Phosphorylation of Skeletal Muscle Pyruvate Dehydrogenase Phosphatase in Response to Insulin Stimulation Jonathan Robert Choptiany, B.Kin. Submitted in partial fulfillment of the requirements for the degree Master of Science in Applied Health Sciences (Kinesiology) Under the supervision of Sandra Peters, Ph.D. Faculty of Applied Health Sciences Brock University St. Catharines, Ontario, Canada Jonathan R. Choptiany© June 2013 M.Sc. Thesis – J. R. Choptiany Brock University Master of Science (2013) Title: Phosphorylation of Skeletal Muscle Pyruvate Dehydrogenase Phosphatase in Response to Insulin Stimulation Author: Jonathan R. Choptiany Supervisor: Dr. Sandra J. Peters Supervisory Committee: Dr. Paul J. LeBlanc Dr. Michael J. Plyley Dr. Brendon Gurd (External Examiner) Number of Pages: 176 ii M.Sc. Thesis – J. R. Choptiany Brock University ABSTRACT Pyruvate dehydrogenase phosphatase (PDP) regulates carbohydrate oxidation through the pyruvate dehydrogenase (PDH) complex. PDP activates PDH, enabling increased carbohydrate flux towards oxidative energy production. In culture myoblasts, both PDP1 and PDP2 undergo covalent activation in response to insulin–stimulation by protein kinase C delta (PKCδ). Our objective was to examine the effect of insulin on PDP phosphorylation and PDH activation in skeletal muscle. Intact rat extensor digitorum longus muscles were incubated (oxygenated at 25°C, 1g of tension) for 30min in basal or insulin–stimulated (10 mU/mL) media. PDH activity increased 58% following stimulation, (p=0.057, n=11). Serine phosphorylation of PDP1 (p=0.047) and PDP2 (p=0.006) increased by 29% and 48%, respectively (n=8), and mitochondrial PKCδ protein content was enriched by 45% in response to stimulation (p=0.0009, n=8). These data suggest that the insulin–stimulated increase in PDH activity in whole tissue is mediated through mitochondrial migration of PKCδ and subsequent PDP phosphorylation. iii M.Sc. Thesis – J. R. Choptiany Brock University ACKNOWLEDGEMENTS To Dr. LeBlanc & Dr. Plyley: a sincere thank you for everything that you have contributed to this thesis. Your expertise and time are valuable commodities, thank you for providing both to me. I am very fortunate to have worked alongside such professionals. To my lab mates: it has been a pleasure to work alongside you over the course of this project. You have all provided fantastic support while watching me pull my hair out on a daily basis in the lab. Thank you for being great friends. I can only hope that I have been as supportive to you, as you have to me. To Sandy: I could not have asked for a better mentor. I will never be able to thank you enough for the opportunity to complete this work, and for your patience throughout its completion. This degree represents a significant period of growth in my life. Thank you for supporting me at every step. iv M.Sc. Thesis – J. R. Choptiany Brock University TABLE OF CONTENTS Chapter 1 Introduction ........................................................................................................................ 1 Literature Review ............................................................................................................... 2 Pyruvate Dehydrogenase: The Background ........................................................... 2 PDH: Structural & Functional Synergy ................................................................. 5 PDH: Individual Reactions .................................................................................... 8 PDH: “Macro” Regulation ................................................................................... 11 PDH: “Micro” Regulation .................................................................................... 13 Rationale: Evidence for the Post–Transformational Regulation of PDP ............. 20 Chapter 2 Statement of the Problem ................................................................................................. 28 Purpose ............................................................................................................................. 28 Hypotheses ....................................................................................................................... 29 Chapter 3 Methods ............................................................................................................................ 30 Study Design ........................................................................................................ 30 Animals ................................................................................................................ 31 Surgeries .............................................................................................................. 32 Muscle Incubations .............................................................................................. 32 Mitochondrial Isolation ........................................................................................ 34 Immunoprecipitation ............................................................................................ 36 Western Blot Analysis ......................................................................................... 37 v M.Sc. Thesis – J. R. Choptiany Brock University Pyruvate Dehydrogenase Assay ........................................................................... 40 Acetyl–CoA Assay ............................................................................................... 41 Citrate Synthase Assay ........................................................................................ 43 Pyruvate Dehydrogenase Phosphatase Assay ...................................................... 44 Statistical Analysis ............................................................................................... 46 Chapter 4 Results .............................................................................................................................. 47 General Observations ........................................................................................... 47 Phospho–Akt Protein ........................................................................................... 48 PDHa Activity ..................................................................................................... 49 Immunoprecipitated PDP Protein ........................................................................ 50 Mitochondrial PKCδ Protein ............................................................................... 52 PDP and CS Activity ........................................................................................... 53 Chapter 5 Discussion ........................................................................................................................ 55 General Summary ................................................................................................ 55 PDHa Activity ..................................................................................................... 55 Immunoprecipitation and PDP Phosphorylation ................................................. 58 Mitochondrial PKCδ Enrichment ........................................................................ 62 Future Directions ................................................................................................. 63 Summary and Perspectives .................................................................................. 64 References ........................................................................................................................ 66 vi M.Sc. Thesis – J. R. Choptiany Brock University Appendix 1: Laboratory Procedures ................................................................................ 78 1a. Skeletal Muscle Tissue Surgical Extraction: Extensor Digitorum Longus .... 77 1b. Isolation of Skeletal Muscle Tissue Subsarcolemmal Mitochondria ............. 92 1c. Homogenization of Whole Skeletal Muscle Tissue ....................................... 98 1d. Quantitative Determination of Proteins: The Bradford Method .................. 101 1e. Immunoprecipitation of Serine–Phosphorylated Proteins ........................... 107 1f. Western Blotting: Polyacrylamide Gel Electrophoresis ............................... 111 1g. PDHa Activity Assay by Measurement of Acetyl–CoA Accumulation ...... 124 1h. Determination of Acetyl–CoA Accumulation ............................................. 131 1i. Citrate Synthase Activity Assay ................................................................... 142 1j. Pyruvate Dehydrogenase Phosphatase Activity Assay ................................. 147 Appendix 2: Calculations ............................................................................................... 154 2a. Dilution of Stock Insulin .............................................................................. 154 2b. Dilution of Stock Kinase Inhibitor ............................................................... 156 2c. Determination of Anti–Phosphoserine Antibody Concentration ................. 158 2d. Statistical Power ........................................................................................... 160 2e. PDH Activity Calculations ........................................................................... 162 2f. Citrate Synthase Calculations ....................................................................... 164 Appendix 3: Citrate Synthase Activity Assay Raw Data ............................................... 166 Appendix 4: Components of Medium199 .....................................................................