The insulin-dependent uptake of glucose by adipose and muscle tissues is accomplished through the regulated vesicle trafficking of the GLUT4 glucose transporter to the plasma membrane. The distal trafficking events comprising the tethering, docking and fusion of GLUT4 vesicles with the plasma membrane are poorly defined, but represent vital steps in this pathway. This dissertation encompasses a series of complementary studies that have provided new insights into how these events are regulated in the adipocyte. The Sec1p homologue Munc18c, is believed to play a central role in the docking of GLUT4 vesicles by controlling SNARE complex assembly. Munc18c was shown to bind the t-SNARE Syntaxin4 and form a stable complex in vivo. Protein binding studies demonstrated that Munc18c interacts with Syntaxin4 via an evolutionarily conserved N-terminal binding mode and the formation of the Munc18c/Syntaxin4 hetero-dimer was shown to promote SNARE complex assembly. In contrast to previous reports, I propose that Munc18c is positive regulator of SNARE assembly and vesicle docking. The exocyst complex is thought to promote the tethering of exocytic GLUT4 vesicles with the plasma membrane. Yeast two-hybrid screens revealed interactions between the exocyst subunits Sec6 and Exo70 and the SNARE-associated proteins Munc18c and Snapin, respectively. Snapin was subsequently shown to have a novel role in GLUT4 trafficking. These interactions suggest Munc18c and Snapin provide a course for cross-talk between the exocyst complex and the SNAREs to stimulate GLUT4 vesicle tethering and docking. In addition to its interactions with Munc18c and Snapin, the exocyst was also found to interact with the GTP-bound form of RalA, a small GTPase regulated by insulin. RalA was almost exclusively localised to the plasma membrane of the adipocyte and a novel role for the RalA/exocyst interaction in GLUT4 trafficking was demonstrated. Specifically, overexpression of a GTP-deficient RalA mutant significantly inhibited insulin-stimulated GLUT4 appearance on the plasma membrane. In addition to its role in GLUT4 trafficking, a novel role for RalA was demonstrated in insulin release from pancreatic -cells, indicating that RalA may represent a universal component of regulated exocytosis. It is becoming increasingly apparent that vesicle trafficking events from yeast to mammals rely on similar protein complexes which communicate through multiple protein interactions, ensuring vesicle transport is highly coupled. Similarly, the Munc18c studies demonstrate that while mammalian cells have evolved to fulfil specialised functions throughout the body, some proteins appear to have retained the biochemical properties of their ancestors, emphasing the importance of this family of proteins throughout eukaryotic vesicle transport. In contrast, proteins such as RalA have evolved only in higher eukaryotes and appear to play a universal role in vesicle transport despite vast differences in the specialised functioning of mammalian cells.

Glucose is a primary and essential energy source for humans. It is broken down from complex carbohydrates in the diet and absorbed across the gut epithelium into the blood stream. Glucose homeostasis is important as hyperglycermia causes damage of pancreatic and peripheral cells. In response to a meal glucose is principally taken up by fat and muscle tissues and this response is activated by insulin release from pancreatic beta cells. Insulin stimulates the translocation of GLUT4 from the intracellular storage vesicles to the plasma membrane in fat and muscle cells. Although many proteins have been implicated in this process, the key insulin-regulated substrate has not been determined yet. In the present study, the phosphoserine/threonine binding protein 14-3-3 was used as a tool to affinity-purify insulin-stimulated phosphoproteins from 3T3-L1 adipocytes. By using mass spectrometry 38 proteins were identified, reflecting the important role of 14-3-3 in mediating many insulin-regulated processes. Among the potential phosphoproteins was Myosin 1C (Myo1c), an actin-associated molecular motor, which has previously been implicated in insulin-stimulated GLUT4 trafficking in adipocytes. I showed that insulin stimulates the activation of CaMKII which phosphorylates Myo1c at S701 in a Ca2+/PI3K-dependent manner. Myo1c phosphorylation induced its interaction with 14-3-3-proteins, reduced calmodulin-binding and stimulated its in vitro ATPase activity. Insulin-dependent stimulation of Myo1c phosphorylation and its ATPase activity were both required for GLUT4 translocation. By using yeast two-hybrid techniques, I identified a candidate ligand of the Myo1c tail, Armcx5, and demonstrated the in vivo interaction in 3T3-L1 adipocytes. The siRNA-mediated knockdown of Armcx5 inhibited insulin-stimulated glucose uptake and GLUT4 translocation. These results suggest that the regulation of Myo1c and its ligand Armcx5 are essential in insulin-regulated GLUT4 trafficking, possibly playing a key role in vesicle fusion.

Several factors may determine the rate. at which exogenous carbohydrate (CHO) is utilised by the human working muscles during prolonged (> 90 min moderate-intensity (63% of peak sustained power output [PPO]) exercise. These include i) the rate of gastric emptying of an ingested fluid, ii) the rate of digestion, absorption and subsequent transport of glucose into the systemic circulation, and iii) the rate of glucose uptake and oxidation by the working muscles. To test the hypothesis that the rate of gastric emptying is the primary factor limiting the rate of CHO delivery to the working muscles during exercise, uniformly labelled ¹⁴carbon (U-¹⁴C) tracer techniques were used in association with conventional gas exchange measurements and post-exercise gastric aspiration to compare the rates of gastric emptying, intestinal CHO delivery and ingested CHO oxidation from 15 g/100 ml solutions of glucose, maltose, a 22 chain-length glucose polymer, and an isocaloric 'soluble' starch preparation. Two groups of six highly-trained male cyclists or triathletes each ingested two of the test drinks which were given as a 400 ml loading bolus immediately before and then as eight 100 ml feedings at 10 min intervals during 90 min of continuous cycling at a work rate of 63% of PPO (~70% of maximal oxygen consumption [VO₂ₘₐₓ]).

Hypothesis: Glucose is known to be protective in moderate low flow ischaemia due to the production of glycolytic ATP. However, it is questioned whether glucose would still be protective in ultra-low flow ischaemia. Firstly, glycolysis is thought to be inhibited, and secondly, deleterious glycolytic metabolites accumulate. Our hypothesis was that in ultra-low flow ischaemia, glucose utilisation is not inhibited at the level of glycolysis, but by delivery. Increased delivery of glucose should result in increased production of protective glycolytic ATP, but the rate of metabolite accumulation would also increase. Using ultra low flow rates, I wished to investigate how to achieve optimal rates of glycolysis, and how such rates would be balanced by any detrimental component of metabolite accumulation. Methods: The isolated Langendorff-perfused rat heart, with a left ventricular balloon to record ischaemic contracture and reperfusion stunning, was used, with severe flow restriction. Glucose concentrations were changed and pre-ischaemic glycogen contents were altered by perfusion with different substrates (acetate - depletion~ glucose + insulin - loading) or by preconditioning, with 5 min ischaemia and 5 min reperfusion prior to sustained ischaemia. Results: Analysis of glucose uptake relative to delivery showed that in severe low flow ischaemia, the extraction of glucose was increased, and glycolysis was thus limited more by substrate supply than by enzyme inhibition. Analysis of metabolites confirmed this concept. The optimal glucose concentration during severe low flow ischaemia was 11 mM, giving maximal recovery on reperfusion. Both lower and higher glucose concentrations increased ischaemic contracture. Changes in pre-ischaemic glycogen levels correlated with the time to onset of contracture, such that a reduction in glycogen accelerated contracture. Prior glycogen depletion or loading did not improve functional recovery. The benefits of preconditioning on reperfusion function following sustained total global ischaemia could not be related to glycogen depletion. If preconditioning were followed by sustained low flow ischaemia, glucose uptake was increased, but no benefit was found, possibly because a low residual flow abolished the effects of preconditioning. Many of the above results are consistent with the hypothesis that too low a rate of glycolysis results. in insufficient ATP production for protection, while excess glycolytic rates lead to excess metabolite accumulation with detrimental effects. Conclusions: Provision of glucose at the correct concentration, when the benefit associated with glycolytic ATP outweighs the detriment associated with moderate metabolite accumulation, is protective to the low-flow ischaemic myocardium, which can upregulate its ability to extract glucose. Improved residual flow enhances this benefit. Prior glycogen depletion is not beneficial, despite a reduced metabolite accumulation. This mechanism cannot be related to the protective effect of preconditioning.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20238号 / 医博第4197号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 河本 宏, 教授 小杉 眞司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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京都大学 / 新制・論文博士 / 博士(医学) / 乙第13489号 / 論医博第2257号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 波多野 悦朗, 教授 羽賀 博典 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Oesophageal adenocarcinoma (OAC) is a leading cause of cancer death in the UK and current systemic therapies are ineffective for the majority of patients. The central aim of this work was to explore strategies to identify novel therapeutic targets. Research has failed, thus far, to identify a dominant oncogene in OAC, although the tumour suppressor p53 is frequently mutated. Inhibiting the mitotic kinase, polo-like kinase 1 (PLK-1), was proposed as a synthetic lethal strategy. PLK-1 was demonstrated to be over-expressed in both verified OAC cell lines and human OAC tissue compared to non-transformed cells and epithelium. Mutation of p53 was associated with over-expression of PLK-1 in both OAC and ovarian cancer tissue. Using a carefully validated viability assay, both an established and novel PLK-1 inhibitor were demonstrated to induce a G2/M arrest and reduce OAC cell proliferation. Relative selectivity was demonstrated for OAC compared to non-transformed cells. This therapeutic window could be enhanced with the induction of cancer cell cytotoxicity by pulsed administration of a short half-life inhibitor. Immunotherapeutics offer potential tumour-selectivity but no OAC-specific proteins have been defined. A comparative proteomic approach was employed to identify OAC-specific proteins as potential therapeutic targets. A tissue resource was established and methods to lyse fresh frozen biopsies optimised. An isobaric quantitative proteomic workflow was applied to OAC and matched normal biopsies and quantitative accuracy confirmed for 6 candidate proteins by immunohistochemistry. Proteome coverage and quantitative dynamic range were compared between isobaric and label-free systematic sequencing proteomic strategies applied to further patients’ tissues. The challenges of combining incomplete datasets were approached with a Bayesian framework to estimate the probability that a protein was missed during an experiment compared to not being present in the sample. This method was applied to generate a complete set of protein identifications and relative tissue expression. To gain insight into the dysregulated cellular processes in human OAC tissue, a network analysis was applied to the quantitative proteomic data. Enriched functional clusters were identified suggesting deranged glucose metabolism, potentially due to the Warburg effect. These findings were duplicated and candidate tumour-specific proteins identified in a further set of biopsies using the optimised quantitative proteomic method. The combined quantitative oesophageal proteomic dataset represents the largest in OAC to date. This thesis demonstrates a hypothesis-driven, synthetic lethal approach can yield cancer-selective therapeutic effects. Novel candidate therapeutic targets are also revealed through the development of quantitative proteomic methods and the application of network analysis.

<p>Understanding the mechanisms that control T cell function and differentiation is crucial to develop new strategies to modulate immune function and prevent autoimmune and inflammatory disease. The balance between effector (Teff; Th1, Th2 and Th17) and regulatory (Treg) T cells is critical to provide an appropriate, but not excessive, immune response and therapies to induce Treg or inhibit Teff are likely promising treatment strategies. It has recently become clear that T cell metabolism is important in both T cell activation and differentiation. T cells undergo a metabolic reprogramming upon activation and not all differentiated T cell subsets utilize the same metabolic fuels or programs.</p><p>These metabolic differences are not trivial, as T cell metabolism is tightly</p><p>regulated and dysregulation can lead to cell death or reduced immunity. An</p><p>understanding of the metabolic differences between Teff and Treg may lead to a new direction for treating inflammatory diseases by modulating the Teff:Treg balance through metabolic inhibition. Previous studies have shown that Teff express higher levels of the glucose transporter Glut1 than Treg, however the role of Glut1, and importantly, the cell-intrinsic role of glucose metabolism in T cell differentiation and inflammation was not previously examined. The work presented here examines the role of Glut1 in T cell differentiation. We show that effector CD4 T cells were dependent on Glut1 for proliferation and function both in vitro and in vivo. In contrast, Treg were Glut1-independent and capable of suppressing colitis in the absence of Glut1 expression.</p><p>Additionally, previous studies have shown broad metabolic differences between Teff and Treg, however the specific metabolic profiles of Teff and Treg are poorly understood. Here, Teff and Treg metabolism is examined to test if dependence on distinct metabolic pathways will allow selective targeting of different T cell populations. We show that pyruvate dehydrogenase kinase 1 (PDHK1) is differentially expressed in the T cell subsets and inhibition of PDHK1 selectively suppresses Th17 and promotes Treg differentiation and function. Because Teff and Treg have distinct metabolic profiles, we hypothesized that the Treg-­specific transcription factor FoxP3 may drive the Treg oxidative metabolic program. We therefore examined the role of FoxP3 in T cell metabolism and determined that FoxP3 promotes glucose and lipid oxidation and suppresses glycolytic metabolism. Importantly, we show that promoting glycolysis with transgenic expression of Glut1 inhibits Treg suppressive capacity. Together, these data suggest that FoxP3 drives an oxidative metabolic program that is critical to Treg function. Overall, this work examines the metabolic phenotypes and regulation of Teff and Treg and potential metabolic targets that could be used to treat autoimmune and inflammatory disease.</p> / Dissertation

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: While antiretroviral therapy decreases HIV/AIDS morbidity and mortality, long-term treatment results in insulin resistance and cardiovascular diseases. A possible cause of such adverse effects may be an increase in oxidative stress resulting from protease inhibitor (PI)-induced mitochondrial dysfunction. We therefore hypothesized that PI treatment, specifically Lopinavir/Ritonavir, results in increases in myocardial reactive oxygen species (ROS), leading to downstream outcomes, i.e. elevated apoptosis. Moreover, we proposed that increased ROS levels in this instance might occur as a result of PI-mediated induction of the non-oxidative glucose pathways (NOGPs). In light of this, we also investigated the effect of PI treatment on the NOGPs by employing both in vitro and in vivo samples. For the in vitro work we employed a rat cardiomyoblast cell line, while tissues (heart, liver) were collected from two separate experimental models, i.e. a) Group A exposed to PIs via mini-osmotic pump for a period of eight weeks, and b) Group B administered PIs via a jelly-based method for 16 weeks. We found that PIs increased mitochondrial ROS levels in vitro but that this was not accompanied by a parallel rise in programmed cell death. Moreover, we found no induction of the NOGPs in response to PI exposure (for both in vitro and in vivo models here employed). However, we found that the AGE pathway was significantly down-regulated in the liver of Group A. Investigation into a proposed mechanism for this observation proved inconclusive and further studies are thus required to clarify the significance in terms of metabolic dysfunction found in the Group A model. Our study thus shows that PIs can increase ROS levels (in vitro) but that compensatory antioxidant mechanisms may prevent this in vivo. Subsequently, downstream effects were limited i.e. we did not observe NOGP induction and programmed cell death. An intriguing finding emerged, however, i.e. that PIs can elicit an impact on the AGE pathway. We propose future studies with modifications to the current rat and cell models in order to evaluate the downstream effects of PIs on the NOGPs and programmed cell death. / AFRIKAANSE OPSOMMING: Terwyl antiretrovirale terapie MIV/VIGS morbiditeit en mortaliteit verlaag, veroorsaak langtermyn behandeling insulienweerstandigheid en kardiovaskulêre siekte. 'n Moonltike oorsaak van sulke newe-effekte kan 'n toename in oksidatiewe stres veroorsaak deur die protease inhibeerder (PI)-geïnduseerde mitochondriale wanfunskionering. Ons hipotetiseer dat PI behandeling, spesifiek Lopinavir/Ritonavir, versoorsaak 'n toename in miokardiale reaktiewe suurstofspesies (ROS), wat aanleiding gee tot afstroom uitkomste, i.e. verhoogde apoptose. Verder, stel ons voor dat verhoogde ROS vlakke in hierdie geval onstaan as gevolg van PI-gemedieerde induksie van die nie-oksidatiewe glukose weë (NOGWe). In die lig hiervan het ons ook die effek van PI behandeling op die NOGWe ondersoek deur beide in vitro en in vivo monsters te gebruik. Vir die in vitro werk het ons van 'n rot kardio-mioblastsellyn gebruik gemaak, terwyl weefsels (hart, lewer) versamel is van twee afsonderlike eksperimentele modelle, i.e. a) Groep A blootgestel aan PIs via mini-osmotiese pomp vir 'n periode van agt weke, en b) Groep B PIs is toegedien via 'n jellie gebaseerde metode vir 16 weke. Ons het bevind dat die die PIs mitochondriale ROS vlakke in vitro verhoog maar dat dit nie vergesel is met 'n paralelle toename in apoptose. Verder is geen induksie van die NOGWe in reaksie op PI blootstelling waargeneem (vir beide in vitro en in vivo modelle). Hoewel ons het bevind dat die AGE weg in die lewer van Groep A beduidend afgereguleer is. Ondersoek na 'n moontlike megansime vir hierdie waarneming was onoortuigend en verdere ondersoek is nodig om die betekenis in terme van die metaboliese wanfunskionering in die Groep A model vas te stel. Ons studie toon dus aan dat PIs, ROS vlakke (in vitro) verhoog, maar dat kompensatoriese anti-oksidant meganismes in die hierdie in vivo model verhoed word. Gevolglik is die afstroom effekte beperk i.e. ons het geen NOGWe induksie en aptoptose waargeneem nie. 'n Interesante bevinding het wel uitgestaan, i.e. PIs kan 'n impak hê op die AGE weg. Ons stel dus voor dat toekomstige studies met modifikasies, tot die huidige rot- en sel-modelle gemaak word om die afstroomeffekte van PIs en apoptose te evalueer.