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Regulation of Glucose Metabolism in Muscles from Dexamethasone Treated Rats

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Regulation of Glucose Metabolism in Muscles from Dexamethasone Treated Rats Regulation of Glucose Metabolism in Muscles from Dexamethasone Treated Rats Effect of insulin, adrenaline and contraction Anders Rasmushaugen Master thesis in pharmacology for the degree Master of Pharmacy Department of Pharmaceutical Biosciences, School of Pharmacy, Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO Spring 2009 2 Regulation of Glucose Metabolism in Muscles from Dexamethasone Treated Rats Effect of insulin, adrenaline and contraction Anders Rasmushaugen The thesis was conducted between July 2008 and May 2009 at the Department for Work-related Muscoskeletal Complaints, National Institiute of Occupational Health (STAMI) Master thesis in pharmacology for the degree Master of Pharmacy Departement of Pharmaceutical Biosciences, School of Pharmacy, Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO Advisors Senior scientist Dr. scient Jørgen Jensen Professor Arild C Rustan 3 Preface This thesis has been my biggest challenge in my years at the Institute of Pharmacy, and I want to thank those who have supported me during it. My advisor, Jørgen Jensen, has directed me through the work of this thesis and I greatly appreciate his efforts. Jørgen is a man that involves himself in his work, I hope this eagerness carries on. Special thanks are directed to the ones of you helping me with the practical work involved in the various analyses. Ada Ingvaldsen, Astrid Bolling and Jorid Thrane Stuenæs have always been accessible for questions as well as discussions. I will also thank STAMI in general for making this thesis possible. The student environment on the 5th floor has been comforting and a source of great fun as well as discussions related the studies. In order of appearance; Stanley, Fang Chin, Monica, Guro, Kristine, Siri, Hanne, Ane, Iselin – I wish you all good luck with your future work and keep your ability of “faglig-sosial dualitet” with you after your work at STAMI is over! I direct a warm gratitude to my family for positive thoughts and support. Friends, both off- and on-campus has also been most supportive. Last, but not least, I thank my boyfriend Michael. Not only am I thanking him for his proof- reading but also for being who he is and the support he has given me in this period. He has also been able to deal with the increased stress levels in the last period and long hours. Good luck in the future! Anders Rasmushaugen Oslo, 14.05.2009 4 Table of Contents Preface ............................................................................................................................................................. 3 Abbreviations ................................................................................................................................................... 7 Abstract ........................................................................................................................................................... 8 1 Background ............................................................................................................................................. 9 1.1 Diabetes .................................................................................................................................................... 9 1.1.1 Prevalence ....................................................................................................................................... 9 1.1.2 Pathophysiology ............................................................................................................................ 10 1.1.3 Diabetic risk factors ....................................................................................................................... 11 1.1.4 Treatment ..................................................................................................................................... 15 1.1.5 Drug-induced hyperglycemia ........................................................................................................ 15 1.2 Glucose and glycogen; metabolism ........................................................................................................ 16 1.3 Insulin signaling ...................................................................................................................................... 17 1.4 Stress hormones ...................................................................................................................................... 20 1.4.1 Anatomy ........................................................................................................................................ 20 1.4.2 Glucocorticoids .............................................................................................................................. 21 1.4.3 Synthetic cortisone; dexamethasone ............................................................................................ 23 1.4.4 Adrenaline ..................................................................................................................................... 24 1.5 The role of skeletal muscles in glucose homeostasis .............................................................................. 25 2 Purpose ................................................................................................................................................. 27 2.1 Experiment I ............................................................................................................................................ 27 2.2 Experiment II ........................................................................................................................................... 27 3 Methods and materials ......................................................................................................................... 28 3.1 Rats ......................................................................................................................................................... 28 3.2 Muscle preparation and incubation ........................................................................................................ 28 3.3 Muscle contraction ................................................................................................................................. 30 3.4 Statistics .................................................................................................................................................. 30 3.5 Glucose uptake analysis .......................................................................................................................... 30 3.6 Glycogen content analysis; muscles ........................................................................................................ 31 5 3.7 Glycogen content analysis; liver and heart ............................................................................................. 32 3.8 Glycolytic flux .......................................................................................................................................... 32 3.9 Glycogen synthesis 14C and 3H ................................................................................................................ 32 3.10 Lactate release in buffer .................................................................................................................... 33 3.11 Western blot....................................................................................................................................... 34 3.11.1 Homogenization of muscle samples ......................................................................................... 34 3.11.2 Gel making ................................................................................................................................ 34 3.11.3 Electrophoresis ......................................................................................................................... 35 3.11.4 Blotting ..................................................................................................................................... 35 3.11.5 Blocking and immune reactions with antibodies ..................................................................... 35 3.11.6 Exposure, interpretation .......................................................................................................... 36 4 Results .................................................................................................................................................. 37 4.1 Experiment I ............................................................................................................................................ 37 4.2 Rat and muscle weight............................................................................................................................ 37 4.3 Glycogen content – epi and sol ............................................................................................................... 37 4.4 Glycogen content - liver and heart .......................................................................................................... 39 4.5 Western blots .......................................................................................................................................... 41 4.5.1 PKB Ser473 phosphorylation ........................................................................................................... 41 4.5.2 PKB Thr308 phosphorylation ..........................................................................................................
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