The purpose of this investigation was to determine the effects of post-exercise carbohydrate-protein feedings on muscle glycogen restoration following exhaustive cycle ergometer exercise. Seven male collegiate cyclist (age=25.6±3.3y, ht.=180.9±8.5cm, wt.=75.4±10.7kg, VO2max=4.20±0.4 1•miri 1) performed three trials, each separated by -lwk, 1) 100% (x-D glucose (CHO), 2) 70% carbohydrate-20% protein-10% fat (CHOPRO), and 3) 86% carbohdyrate-14% amino acid (CHO-AA). All feedings were eucaloric, based upon 1.0 g•kgb.W.'1•hr"1 of carbohydrate, and administered every half hour during a four hour muscle glycogen restoration period in an 18% wt./vol. solution. Muscle biopsies were obtained immediately and four hours post exercise. Following the exhaustive exercise and every half hour for four hours a blood sample was drawn. Muscle glycogen concentrations increased 53%, 47%, and 57% for the CHO, CHO-PRO, and CHO-AA feedings, respectively, however no differences among the feedings were apparent in muscle glycogen restoration. The plasma glucose and insulin concentrations demonstrated no differences throughout the restoration period among the three feedings. These results suggest that muscle glycogen restoration does not appear to be enhanced with the addition of either protein or amino acids to an eucaloric carbohydrate feeding following an exhaustive cycle exercise. However, it appears that if adequate amounts of carbohydrates are consumed (greater than 0.70 g•kgb,W,."'•hf' carbohydrate) following exhaustive exercise, maximal muscle glycogen restoration occurs. / School of Physical Education

by Judy, Yuet-wa Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 107-111). / Acknowledgements --- p.i / Contents --- p.ii-iv / Abstract --- p.v-vii / Abbreviations --- p.ix / List of figures and tables --- p.x-xii / Chapter Chapter One: --- Introduction --- p.1-20 / Chapter 1.1 --- Facilitative Glucose Transporter Family (GLUT) / Chapter 1.2 --- Sequence and characterization of GLUT / Chapter 1.3 --- Overexpression of GLUT 1 in human cancer cells / Chapter 1.4 --- Inhibition of gene expression by antisense nucleic acid / Chapter 1.5 --- Types of antisense nucleic acids / Chapter 1.5.1 --- Nuclear expression of RNA by engineered antisense genes / Chapter 1.5.2 --- Antisense oligonucleotides / Chapter 1.6 --- Use of antisense oligomers in cell culture system / Chapter 1.7 --- Modification of antisense oligonucleotides / Chapter 1.8 --- Length and sequence selection of antisense oligomers / Chapter 1.9 --- Controls for measuring antisense effect / Chapter 1.10 --- Internalization and targeting of oligonucleotides / Chapter 1.11 --- Possible action mechanisms of antisense nucleotides / Chapter 1.12 --- Clinical applications of antisense approach / Chapter 1.13 --- Aim of the project / Chapter Chapter Two: --- Materials and Methods --- p.21-45 / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Cell line and culture medium / Chapter 2.1.1a --- Cell line / Chapter 2.1.1b --- Culture medium / Chapter 2.1.2 --- Reagents and Buffers / Chapter 2.1.2a --- Phosphate-Buffered Saline (PBS) / Chapter 2.1.2b --- 50XTAE Buffer / Chapter 2.1.2c --- Tris-EDTA Buffer / Chapter 2.1.2d --- MTT solution / Chapter 2.1.2e --- Lipofectin Reagent / Chapter 2.1.3 --- Reagents for Northern Analysis / Chapter 2.1.3a --- DEPC-treated water (0.1% DEPC) / Chapter 2.1.3b --- 20X SSC / Chapter 2.1.3c --- 20X SSPE / Chapter 2.1.3d --- 10X Formaldehyde gel-running buffer / Chapter 2.1.3e --- Formaldehyde gel-loading buffer / Chapter 2.1.3f --- Prehybridization buffer / Chapter 2.1.3g --- Hybridization buffer / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Synthesis of oligonucleotides and phosphorothioated oligonucleotides / Chapter 2.2.2 --- Cloning of human GLUT 1 cDNA into pRc/CMV expression vector at sense and antisense orientation / Chapter 2.2.2a --- Primer designed for cloning of sense and antisense GLUT 1 cDNA / Chapter 2.2.2b --- Isolation of sense and antisense GLUT 1 clone by PCR / Chapter 2.2.2c --- Restriction Digestion / Chapter 2.2.2d --- Purification of Restriction Digested DNA / Chapter 2.2.2e --- DNA Ligation / Chapter 2.2.2f --- Preparation of competent bacterial cells for transformation / Chapter 2.2.2g --- Plasmid DNA Transformation / Chapter 2.2.3 --- Large scale preparation of plasmid DNA / Chapter 2.2.4 --- Formation of Lipofectin-encapsulated oligonucleotides / Chapter 2.2.5 --- [32P]-labeled oligonucleotides uptake assay / Chapter 2.2.6 --- Methods to monitor antisense effect / Chapter 2.2.6a --- MTT assay / Chapter 2.2.6b --- Northern Analysis / Chapter (i) --- Preparation of radiolabeled probe / Chapter (ii) --- Isolation of total RNA from HL-60 cells / Chapter (iii) --- Separation of total RNA by eletrophoresis and blotting onto a membrane / Chapter (iv) --- Prehybridization of the Northern blot / Chapter (v) --- Hybridization of the Northern blot / Chapter 2.2.6c --- [3H]-deoxyglucose uptake assay / Chapter Chapter Three: --- Results --- p.46-88 / Chapter 3.1 --- Synthesis of Oligonucleotides / Chapter 3.2 --- Multiple alignment of cDNA sequence of Glucose Transporter isoforms / Chapter 3.3 --- [32P]-labeled oligonucleotide uptake assay / Chapter 3.4 --- Antisense oligonucleotides designed against different regions of GLUT 1 cDNA sequence / Chapter 3.4.1 --- Effects on HL-60 cell proliferation / Chapter 3.4.2 --- Effects on GLUT 1 mRNA level / Chapter 3.5 --- The effects of different oligonucleotide concentrations on HL- 60cell proliferation / Chapter 3.6 --- The effects of modified oligonucleotides on HL-60 cell proliferation / Chapter 3.7 --- The effects of different oligonucleotide lengths on HL-60 cell proliferation / Chapter 3.8 --- [3H]-deoxyglucose uptake assay / Chapter 3.9 --- Cloning of sense and antisense GLUT 1 cDNA into pRc/CMV vector / Chapter 3.10 --- Inhibition of GLUT 1 gene expression by expressed antisense nucleotides / Chapter Chapter Four: --- Discussion --- p.89-106 / Chapter 4.1 --- Importance of GLUT 1 gene / Chapter 4.2 --- HL-60: the target cancer cell line / Chapter 4.3 --- "Importance of ""Antisense Approach""" / Chapter 4.4 --- Optimization of condition for antisense inhibition by oligonucleotides / Chapter 4.4.1 --- Oligonucleotide length / Chapter 4.4.2 --- Oligonucleotide Modification / Chapter 4.4.3 --- Sequence selection / Chapter 4.4.4 --- Uptake efficiency / Chapter 4.5 --- Intracelluar distribution of oligonucleotides / Chapter 4.6 --- Inhibition of GLUT 1 gene expression by expressed antisense nucleotides / Chapter 4.7 --- Mechanisms for antisense inhibition of gene expression / Chapter 4.8 --- Further Directions / References --- p.107-117