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Proglycogen and Macroglycogen in Equine Skeletal Muscle

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Proglycogen and Macroglycogen in Equine Skeletal Muscle PROGLYCOGEN AND MACROGLYCOGEN IN EQUINE SKELETAL MUSCLE A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by JOHAN BROJER In partial fulfillment of requirements for the degree of Master of Science April, 2001 O Johan Brojer, 2001 Nationai Library Bibliothèque nationale I*I of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services sewices bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON KIA ON4 Ottawa ON KIA ON4 Canada Canada Your fiie Vorre référence Our file Notre reférence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d7auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author7s ou autrement reproduits sans son permission. autorisation. ABSTRACT PROGLYCOGEN ANli) MACROGLYCOGEN IN EQULNE SKELETAL MUSCLE Johan Brojer Advisor: uni ver si^ of Guelph, 2001 Dr. Henry R. St-pfli The objectives of this thesis were to investigate the existence of proglycogen (PG) and macroglycogen (MG) in equine skeletal muscle; to compare the analysis of total glycogen by two methods (acid hydrolysis and MGtPG determination) for total muscle glycogen fiom biopsy sized sarnples and to evaluate the effect of extraction time and perchloric acid (PCA) concentration on the recovery of MG and PG. The first study indicated that extraction time had very little effect on the recovery of PG and MG but there was an effect of PCA concentration. Extraction with 0.5 and 1.5 M PCA yielded similar values for PG and MG but extraction in 3.0 M PCA yieIded lower PG and higher MG concentrations (W0.05). The results f?om the second study showed that PG and MG exist in equine skeletal muscle and the measurement techniques for total glycogen analyzed by the two methods were very comparable. The present study was conducted at the Departrnent of Clinical Studies - Large Animal Interna1 Medicine, Ontario Veterinary College, University of Guelph, Canada. These studies were generously supported by the EP Taylor Equine Trust Fund. During the the spent completing this work, nurnerous people have been involved, and 1 would like to express my thanks to au who have contributed in various ways. 1wish to express my sincere gratitude to: Dr. Henry Stampfli, my supervisor, for introducing me to the interesting field of muscle physiology and for stirnulating my interest in large animal interna1 medicine. You have been such a nice person to work with - both in the large animal chic and in the area of research. Thank you for your guidance, patience and your unfailing encouragement and interest and for aIways keeping me cheerfül when progress was slow. 1 consider you and your family as very good fnends and 1 am very happy for a11 the dimers you provided me and my wife during our time in Canada. The members of my research cornmittee: Dr. George Heigenhauser and Dr. Tim Lumsden, for your constructive criticism on the manuscript, for interesting scientific discussions during the cornmittee meetings and for offerhg valuable advice conceming the thesis. Dr. Terry Graham, rny CO-author,for your excellent guidance in the field of pro- and macroglycogen and for your always optirnistic and friendly attitude. Your constructive criticism and comments on the manuscripts have always been appreciated. Thanks a lot for letting me have access to your research Iaboratory. The owners of the valuable endurance ride horses, for giving me the opportunity to study their fine horses. Dr. Birgitta Essén-Gustavsson for your knowledge, enthusiasm, support and for al1 interesting scientific discussions. To write a thesis having the advisors and CO-authoron the other side of the Atlantic sea does not make things easier. Therefore, 1 am gratefb! for a11 your help - to have a person at the same department who 1could ask questions to right away. Premila Sathasivam for al1 your help, support and knowledge. You had al1 the answers 1 needed when prob1ems suddenly appeared in the laboratory. Kristi Adamo and Jane Shearer for introducing me to the analytical methods of pro- and macrogfycogen. My colleagues at the department of Equine Interna1 Medicine, Swedish Veterinary College, Susanne Demmers, Pia Funkquist, Agneta Gustafsson, John Pringle, Miia Riihimaki and Katarina Schuback for your help with the schedule so 1 finally got the time off for writing the thesis, Kim Kultiman for a11 your knowledge about statistics and your help with the computer program Minitab. Caroline, my wife and very best fiend, for being the most important person in my life and for sharing rny interest in hunting and the outdoors. Thank you for your love, help, support and encouragement but most of al1 for being my wife. Rebecka, my newborn daughter, for helping me remember what the important things are in life. My four-footed fiends Smaragd and Amethist (my horses) for sharing al1 those nice moments in the countryside and Siska (my dog) for being a nice hunting partner but also for being a Company during my thesis-writing at night. TABLE OF CONTENTS TABLE OF CONTENTS................................................................... iii DECLARATION OF WOEX PERFORMED ......................................... v LIST OF TABLES ............................................................................ vi ... LIST OF FIGURES .......................................................................... vlzl CHAPTER 1 1. 0 General Introduction ................................................................... -1 1.1 Statement of Goals and Hypotheses.................................................. 5 1.2 General Literature Review 1.2.1 Introduction ............................................................................. -7 1.2.2 Glycogen ................................................................................. 7 1.2.3 Glycogenin .............................................................................. -8 1.2.4 Proglycogen and macroglycogen ...... .. ............................................. 9 1.2.5 Theoretical modefing of the glycogen structure................................... 12 1.2.6 Storage of glycogen in skeletal muscle - the relationship between pro- and macroglycogen .................................................. -13 1.2.7 Synthesis and glycogenolysis of pro- and macroglycogen...................... -15 1.2.8 Reference list ........................................................................... 23 CHAPTER 2 The effect of extraction time and perchloric acid concentration on the recovery of pro- and macroglycogen from muscle biopsies from the horse 2.0 Abstract ................................................................................. 30 2.1 Introduction ............................................................................ 30 2.2 Material and Methods ................................................................ 32 2.3 Results .................................................................................. 35 2.4 Discussion .............................................................................. 37 2.5 Acknowledgments.................................................................... 40 2.6 References ............................................................................. -45 iii DECLARATION OF WORK PERFORMED I declare that al1 work reported in this thesis was performed by me. Biopsy sarnpling procedures were undertaken with the assistance of Dr. Henry R, Stiimpfli. LIST OF TABLES TABLE Theoretical increase in glycogen levels above basal values.. .................. .22 Mean pro-, macro- and total glycogen concentrations for different extraction times.. .................................................................................. 41 Mean proglycogen concentration for different combinations of extraction tirne and perchloric acid concentration.. .......................................... -42 Mean rnacroglycogen concentration for different combinations of extraction time and perchloric acid concentration.. .......................................... .43 Mean total glycogen concentration for different combinations of extraction time and perchloric acid concentration.. .......................................--. -44 Reproducibily for total glycogen in duplicate analyses from the same muscle biopsy for the acid hydrolysis and the pro- and macroglycogen technique.. -65 Reproducibilty in duplicate biopsies obtained at the sarne site in the glzlteza medius muscle. ........................................................................ 66 The effect of sarnpling dep th in the glutetcs medius muscle. ................... -66 Individual data for proglycogen (PG), macroglycogen (MG), PWMG and acid hydrolysis (AC) fiom 45 individual muscle biopsies.. ............................. 94 Individual data for proglycogen (PG), macroglycogen (MG) and PG+MG £kom biopsies obtained at different sarnpling depths.. ................................. ..96 Individual data for proglycogen (PG),
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