Akt activation mediates increases in glycogen synthesis in response to insulin in humans, while extracellular signal-regulated kinase (ERK) activation increases gene transcription and protein translation in response to endurance and resistance exercise. Akt activation increases only in response to intense muscle contractions and during hypertrophy in rats. No study has examined Akt and ERK activation with increasing numbers of intense muscle contractions in humans. Therefore, the primary objectives of this investigation were to determine if Akt activation increases in response to resistance exercise in humans, and to compare the changes in Akt and ERK activation in response to increasing numbers of muscle contractions.Akt and ERK activation were compared in muscle biopsy samples from 7 men before (Pre) and after (Post) knee extension and control protocols using enzyme linkedimmunosorbent assays. Baseline information was obtained including body composition and maximal strength (1-RM). Subjects were familiarized with knee extensions performed at 70% of 1-RM and a specified repetition cadence (2sec up, 2sec down). Once/wk, subjects performed one protocol in random order: 1 repetition (rep), 10reps, 3 sets of l0reps (3x10), or 6min of sitting. Akt activation decreased 42%, while ERK activation increased 108% in response to 3x10 (p<0.05). Akt and ERK activation did not change with 1 and 10reps, and thus their responses were not dose-dependent with resistance exercise in humans. The findings from this study represent the first indication that Akt activation is reduced in response to resistance exercise in human skeletal muscle, possibly to help mediate reductions in glycogen synthesis. / Human Performance Laboratory

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

Exercise induces morphological and metabolic adaptations that are highly specific to the mode of exercise training. These specific phenotypical changes are due to an equally specific molecular response that may depend on the activation and coordination intramuscular signaling pathways. Just as metabolic and morphological changes are influenced by the mode of exercise training, the signaling pathways that mediate exercise adaptation may also be directly related to the training status of skeletal muscle. For example, pre-conditioned skeletal muscle may exhibit a specific intracellular signaling response to an acute bout of exercise that is dependent on past training history. Both Akt (protein kinase B) and extra-cellular signal-related kinase (ERK 1 /2) have been shown to be phosphorylated in response to an acute bout of resistance exercise in human skeletal muscle and have been suggested to mediate the adaptive response to exercise. The purpose of this investigation was to examine the response of Akt and ERKI/2 to an acute bout of resistance exercise in three groups with distinctly different exercise training backgrounds. Twenty one subjects performed 3 sets of 10 repetitions of knee extension exercise at 70% 1-RM. The subjects consisted of a resistance-trained group (RE) (n=7), endurance trained group (END) (n=7) and a sedentary group (SED) (n=7). Muscle biopsies were taken from the vastus lateralis muscle before, immediately after, and 10 min post-exercise and were analyzed for phosphorylation of Akt and ERK1/2. ERK1/2 phosphorylation increased 47%, and 54% from pre-exercise to immediately post-exercise in the SED and RE groups respectively (p < 0.05). ERK1/2 phosphorylation increased 95%, 196%, and 47% from pre-exercise to 10 min post-exercise in the SED, RE, and END groups, respectively. (p < 0.05). The magnitude of ERK1/2 phosphorylation 10 min post-exercise was different between each group and may be linked to the group's training status. (p < 0.05) Akt phosphorylation decreased 42% and 37% from pre-exercise to immediately post-exercise in the SED and END group, respectively (p < 0.05). There was a 40 % increase in Akt phosphorylation from immediate post-exercise to 10 min post-exercise in the END group. In conclusion, training status appears to influence the magnitude and time course of activation of both Akt and ERK1/2 in response to an acute bout of resistance exercise. The immediate response of both ERK1/2 and Akt may play a key role in the adaptive response of skeletal muscle ultimately resulting in metabolic and morphological changes that are dependent on the past training history of the individual. / School of Physical Education, Sport, and Exercise Science

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.

Chan Siu Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 149-181). / Abstracts in English and Chinese. / Abstract --- p.ii / Chinese Abstract (摘要) --- p.v / Acknowledgements --- p.viii / Publications --- p.x / Table of contents --- p.xii / List of Figures --- p.xvi / List of Table and Diagram --- p.xx / Abbreviations --- p.xxi / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Major objectives and long-term significance --- p.1 / Chapter 1.2 --- Biological significance of apoptosis --- p.1 / Chapter 1.3 --- Importance of apoptosis in the study of the female reproductive system --- p.3 / Chapter 1.4 --- Specific aims of the present project --- p.3 / Chapter 1.5 --- Experimental approaches --- p.8 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- General experimental methods --- p.11 / Chapter 2.1.1 --- Culture of cells --- p.11 / Chapter 2.1.1.1 --- Culture of rabbit immortalized uterine epithelial cells --- p.11 / Chapter 2.1.1.2 --- Culture of primary mouse uterine epithelial cells --- p.12 / Chapter 2.1.1.3 --- Culture of human ovarian epithelial cancer cells --- p.13 / Chapter 2.1.2 --- Assessment of apoptotic DNA fragmentation --- p.13 / Chapter 2.1.2.1 --- DNA extraction --- p.14 / Chapter 2.1.2.2 --- Assessment of apoptotic DNA --- p.14 / Chapter 2.1.2.3 --- Assessment of apoptotic DNA by CE-LIF --- p.15 / Chapter 2.1.2.4 --- Assessment of apoptosis by Nuclear Hoechst 33248 Staining / Chapter 2.1.3 --- Assessement of protein content --- p.16 / Chapter 2.1.3.1 --- Protein extraction and western blot analysis --- p.16 / Chapter 2.1.4 --- Adenoviral infection of A2780s cells --- p.18 / Chapter 2.2 --- Preparation of solutions --- p.18 / Chapter 2.3 --- Animals and cell lines --- p.25 / Chapter 2.4 --- Statistical analysis --- p.25 / Chapter Chapter 3: --- Literature Review / Chapter 3.1 --- Morphological analysis of physiological cell death --- p.26 / Chapter 3.1.1 --- Characteristics of apoptosis --- p.27 / Chapter 3.2 --- Methods of detecting apoptosis --- p.31 / Chapter 3.3 --- Molecules controlling apoptosis --- p.33 / Chapter 3.3.1 --- Caspases --- p.33 / Chapter 3.3.2 --- The Bcl-2 family proteins --- p.34 / Chapter 3.4 --- Apoptosis signalling --- p.36 / Chapter 3.4.1 --- The death receptor-dependent pathway --- p.36 / Chapter 3.4.2 --- The mitochondria-dependent pathway --- p.38 / Chapter 3.4.3 --- The endoplasmic-reticulum-dependent pathway --- p.39 / Chapter 3.5 --- Importance of apoptosis in the female reproductive system --- p.40 / Chapter 3.5.1 --- Apoptosis in uterus epithelial cells --- p.40 / Chapter 3.5.2 --- Apoptosis in ovarian cancer cells --- p.42 / Chapter 3.6 --- Regulation of apoptosis by nitric oxide/cGMP/protein kinase G --- p.44 / Chapter 3.6.1 --- Regulation of apoptosis by nitric oxide --- p.44 / Chapter 3.6.2 --- Regulation of apoptosis by cGMP --- p.48 / Chapter 3.6.3 --- Regulation of apoptosis by soluble guanyly cyclase activator --- p.50 / Chapter Chapter 4: --- "Apoptotic DNA fragmentation caused by sodium nitroprusside, a nitric oxide donor, in uterine epithelial cells: ultrasensitive quantitation using the new capillary electrophoresis/laser-induced fluorescence (CE-LIF) technology" / Chapter 4.1 --- Abstract --- p.52 / Chapter 4.2 --- Introduction --- p.53 / Chapter 4.3 --- Results --- p.57 / Chapter 4.4 --- Discussion --- p.61 / Chapter 4.5 --- Figures of Chapter 4 --- p.66 / Chapter Chapter 5: --- Guanylyl-cyclase inhibitors NS2028 and ODQ and protein-kinase-G inhibitor KT5823 trigger apoptotic DNA fragmentation in an immortalized uterine epithelial cell line: anti-apoptotic effects of basal cGMP/PKG / Chapter 5.1 --- Abstract --- p.74 / Chapter 5.2 --- Introduction --- p.75 / Chapter 5.3 --- Results --- p.80 / Chapter 5.4 --- Discussion --- p.83 / Chapter 5.5 --- Figures of Chapter 5 --- p.89 / Chapter Chapter 6: --- "Direct, prolonged activation of soluble guanylyl cyclase by YC-1 or protein kinase G by cGMP analogs enhances the level of apoptosis in an immortalized uterine epithelial cell line, HRE-H9 cells" / Chapter 6.1 --- Abstract --- p.100 / Chapter 6.2 --- Introduction --- p.101 / Chapter 6.3 --- Results --- p.105 / Chapter 6.4 --- Discussion --- p.107 / Chapter 6.5 --- Figures of Chapter 6 --- p.114 / Chapter Chapter 7: --- "ODQ，an inhibitor of soluble guanylyl cyclase, down-regulates XIAP expression and induces apoptosis in human ovarian cancer cells" / Chapter 7.1 --- Abstract --- p.124 / Chapter 7.2 --- Introduction --- p.125 / Chapter 7.3 --- Results --- p.129 / Chapter 7.4 --- Discussion --- p.132 / Chapter 7.5 --- Figures of Chapter 7 --- p.138 / Chapter Chapter 8: --- Overall Conclusion --- p.145 / Chapter Chapter 9: --- References --- p.149