Pimplikar, and Dr. Alan Tartakoff contributed their time, expertise, and guidance for which I am grateful. The department of Pathology, led first by Dr. Micheal Lamm and succeeded by Dr. George Perry, has been a great place for my training. I have had the pleasure of learning from two laboratories during the course of my graduate education and both provided numerous experiences which have helped my development as a scientist tremendously. I also thank my immediate family for their unwavering support during this time in my career as a student. Of my friends, I especially thank Michael Payne for his support. Finally, I acknowledge the staff and director of the Medical Scientist Training Program, especially former program coordinator Felicite Katz and director emeritus Dr. John Nilson, for their support. ix List of Abbreviations Homog homogenization SDH succinic dehydrogenase M-J McKeel and Jarret DRM detergent – resistant membrane PM plasma membrane LDM low density membrane HDM high density membrane ETC electron transport chain 3H tritium x ACUTE REGULATION OF GLUT1 FUNCTION: THE ROLE OF DETERGENT-RESISTANT MEMBRANE DOMAINS Abstract by DARRELL RUBIN Identifying which processes and proteins control glucose transport could provide important clues to understanding and treating a number of clinical entities including diabetes and some cancers. Glucose transport across the plasma membrane occurs by either sodium-dependent or independent glucose transporters. In order to study the mechanisms which control acute changes in glucose transport by sodium-independent glucose transporters, we use the non-transformed rat liver – derived Clone 9 cell line. These cells respond to the acute inhibition of oxidative phosphorylation by azide with a 4-6 fold stimulation of glucose transport and a 1.8 fold increase in the amount of glucose transporter 1 (Glut1) in the plasma membrane. In Clone 9 cells under basal conditions, ~38 % of Glut1 in the post-nuclear lysate is localized to the detergent-resistant membrane (DRM) microdomains. Acute exposure to azide decreased this figure by ~40 %. In order to examine the effects of azide on Glut1 localization to the plasma membrane of the Clone 9 cell, we performed subcellular fractionation of the post-nuclear homogenates. Approximately 30 % of the Glut1 in the post-nuclear homogenate was recovered in the plasma membrane (PM) compartment and 50 % of this PM Glut1 localized to the DRM fraction. Acute inhibition of oxidative phosphorylation with azide xi resulted in a 1.6-fold increase in the total abundance of Glut1 in the PM and was associated with a 2.9 fold increase in the abundance of Glut1 in the non-DRM fraction but no significant change in the content of Glut1 in the DRM fraction. We conclude that in the Clone 9 cell Glut1 localizes to detergent-resistant membrane microdomains in the plasma membrane. Moreover, in these cells the azide – induced increase in glucose transport is associated with an increase of Glut1 abundance in the non – DRM fraction of the plasma membrane and a decrease of Glut1 association with the DRM fraction of a membrane compartment other than the plasma membrane. These findings indicate that the distribution of Glut1 to the DRM and non-DRM domains of the cellular membrane compartments contribute, in part, to the mechanisms of glucose transport regulation in response to the acute inhibition of oxidative phosphorylation. xii Chapter 1 Introduction Abnormal responses to increased blood glucose are an important clinical signal suggesting the patient may be at risk for or already suffer from pathological processes leading to diabetes (Bjornholt et al., 2001). It has also been suggested that the prognosis of patients with certain tumors correlates with the expression of Glut1 (Kurokawa et al., 2004; Vordermark et al., 2003). Identifying which processes and proteins control glucose transport may facilitate the development of improved therapies for managing the course of disease in these patients. Members of the Glucose Transporter Family There are many members of the facilitative glucose transporter family (gene name Solute Carrier (SLCA) 2A) designated Glut1 – 14 including the proton/myoinositol co- transporter (HMIT) (Table 1.1) (Joost et al., 2001; Wood et al., 2003b). These integral membrane proteins have 12 transmembrane domains and a glycosylation site present in one extracellular loop. The most recent additions to the Glut family were identified based on the greater than 28% identity of their primary sequences with that of Glut1 and the presence of the so-called “sugar transporter signature” sequences (Figure 1.1) which are conserved among Glut1 – 5 (Joost et al., 2001). The family was subdivided into three classes based on sequence identity (Figure 1.2). The following short discussion regarding the Glut family summarizes the