Insulin stimulates glucose transport into muscle cells and adipocytes via a process that involves the translocation of GLUT4 proteins from intracellular stores to the cell membrane. The pathway by which this translocation takes place has not been fully elucidated. The purpose of this study was to determine the effect of the calciumdependent calmodulin protein kinase II (CAMKII) inhibitor KN-62 on insulin stimulated 3-0-methylglucose transport in isolated rat epitrochlearis muscles. The primary finding of this investigation was that KN-62 decreased insulin stimulated glucose transport by -35%. KN-04, a structural analogue of KN-62, did not affect insulin stimulated glucose transport. Additional experiments showed that the L-type calcium (Ca 2+) channel inhibitor nifedipine inhibited glucose transport to a similar extent as KN-62 (-29%). Furthermore, no additive inhibitory effect was seen when KN-62 and nifedipine were used in combination. The results of this investigation suggest that CAMKII has a critical role in insulin stimulated glucose transport, and this role may be dependent upon L-type Cat- channel activation. / School of Physical Education

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Wong Hei Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 157-175). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract 摘要 --- p.iv / List of Figures --- p.vi / List of Tables --- p.ix / List of Abbreviations --- p.x / Table of Contents --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Importance of Glucose in Biological System --- p.1 / Chapter 1.2 --- Glucose Transporter Families --- p.2 / Chapter 1.2.1 --- Na+-Dependent Glucose Transporters --- p.2 / Chapter 1.2.2 --- Facilitative Glucose Transporters --- p.3 / Chapter 1.3 --- Glucose Transporter Type1 --- p.7 / Chapter 1.3.1 --- Primary Structure --- p.7 / Chapter 1.3.2 --- Secondary Structure --- p.8 / Chapter 1.3.3 --- Membrane Topology --- p.8 / Chapter 1.3.4 --- Tertiary Structure --- p.9 / Chapter 1.3.5 --- Kinetics Properties --- p.11 / Chapter 1.3.6 --- Affinity Reagents --- p.12 / Chapter 1.3.7 --- Tissue Distribution --- p.13 / Chapter 1.3.8 --- Multifunctional Property --- p.14 / Chapter 1.3.9 --- Characterization of GLUT1 Gene --- p.14 / Chapter 1.3.10 --- Regulation of GLUT1 Expression --- p.15 / Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.17 / Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome --- p.20 / Chapter 1.5.1 --- Background of GlutlDS --- p.20 / Chapter 1.5.2 --- Clinical Features of GlutlDS --- p.23 / Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24 / Chapter 1.5.4 --- Diagnosis --- p.26 / Chapter 1.5.4.1 --- Erythrocyte Glucose Transporter Activity --- p.26 / Chapter 1.5.4.2 --- Molecular Genetic Testing of GLUT1 Gene --- p.27 / Chapter 1.5.4.3 --- Glucose Concentration --- p.27 / Chapter 1.5.5 --- Management --- p.28 / Chapter 1.5.5.1 --- Ketogenic Diet --- p.28 / Chapter 1.5.5.2 --- Medication --- p.29 / Chapter 1.5.5.2.1 --- Glutl Activator --- p.29 / Chapter 1.5.5.2.2 --- Glutl Inhibitor --- p.29 / Chapter 1.6 --- Hypothesis and Objectives --- p.31 / Chapter Chapter 2: --- Identification of the First Two Asian GlutlDS Cases --- p.33 / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patients --- p.34 / Chapter 2.1.2 --- Blood Samples --- p.35 / Chapter 2.1.3 --- Reagents for Zero-trans Influx of 3-OMG Uptake in Erythrocytes --- p.35 / Chapter 2.1.4 --- Reagents for Zero-trans Efflux of 3-OMG Uptake in Erythrocytes --- p.37 / Chapter 2.1.5 --- Reagents for Glutl Gene Analysis --- p.37 / Chapter 2.1.6 --- Reagents and Buffers for Reverse Transcription --- p.38 / Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.39 / Chapter 2.1.8 --- Reagents for Erythrocytes Membrane Preparation and Detection --- p.41 / Chapter 2.2 --- Methods --- p.46 / Chapter 2.2.1 --- Zero-trans Influx of 3-OMG Uptake in Erythrocytes --- p.46 / Chapter 2.2.2 --- Zero-trans Efflux of 3-OMG out of Erythrocytes --- p.47 / Chapter 2.2.3 --- Glutl Protein Expression --- p.48 / Chapter 2.2.4 --- GLUT1 Gene Analyses --- p.51 / Chapter 2.2.5 --- Statistics --- p.58 / Chapter 2.3 --- Results --- p.59 / Chapter 2.4 --- Discussions and Conclusions --- p.69 / Chapter Chapter 3: --- Pathogenicity of GLUT1 Mutations --- p.78 / Chapter 3.1 --- Materials --- p.79 / Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.79 / Chapter 3.1.2 --- Cell Lines --- p.80 / Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.81 / Chapter 3.1.4 --- Cell Culture Wares --- p.83 / Chapter 3.1.5 --- Reagents for Transfection --- p.83 / Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.83 / Chapter 3.1.7 --- Reagents and Buffers for Flow Cytometry --- p.84 / Chapter 3.1.8 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.84 / Chapter 3.1.9 --- Reagents and Consumables for Confocal Microscopy --- p.85 / Chapter 3.2 --- Methods --- p.86 / Chapter 3.2.1 --- Cell Culture Methodology --- p.86 / Chapter 3.2.2 --- Construction of Glutl-Encoding Vectors --- p.87 / Chapter 3.2.3 --- Construction of Glutl Mutants --- p.91 / Chapter 3.2.4 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.92 / Chapter 3.2.5 --- Glucose Influx Assays in CHO-K1 Cells --- p.96 / Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.97 / Chapter 3.2.7 --- Statistics --- p.98 / Chapter 3.3 --- Results --- p.99 / Chapter 3.4 --- Discussions and Conclusions --- p.112 / Chapter Chapter 4: --- Effects of Anticonvulsive Compounds on Cellular Glucose Transport --- p.117 / Chapter 4.1 --- Materials --- p.118 / Chapter 4.1.1 --- Cell Lines --- p.118 / Chapter 4.1.2 --- Cell Culture Media --- p.118 / Chapter 4.1.3 --- Blood Sample --- p.119 / Chapter 4.1.4 --- Anticonvulsive Compounds --- p.119 / Chapter 4.1.5 --- Reagents for Zero-trans Influx of 3-OMG Uptake in Fibroblasts --- p.120 / Chapter 4.1.6 --- Reagents for Zero-trans Influx of 2-DOG Uptake in Primary Astrocytes --- p.120 / Chapter 4.1.7 --- Reagents for Total RNA Isolation --- p.121 / Chapter 4.1.8 --- Reagents and Consumables for Real-Time PCR --- p.122 / Chapter 4.2 --- Methods --- p.123 / Chapter 4.2.1 --- Cell Culture --- p.123 / Chapter 4.2.2 --- Drug Concentrations --- p.123 / Chapter 4.2.3 --- Zero-trans Influx of 3-OMG Uptake in Erythrocytes --- p.123 / Chapter 4.2.4 --- Zero-trans Influx of 3-OMG Uptake in Fibroblasts --- p.124 / Chapter 4.2.5 --- Zero-trans Influx of 2-DOG Uptake in Primary Astrocytes --- p.125 / Chapter 4.2.6 --- Gene Expression Study --- p.127 / Chapter 4.2.7 --- Statistics --- p.130 / Chapter 4.3 --- Results --- p.131 / Chapter 4.4 --- Discussions and Conclusions --- p.148 / Chapter Chapter 5: --- General Conclusions and Future Perspectives --- p.154 / References --- p.157

Insulin-like growth factor-I (IGF-I) is a known stimulator of glucose transport. IGF binding protein 1 (IGFBP1) is a protein that regulates the actions of IGF-I by binding to IGF-I which alters it's ability to bind to the IGF-I receptor. Diet and exercise may influence this system. While IGFBP1 levels increase with fasting or prolonged exercise, feeding will reverse this elevation. The intent of this study was to determine if an in vivo manipulation of IGFBP1 affects in vitro glucose transport in the rat soleus. Sixteen male Spaque Dawley rats were fasted for 12 hours. Half of the animals were then allowed a two hour ad libitum refeeding period. Animals were anesthetized and had their soleus muscles removed. Muscles were then randomly assigned to one of four treatment groups. Treatments involved an incubation in either 4 or 8 mM glucose in either the presence or absence of IGF-I (75 ng x ml"'). Final incubation for all treatment groups included [3H]-3-O-methylglucose (437 µCi x mM-) for the measurement of glucose transport. Following incubation, muscles were weighed, homogenized in 1 ml of 10% trichloroacetic acid, and centrifuged to precipitate out protein. 100 µl of the supernatant was added to 3 ml of scintillation fluid and analyzed in a scintillation counter. Glucose transport was determined by 3H activity. A statistical analysis of the various groups shows that there is no significant difference between fasted and refed animal for any specific treatment. However, when all the fasted and refed animals area grouped, glucose transport rate is significantly greater (p<0.05) in fasted (3.59 ± 0.44 µM x ml"' x hr) animals than in refed animals (2.56 ± 0.27 µM x ml"' x hr'). Additionally, muscles that were treated with IGF-I in 8 mM glucose demonstrated a greater rate of glucose transport (5.12 ± 0.68 µM x ml-1 x hr') than all other treatments (2.13 ± 0.39 to 2.90 ± .33 µM x ml-' x hr'). This study showed that IGF-I is a stimulator of glucose transport in an 8 mM glucose media. Additionally, the results show that glucose transport is greater if the animals are fasted. The differences between fasted and refed animals demonstrated in this study supports the hypothesis that diet manipulated IGFBP1 levels are able to alter the biological effects of IGF-I. / School of Physical Education

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Introduction: Exercise increases skeletal muscle glucose uptake via AMP-activated protein kinase (AMPK) activation and GLUT4 translocation from cytosol to cell membrane. It also promotes glucose utilisation in type 2 diabetic patients via increased insulin sensitivity. Insulin stimulates GLUT4 translocation by activating P13- kinase and protein kinase B (PKB/Akt). We therefore postulated that a connection exists between these two pathways upstream of GLUT4 translocation. Understanding this connection is important in the development of treatment strategies for type 2 diabetes. This exercise-induced increase in AMP-activated protein kinase (AMPK) activation can be mimicked by a pharmacological agent, 5'-aminoimidazole-4- carboxamide ribonucleoside (AlGAR), which is converted intracellularly into 5'- aminoimidazole-4-carboxamide-ribonucleosidephosphate (ZMP), an AMP analogue. Aim: To investigate the effect of two pharmacological AMPK-activating compounds, ZMP and AlGAR, on the phosphorylation of AMPK, the phosphorylation of PKB/Akt as well as possible feedback on insulin-stimulated glucose uptake and GLUT4 translocation. Materials and Methods: Adult ventricular cardiomyocytes were isolated from male Wistar rats by collagenase perfusion and treated with 1 mM AlGAR or 1 mM ZMP in the presence or absence of 100 nM insulin or 100 nM wortmannin, an inhibitor of P13- kinase. Glucose uptake was measured via eH]-2-deoxyglucose (2DG) accumulation. PKB/Akt and AMPK phosphorylation and GLUT4 translocation was detected by Western blotting. Purinergic receptors were blocked with 8-cyclopentyl-1,3- dipropylxanthine (8CPT) and the effect on AMPK phosphorylation noted. Certain results were confinned or refuted by repeating experiments using the isolated rat heart model. Results: AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but in fact inhibited basal glucose uptake, although GLUT4 translocation from cytosol to membrane occurred. Both compounds also attenuated insulin stimulated glucose uptake. Wortmann in abolished glucose uptake and PKB/Akt phosphorylation elicited by insulin while, in the presence of wortmannin, AICAR and ZMP increased levels of PKB/Akt phosphorylation. Although AICAR and ZMP increased glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. However both compounds increased GLUT4 translocation, clearly demonstrating that translocation and activation of GLUT4 are separate processes. 8CPT had no effect on the phosphorylation of AMPK by either AICAR or ZMP indicating that there was no involvement of the purinergic receptors. Conclusion: Although AICAR and ZMP increase glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. Conversely, both compounds inhibited both basal and insulin stimulated glucose uptake despite increasing GLUT4 translocation. Inhibition of PI3-kinase in presence or absence of insulin unmasked hitherto unknown effects of AICAR and ZMP on PKB phosphorylation. / AFRIKAANSE OPSOMMING: Agtergrond: Oefening verhoog skeletspier glukose opname via AMP-geaktiveerde protein kinase (AMPK) aktivering en GLUT4 translokering vanaf die sitosol na die selmembraan. Dit verbeter ook glukose verbruik in tipe 2 diabetes pasiënte via verhoogde insulien sensitiwiteit. Insulien stimuleer GLUT4 translokering deur P13- kinase en protein kinase B (PKB/Akt) te aktiveer. Dit word dus gepostuleer dat daar 'n verbinding tussen hierdie twee paaie, wat beide betrokke is by GLUT4 translokering, bestaan. Dit is belangrik om hierdie verbinding te verstaan aangesien dit in behandelingstrategieë van tipe 2 diabetes geteiken kan word. Die oefening geïnduseerde verhoging in AMPK aktivering, kan deur 'n farmakologiese middel 5'- aminoimidasool-4-karboksamied ribonukleosied (AICAR), wat intrasellulêr omgesit word na 5'-aminoimidasool-4-karboksamied-ribonukleosiedfosfaat (ZMP), 'n AMP analoog, nageboots word. Doel: Om die effek van twee farmakologiese AMPK-aktiveringsmiddels, AICAR en ZMP, op die fosforilering van AMPK en PKB/Akt, sowel as moontlike effekte daarvan op insulien-gestimuleerde glukose opname en GLUT4 translokering, te ondersoek. Materiale en Metodes: Volwasse ventrikulêre kardiomiosiete is uit manlike Wistar rotharte geïsoleer d.m.v kollagenase perfusies en behandel met 1 mM AICAR of 1 mM ZMP in die teenwoordigheid of afwesigheid van 100 nM insulien of 100 nM wortmannin. Glukose opname is gemeet via intrasellulêre [3H]-2-deoksiglukose akkumulasie; PKB/Akt en AMPK fosforilering sowel as GLUT4 translokering is bepaal deur Western blot analises. Purinergiese reseptore is geblokkeer met 8-siklopentiel- 1,3-dipropielxanthien (8CPT) en die effek daarvan op AMPK fosforilering genoteer. Ten einde resultate wat in die geïsoleerde kardiomiosiet-model verkry is, te bevestig, is sekere eksperimente in die geïsoleerde rothart herhaal. Resultate: Beide AIGAR en ZMP stimuleer AMPK fosforilering. Die middels kan nie glukose opname verhoog nie, inteendeel, basale glukose opname is onderdruk alhoewel GLUT4 translokering vanaf die sitosol na die selmembraan wel plaasgevind het. Wortmannin kon insulien gemedieerde glukose opname en PKB/Akt fosforilering onderdruk. In die teenwoordigheid van wortmannin het beide AIGAR en ZMP PKB/Akt fosforilering verhoog. Alhoewel beide AIGAR en ZMP glukose opname in skeletspier verhoog, was dit nie die geval in kardiomiosiete nie. Beide middels het wel GLUT 4 translokering verhoog, wat duidelik demonstreer dat die translokering en aktivering van GLUT4, verskillende prosesse is. 8GPT het geen effek gehad op die fosforilering van AMPK deur AIGAR of ZMP nie, wat bewys dat daar geen betrokkenheid van die purinergiese reseptore was nie. Gevolgtrekking: Alhoewel AIGAR en ZMP glukose opname in skeletspier verhoog is dit nie die geval in kardiomiosiete nie. Beide middels inhibeer basale en insuliengestimuleerde glukose opname maar stimuleer GLUT4 translokeering. Inhibisie van PI3-kinase in die teenwoordigheid of afwesigheid van insulien, ontmasker voorheen onbekende effekte van AIGAR en ZMP op PKB/Akt fosforilering.

The glucose toxicity has been recognized over the last several years as a factor contributing to both impaired insulin secretion and insulin resistance in patients with diabetes. However, the molecular mechanisms that underlie the changes in glucose transport activity induced by hyperglycemia have not been fully understood. The purpose of the present investigation is to determine if acute hyperglycemia affects an activation of glucose transport and also if hyperglycemic-induced change in insulinstimulated glucose transport is mediated via a PKC-dependent signaling system. Animals were anesthetized, and the soleus (SOL) muscles were isolated and clamped at their resting length. After a 10 minute recovery period the muscles were transferred to preincubation vials containing KHB supplemented with 4 or 16 mmol of glucose and 16 mmol/1 mannitol with or without insulin and/or inhibitors for 30 minutes. Following an incubation series to prepare the muscle, the muscle was incubated in radioactive 3-0- [3H] methylglucose and [14C] mannitol for 10 min. in the presence/absence of insulin and inhibitors, and the amount of glucose transport was measured. A total of 100µU/ml insulin with 4 mM glucose led to increase glucose transport by 155%, whereas the same amount of insulin with 16 MM glucose led to 80% increment in glucose transport. Also, 16 mM glucose in the absence of insulin induced an increase of glucose uptake by apporoximately 50% compared with 4 MM glucose. However, the addition of insulin reduced that difference to 5.3%. The conventional PKC inhibitor GF 109203X in the muscle incubated with 16 MM glucose led to a decrease in insulin-stimulated glucose transport (1l%), whereas the inhibitor with 4 mM glucose induced a decrease in insulin-stimulated glucose transport (24%). These findings suggest that glucose can directly regulate glucose transport activity by a mechanism that possibly involves a facilitated GLUT1 transporter activity. In addition to the mass action of glucose, the hyperglycemic-induced increase in insulin stimulated glucose transport may be partially mediated via a PKC-dependent signaling system. / School of Physical Education

Tsang Po Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 135-152). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract 摘要 --- p.iv / List of Figures --- p.vi / List of Tables --- p.viii / List of Abbreviations --- p.ix / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The Role of Glucose in Biological System --- p.1 / Chapter 1.2 --- Glucose Transporter Families --- p.1 / Chapter 1.2.1 --- Na+-Dependent Glucose Transporters --- p.2 / Chapter 1.2.2 --- Facilitative Glucose Transporters --- p.3 / Chapter 1.3 --- Glucose Transporter Type1 --- p.7 / Chapter 1.3.1 --- Primary Structure of the Glutl Protein --- p.7 / Chapter 1.3.2 --- Secondary Structure --- p.8 / Chapter 1.3.3 --- Tertiary Structure --- p.8 / Chapter 1.3.4 --- Kinetics Properties --- p.11 / Chapter 1.3.5 --- Tissue Distribution --- p.12 / Chapter 1.3.6 --- Multifunctional Property --- p.13 / Chapter 1.3.7 --- Characterization of GLUT1 Gene --- p.13 / Chapter 1.3.8 --- Regulation of GLUT1 Expression --- p.14 / Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.16 / Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome (GIutlDS) --- p.19 / Chapter 1.5.1 --- Backgronnd of GIutlDS --- p.19 / Chapter 1.5.2 --- Clinical Features of GIutlDS --- p.23 / Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24 / Chapter 1.5.4 --- Diagnosis --- p.26 / Chapter 1.5.5 --- Manage nent --- p.27 / Chapter 1.5.5.1 --- Ketogenic Diet --- p.27 / Chapter 1.6 --- Hypothesis and Objectives --- p.29 / Chapter Chapter 2: --- Biochemical and Molecular Analysis of GLUT1 in a Suspected GlutlDS Case --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patient --- p.32 / Chapter 2.1.2 --- Blood Samples --- p.32 / Chapter 2.1.3 --- Reagents and Buffers for Reverse Transcription --- p.32 / Chapter 2.1.4 --- Reagents and Buffers for TA Cloning --- p.34 / Chapter 2.1.5 --- Reagents for Genomic DNA Extraction --- p.34 / Chapter 2.1.6 --- Reagents and Buffers for Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.35 / Chapter 2.1.8 --- Reagents for Zero-trans 3-OMG Influx in Erythrocytes --- p.37 / Chapter 2.1.9 --- Reagents for Zero-trans 3-OMG Efflux from Erythrocytes --- p.38 / Chapter 2.1.10 --- Reagents for Erythrocytes Membrane Extraction and Detection --- p.39 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- GLUT1 Gene Analysis --- p.44 / Chapter 2.2.2 --- Zero-trans 3-OMG Influx into Erythrocytes --- p.51 / Chapter 2.2.3 --- Zero-trans 3-OMG Efflux from Erythrocytes --- p.52 / Chapter 2.2.4 --- Glutl Protein Expression --- p.54 / Chapter 2.2.5 --- Statistics --- p.57 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Molecular Analysis of the GLUT1 Gene of a Suspected GlutlDS Patient --- p.58 / Chapter 2.3.2 --- Functional Analysis of the GlutlDS Patient's Glutl Protein --- p.61 / Chapter 2.3.3 --- Glutl Protein Expression in the GlutlDS Patient --- p.64 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3: --- Pathogenicity Studies of GLUT1 Mutations --- p.71 / Chapter 3.1 --- Materials --- p.72 / Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.72 / Chapter 3.1.2 --- Cell Lire --- p.73 / Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.73 / Chapter 3.1.4 --- Cell Culture Wares --- p.75 / Chapter 3.1.5 --- Reagents for Transfection --- p.75 / Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.76 / Chapter 3.1.7 --- Consumables for Confocal Microscopy --- p.77 / Chapter 3.1.8 --- Reagents and Buffers for Flow Cytometry --- p.77 / Chapter 3.1.9 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.77 / Chapter 3.2 --- Methods --- p.79 / Chapter 3.2.1 --- Cell Culture Methodology --- p.79 / Chapter 3.2.2 --- Construction of GLUT1 Mutants --- p.80 / Chapter 3.2.3 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.84 / Chapter 3.2.4 --- Protein Expression Study --- p.85 / Chapter 3.2.5 --- 2-DOG Influx Assay in CHO-K1 Cells --- p.87 / Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.89 / Chapter 3.2.7 --- Statistics --- p.90 / Chapter 3.3 --- Results --- p.91 / Chapter 3.3.1 --- Molecular Analysis of 1034-1035Insl2 Mutation --- p.91 / Chapter 3.3.2 --- Expression of the Wild Type and Mutant GFP-Glutl Fusion Proteins --- p.92 / Chapter 3.3.3 --- Functional Analysis of the 1034-1035Insl2 Mutant --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter Chapter 4: --- GLUT1 Promoter Study --- p.100 / Chapter 4.1 --- Materials --- p.101 / Chapter 4.1.1 --- Construction of GLUT1 Promoter Vectors --- p.101 / Chapter 4.1.2 --- Cell Lines --- p.102 / Chapter 4.1.3 --- Cell Culture Media and Other Reagents --- p.103 / Chapter 4.1.4 --- Dual Luciferase Reporter Assay System --- p.103 / Chapter 4.2 --- Methods --- p.105 / Chapter 4.2.1 --- Bioinformatics --- p.105 / Chapter 4.2.2 --- Cell Culture --- p.105 / Chapter 4.2.3 --- Construetion of GLUT1 Promoter Vectors --- p.105 / Chapter 4.2.4 --- 5'-Deletion Analysis of GLUT1 Promoter --- p.108 / Chapter 4.2.5 --- Determination of the Activities of GLUT1 Promoter Fragments --- p.110 / Chapter 4.2.6 --- Statistics --- p.113 / Chapter 4.3 --- Results --- p.114 / Chapter 4.3.1 --- Determination of the Promoter Activity of the 5'-deletion Fragments --- p.114 / Chapter 4.3.2 --- Prediction of Transcription Factors in the 5'-deletion Fragments --- p.119 / Chapter 4.4 --- Discussion --- p.121 / Chapter Chapter 5: --- General Conclusion and Future Perspectives --- p.133 / References --- p.135

Chung Ka Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 151-162). / Abstracts in English and Chinese. / Contents --- p.i / Acknowledgements --- p.v / Abstract --- p.vi / 論文摘要 --- p.ix / List of Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Breast Cancer --- p.2 / Chapter 1.1.1 --- Incidence Rate of Breast Cancer --- p.2 / Chapter 1.1.2 --- Risk Factors Lead to Breast Cancer --- p.5 / Chapter 1.1.3 --- Conventional Treatments --- p.5 / Chapter 1.2 --- Relationship between Breast Cancer and Glucose Transporters --- p.7 / Chapter 1.2.1 --- Importance of Glucose and Fructose --- p.7 / Chapter 1.2.2 --- Facilitative Glucose Transporters (Gluts) and The Relationship with Breast Cancer --- p.7 / Chapter 1.3 --- Antisense Oligonucleotides --- p.13 / Chapter 1.3.1 --- Characteristics of Antisense Oligonucleotides --- p.13 / Chapter 1.3.2 --- Action Mechanism of Antisense Oligonucleotides --- p.15 / Chapter 1.3.3 --- Sequence Selection --- p.19 / Chapter 1.3.4 --- Chemical Modifications of Antisense Oligonucleotides --- p.20 / Chapter 1.3.5 --- Uptake and Delivery Means of Antisense Oligonucleotides --- p.24 / Chapter 1.4 --- Objectives of Present Study --- p.26 / Chapter Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.32 / Chapter 2.1.2 --- Buffers and Reagents --- p.33 / Chapter 2.1.3 --- Reagents for Transfection --- p.34 / Chapter 2.1.4 --- Reagents for D-[U14C]-Fructose and 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.35 / Chapter 2.1.5 --- Reagents for ATP Assay --- p.35 / Chapter 2.1.6 --- Reagents for RT-PCR --- p.36 / Chapter 2.1.6.1 --- Reagents for RNA Extraction --- p.36 / Chapter 2.1.6.2 --- Reagents for Reverse Transcription --- p.36 / Chapter 2.1.6.3 --- Reagents for Gel Electrophoresis --- p.37 / Chapter 2.1.7 --- Reagents for Real Time-PCR --- p.38 / Chapter 2.1.8 --- Reagents and Chemicals for Western Blotting --- p.39 / Chapter 2.1.8.1 --- Reagents for Protein Extraction --- p.39 / Chapter 2.1.8.2 --- Reagents for SDS-PAGE --- p.39 / Chapter 2.1.9 --- Reagents for Flow Cytometry --- p.42 / Chapter 2.1.10 --- In Vivo Study --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Oligonucleotide Design --- p.44 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.47 / Chapter 2.2.3 --- Transfection --- p.47 / Chapter 2.2.4 --- MTT Assay --- p.47 / Chapter 2.2.5 --- D-[U14C]-fructose and 2-deoxy-D-[l-3H] Glucose Uptake Assay --- p.48 / Chapter 2.2.6 --- Detection of Intracellular ATP Concentration --- p.49 / Chapter 2.2.7 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.2.7.1 --- RNA Extraction by TRIzol Reagent --- p.51 / Chapter 2.2.7.2 --- Determination of RNA Concentration --- p.51 / Chapter 2.2.7.3 --- Reverse Transcription --- p.52 / Chapter 2.2.7.4 --- Polymerase Chain Reaction (PCR) --- p.52 / Chapter 2.2.8 --- Real-Time PCR --- p.55 / Chapter 2.2.8.1 --- Analysis of the Real-Time PCR Data --- p.57 / Chapter 2.2.9 --- Western Blot Analysis --- p.58 / Chapter 2.2.9.1 --- Protein Extraction --- p.58 / Chapter 2.2.9.2 --- Protein Concentration Determination --- p.58 / Chapter 2.2.9.3 --- Western Blotting --- p.60 / Chapter 2.2.10 --- Flow Cytometry --- p.62 / Chapter 2.2.10.1 --- Detection of Cell Cycle Pattern with PI --- p.62 / Chapter 2.2.10.2 --- Detection of Apoptosis with Annexin V/PI --- p.62 / Chapter 2.2.11 --- In Vivo Study --- p.63 / Chapter 2.2.11.1 --- Establishment of Tumor-Bearing Animal Model --- p.63 / Chapter 2.2.11.2 --- Treatment Schedule --- p.63 / Chapter 2.2.11.3 --- Toxicity of Antisense Oligonucleotides --- p.64 / Chapter Chapter 3 --- Results --- p.66 / Chapter 3.1 --- In Vitro Study --- p.67 / Chapter 3.1.1 --- Effect of Tamoxifen on MCF-7 cells and MDA-MB-231 cells --- p.67 / Chapter 3.1.2 --- Cytotoxicity of Antisense Oligonucleotides against Glut 5 on MCF-7 cells and MDA-MB-231 cells by MTT Assay --- p.69 / Chapter 3.1.3 --- Effect of Antisense Oligonucleotides against Glut 5 on Fructose and Glucose Uptake of MCF-7 cells and MDA-MB-231 cells by D-[U14C]-Fructose & 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.77 / Chapter 3.1.4 --- Effect of Antisense Oligonucleotides against Glut 5 on Intracellular ATP Content of MCF-7 cells and MDA-MB-231 cells by ATP Assay --- p.81 / Chapter 3.1.5 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 RNA Expression of MCF-7 cells and MDA-MB-231 cells by RT-PCR and Real-Time PCR --- p.83 / Chapter 3.1.5.1 --- RT-PCR --- p.83 / Chapter 3.1.5.2 --- Real-Time PCR --- p.87 / Chapter 3.1.6 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 Protein Expression of MCF-7 cells and MDA-MB-231 cells by Western Blot Analysis --- p.89 / Chapter 3.1.7 --- "Effect of Antisense Oligonucleotides against Glut 5 on Change in Cell Cycle Pattern of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using PI Stainning" --- p.93 / Chapter 3.1.8 --- "Effect of Antisense Oligonucleotides against Glut 5 on Induction of Apoptosis of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using Annexin V-FITC Stainning" --- p.98 / Chapter 3.2 --- In Vivo Study --- p.101 / Chapter 3.2.1 --- Animal Model: Nude Mice --- p.101 / Chapter 3.2.2 --- Effect of Antisense Oligonucleotides against Glut 5 on the MCF-7 cells-Bearing Nude Mice --- p.101 / Chapter 3.2.2.1 --- Change of Weight of the Tumor-Bearing Nude Mice --- p.101 / Chapter 3.2.2.2 --- Tumor Growth Rate --- p.105 / Chapter 3.2.2.3 --- Glut 5 RNA Expression by Real-Time PCR --- p.109 / Chapter 3.2.2.4 --- Glut 5 RNA Expression by Western Blotting --- p.111 / Chapter 3.2.3 --- "Assessment of Side Effects of Antisense Oligonucleotides against Glut 5, by Measuring the Plasma Enzyme Level" --- p.113 / Chapter Chapter 4 --- Discussion --- p.118 / Chapter 4.1 --- Antisense Oligonucleotides against Glut 5 on Human Breast Cancer --- p.119 / Chapter 4.1.1 --- Antisense Oligonucleotides Strategy --- p.119 / Chapter 4.1.2 --- Role of Glut 5 in Breast Cancer --- p.123 / Chapter 4.1.3 --- Effects of Tamoxifen on MCF-7 and MDA-MB-231 --- p.126 / Chapter 4.2 --- In Vitro Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.127 / Chapter 4.3 --- In Vivo Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.135 / Chapter 4.3.1 --- Effects of Antisense Oligonucleotides against Glut 5 on Body Weight and Tumor Size --- p.137 / Chapter 4.3.2 --- Expression Level of Glut 5 of the Tumor --- p.138 / Chapter 4.3.3 --- Assessment of Side Effects of Antisense Oligonucleotides against Glut 5，by Measuring the Plasma Enzymes Level --- p.140 / Chapter 4.4 --- Possible Mechanism of Antisense Oligonucleotides against Glut 5 on Breast Cancer --- p.141 / Chapter Chapter 5 --- Future Prospectus and Conclusions --- p.143 / Chapter 5.1 --- Future Prospectus of Antisense Oligonucleotides --- p.144 / Chapter 5.1.1 --- Antisense Oligonucleotides and Treatment of Breast Cancer --- p.144 / Chapter 5.1.2 --- Role of Glut 5 in Breast Cancer --- p.147 / Chapter 5.2 --- Conclusions and Remarks --- p.148 / References --- p.151

Dietary fats have been proposed to alter the amount of glucose transporters in various tissues. This study examined how diets containing linoleic or palmitic fatty acids affected the amount of the major insulin-responsive glucose transporter protein, GLUT-4, in red vastus muscle of mice. At 8 weeks of age, 28 healthy female mice were separated into 3 dietary groups, one control group (5% corn oil fat) and two high fat (15% fat) groups. One of the high fat diets was a linoleic acid rich diet (76% linoleic polyunsaturated fat), while the other was a palmitic acid rich diet (95% palmitic saturated fat). The mice remained on their respective diets for 12-13 weeks until sacrifice. Red vastus muscle samples were removed and prepared for GLUT-4 protein analysis. Homogenized red vastus muscle samples were separated by SDSPAGE, transfered to membrane paper, and immunoblotted. scanning densitometry determined the relative quantity of GLUT-4 from each sample. TAP GLUT-4 protein in the group fed the linoleic acid rich diet was 9% higher than the group fed the low fat diet, and 37% higher than the group fed the palmitic acid rich diet. These data suggest that a prolonged high fat diet consisting of linoleic or palmitic fatty acids play a role in the regulation of GLUT-4 protein content. / School of Physical Education

There is no abstract available for this dissertation. / School of Physical Education