ENGAGEMENT OF THE INSULIN-SENSITIVE PATHWAY IN THE STIMULATION OF GLUCOSE TRANSPORT BY a-LIPOIC ACID. Karen Lynne Yaworsky A M. Sc. thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto O Copyright by Karen Yaworsky 1999 National Library Bibliothèque nationale of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services senrices bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON KIA ON4 OtiawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distibute or sel1 reproduire, prêter, distribuer ou copies of ths thesis in microfonn, vendre des copies de cette thèse sous paper or electronic fomats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur consenre la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be prhted or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ENGAGEMENT OF THE INSULIN-SENSITIVE PATHWAY IN THE STIMULATION OF GLUCOSE TRANSPORT BY a-LIPOIC ACID. A MeSc. thesis by Karen Lynne Yaworsky submitted in conformity with the requirements for the degree of Master of Science, Graduate Department of Biochemistry, University of Toronto, 1999. ABSTRACT A pnmary metabolic response to insulin is the acute stimulation of glucose transport in muscle and adipose tissue. Activation of an intracellular signalling cascade by insulin results in recruitment of glucose transporters to the plasma membrane; a process impaired in type 2 diabetes. In search of other relevant insulin-mimetic agents our attention has focused on a-lipoic acid. a- Lipoic acid, a cofactor of oxidative rnetabolism and a potent antioxidant, was shown to enhance insulin-stimulated glucose rnetabolism and to stimulate glucose transport. Therefore, in an attempt to undentand the mechanism underlying the stimulation of glucose transport by a-lipoic acid, the effect of this compound on glucose transporter localization and intracellular signals involved in the stimulation of glucose transport were examined. The results presented in this thesis suggest that a-lipoic acid stimulates glucose transport in a unique manner, by directly targetting elements of the insulin-sensitive signalling pathway. The work presented in this M.Sc. thesis was perfonned from 1997- 1999 in the Programme in Ce11 Biology, The Hospital for Sick Children, Toronto, ON, Canada, under the supervision of Dr. Amira Klip. Financial support was provided by the Canadian Diabetes Association and ASTA Medica, Germany. The results of this Thesis have been presented in one publication: K. Yaworsky, R. Somwar, T. Ramlal, H.J.Triischler, and A. Klip. 1999. Unique action of an anti-diabetic agent: Engagement of the insulin-sensitive pathway in the stimulation of glucose ansport by a-lipoic acid. Diabetologia -- Submitted. (Chapter one) ACKNOWLEDGMENTS A mere note of thanks cannot justifiably express my profound gratitude to everyone who has helped me with this degree. To begin with, 1 would like to thank the members of the Klip lab, past and present, for making the everyday so enjoyable. Special thanks to my 'big sister' Celia for continual support; Leonard, for your patience when explaining how a computer actually works; Toolsie, for laughter and for always having a solution; and to Rome1 for answering my endless questions. 1 would also like to thank the rnembers of my advisory committee and especially rny supervisor, Amira Klip. Arnim's enthusiasm, generosity and continual support are unparalleled. Lastly, to my greatest supporters, Jonathan, Dodie, Kim and Alberta - words cannot even begin to express my sincere appreciation. 1 really could not have achieved this without you. TABLE OF CONTENTS ABSTRACT ...................................................................................II PREFACE..................................................................................... III ACKNOWLEDGMENTS .................................................................IV TABLE OF CONTENTS................................................................. V LIST OF TABLES .........................................................................IX LIST OF FIGURES .......................................................................X LIST OF ABBREVIATIONS ...........................................................XII BACKGROUND ...............................................................................1 TYPE 2 DIABETES .....................................................................1 BIOLOGICAL ACTIONS OF WSULIN .............................................4 Glucose Transport And Transporters .........................................4 Structure. Function and Tissue-Specific Expression of Glucose Transporter Isoforms.................................................. 6 GLUT 1 .........................................................6 GLUT4 .........................................................6 Acute Regulation of Glucose Transport by Insulin ................. 9 SIGNALLING MECHANISMS REGULATING INSULIN-STIMULATED GLUCOSE TRANSPORT ..............................................................10 The Insulin Receptor ............................................................12 Insulin Receptor Subsuate Proteins ...........................................15 Insulin Receptor Substrate-1 (IRS- 1)................................ 16 lnsulin Receptor Substrate-2 (ES-2) ................................20 Insulin Receptor Substrate-3 (IRS-3) ................................21 Insulin Receptor Substrate-4 (IRS-4) ................................21 Role of lnsulin Receptor Substrate Proteins in Insulin-Stimulated Glucose Transport ......................................................22 Phosphatidylinositol 3-Kinase (PI 3-kinase) ................................. 24 Pharmacological Inhibitors of PI 3-Kinase .......................... 26 The Role of PI 3-kinase in Insulin-Regulated Glucose Transport. 27 Signals Downstream of PI 3-kinase: lnvolvement of Senne and Threonine Kinases ...........................................................................30 Akt ......................................................................-30 Role of Akt in the Stimulation of Glucose Transport by Insulin .........................................................-35 Atypical PKC's: Emerging Role in Insulin-Stimulated Glucose Transport ................................................................36 GLUCOSE TRANSPORTERS IN TYPE 2 DIABETES ...........................39 GLUT4 Expression ............................................................-39 GLUT4 Translocation ..........................................................41 Insulin Signalling ................................................................41 Factors That May Trigger Insulin Resistance ...............................$43 Anti-Diabetic Drugs: Potential nierapies for Insulin Resistance and Type 2 Diabetes ...........................................................................46 a-Lipoic Acid ..........................................................-47 CHAPTER ONE................................................................................ 52 RATION ALE AND HYPOTHESIS ...................................................53 EXPERIMENTAL PROCEDURES ...................................................55 Materials ..........................................................................55 Methods .........................................................................-56 Cell Culture and Incubations ..........................................56 2-~eox~-3~-~-~lucoseUptake ...................................... 56 SubcelluIar Fractionation of 3T3-L1Adipocytes ...................57 Immunoprecipitation and Assay of Phosphatidylinositol 3-Kinase Activity ..................................................................57 Immunoprecipitation and Assay of Aktl Protein Kinase Activity . 58 Detection of Insulin Receptor Substrate- 1 Phosphorylation....... 59 Detection of Insulin Receptor Phosphorylation .....................60 Insulin Receptor Autophosphorylation ..............................60 Statistical Analysis..................................................... 61 RESULTS ................................................................................-62 a-Lipoic Acid Stimulates Glucose Uptake in 3T3-L1 Adipocytes .......-62 The Effeci of a-Lipoic acid on Insulin-Stimulated Glucose Uptake in 3T3- Ll Adipocytes................................................................... 64 a-Lipoic Acid Stimulates the Translocation of GLUTl and GLUT4 to the Plasma Membrane ...............................................................66 Wortmannin Prevents the Stimulation of Glucose Transport by cc-Lipoic Acid in 3T3-L1 Adipocytes................................................... -69 Activation of PI 3-Kinase by a-Lipoic Acid in 3T3-L 1 Adipocytes...... 71 Effect of a-Lipoic Acid on Akt 1 Activity in 3T3-L1 Adipocytes .......... 73 Effect of Erbstatin on a-Lipoic Acid-Stimulated Glucose Uptake in 3T3-LI Adipocytes.....................................................................