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.

Obesity and type 2 diabetes are the most prevalent metabolic diseases affecting over 50% of people in the western world. Although the pathogenesis of type 2 diabetes is not fully understood, growing evidence links this disease to a state of chronic inflammation, which occurs in metabolically active tissue such as the liver, adipose tissue and skeletal muscle and results in the secretion of inflammatory cytokines, of which interleukin-6 (IL-6) is one. It is generally accepted that elevations in the plasma and/or tissue of this family of cytokines have a negative effect on whole body glucose homeostasis. While there is compelling evidence for the negative effects of resistin and TNF-á on insulin sensitivity, the role of IL-6 in the etiology of insulin resistance is not fully understood. The notion of negative effects of IL-6 in metabolic processes is further confounded by the marked elevations of IL-6 which occur in conjunction with the beneficial activity of exercise. We firstly sought to examine the effect of the lipolytic hormone adrenaline on IL-6 expression and release in order to establish whether IL-6 acts independently of adrenaline in the regulation of fat metabolism. Reporting the absence of an effect of adrenaline on IL-6, we then investigated the role of IL-6 on metabolic processes in humans at rest and during exercise in circumstances where lipolysis was inhibited. Marked increases in IL-6 circulating protein and tissue gene expression were observed with exercise and further so with fatty acid suppression. In a mouse model of IL-6 depletion marked insulin sensitivity was observed, which was reversed with IL-6 treatment. In a mouse model with normal endogenous IL-6 levels IL-6 treatment also impaired glucose tolerance. Contrastingly, in a rat model both chronic and acute IL-6 treatment improved glucose tolerance In summary, studies from this thesis suggest that, rather than being causally related to insulin resistance, the cytokine IL-6 increases lipolysis, fat oxidation, and glucose metabolism in insulin sensitive tissues in humans. This does not appear to be the case in the mouse, where contrasting actions are observed, perhaps due to differences in the reliance of various parameters for metabolic processes between the species.

This thesis describes an investigation of the effects of vitamin A deficiency on gut function, The central hypothesis to be tested was that acute vitamin A deficiency affects glucose uptake from the small intestine- The hypothesis was tested using a system involving perfusion of isolated segments of the small intestine in the anaesthetized rat. The system was used to study effects on glucose uptake under steady-state conditions. In the initial part of the study, experiments were diverted towards setting up the system for measuring steady-state uptake, and determining the relative contributions of active uptake and diffusion. Phenol red was found to be a reliable non-absorbable marker for determining net water movement. Phlorizin, generally at 1 mmol/L, was used as a competitive (reversible) inhibitor of active uptake. It is difficult however to confirm complete inhibition of active uptake by phlorizin because of the limited solubility of the inhibitor. The kinetics of glucose uptake f ram intra-luminal maltose were found to be, in general, not significantly different from those applying to the uptake of glucose from an equivalent glucose solution. Maltase activity in the perfused gut segment was found to be sufficient to hydrolyse most of the maltose (80 per cent or more) in the solution being perfused, a much greater proportion than was absorbed. Glucose absorptive capacity, measured on an intestinal dry weight basis, was greatest in the duodenum and progressively less in the jejunum and ileum. The rate of water uptake f ran the gut was increased by the presence of glucose in the lumen, and was linked to glucose uptake as shown by the inhibition of water uptake by phlorizin. Uptake of glucose by solvent drag was demonstrated by showing an increased rate of glucose uptake when the rate of water uptake was increased by perfusing a solution of reduced osmotic pressure. In the experiment a low intra-luminal glucose concentration was used to preclude net uptake by diffusion and active uptake was blocked with phlorizin. This process was further investigated using streptozotocin-diabetic rats in which the diabetes establishes a hyperosomotic blood with hyperglycaemia. Uptake by solvent drag was more obvious in diabetic animals. A back-diffusion (exsorption) of glucose from the tissues to the lumen was also shown; the rate being proportional to plasma glucose concentration. Vitamin A deficiency was established in weanling rats after 6-7 weeks feeding on a diet based on wheat starch, coconut oil, and casein washed with hot ethanol, together with vitamins and minerals. The vitamin A deficiency led to classic eye signs and was reversed by the addition to the diet of retinoic acid (5 g/g diet). Vitamin A deficiency decreased intestinal mucus production (dry weight) but had no detectable effect on the histology of the villous epithelium as shown under the light microscope. Using perfusion experiments it was shown that vitamin A deficiency had no significant effect on the rate of active uptake of glucose, but that deficiency increased the rate of passive uptake.

This thesis involves an investigation in three areas; first, a study of an enzymatic-gravimetric method for the analysis of dietary fibre; second, a survey of dietary fibre intake in an area of a developing country, and finally, some observations on the functional aspects of gel-forming dietary fibre in the rat. A simple and rapid enzymatic-gravimetric assay for both soluble and insoluble dietary fibre has been critically investigated. Reference samples were also analysed by a more comprehensive, enzymatic gas chromatographic method to allow testing of the relative accuracy of the enzymatic-gravimetric method. The enzymatic-gravimetric method was found to be highly reproducible but gave a slightly higher value for total dietary fibre than the more comprehensive method. This discrepancy is probably due to the presence of small quantities of resistant starch and protein residue which are recovered in the enzymatic-gravimetric method. In the enzymatic-gas chromatographic method, protein residue is not measured, and resistant starch is estimated, but not counted as dietary fibre. The enzymatic-gravimetric method was applied to the analysis of foods commonly consumed in the Padang region of West Sumatra in Indonesia, in order to estimate dietary fibre intake in the region. Daily intakes of usual foods were estimated by use of a 24-hour recall procedure aided by food photographs to assist in the estimation of portion size. Samples of approximately 60 of the most commonly consumed foods were collected and analysed for dietary fibre. These appear to be the first data which report values for dietary fibre in Indonesion foods and they represent a significant improvement upon the existing data on crude fibre content. Knowledge of the amounts of foods usually consumed and their dietary fibre content allowed an estimation of usual intakes of dietary fibre. Fibre intake was found to be lower than in the developing countries of Africa and was comparable to intakes measured in the U.K. This is the first study to show that in this part of South East Asia, a developing country area using polished rice as a staple food, dietary fibre intakes are as low as in Western countries. Low intakes of fibre are believed to be related to the prevalence of a range of diseases and, in this study, preliminary data on the rates of non-infective, chronic diseases were collected from the two main hospitals in West Sumatra. Chronic, non-infectious diseases such as inguinal hernia, appendicitis, haemorrhoids, diabetes mellitus, hypertension and malignant neoplasms of the rectum are relatively frequent in West Sumatra. While no firm conclusions can be drawn from these data, they do show the possibility of a relationship between low intakes of dietary fibre and the prevalence of these diseases, and suggest that further investigation is necessary. Some observations were made of the effect of gel-forming dietary fibre on stomach emptying and intestinal transit rate in the rat. Xanthan gum was added to iso-osmotic solutions to produce increased viscosity and phenol sulphonphthalein (phenol red) was used as a non-absorbable marker. Gavage feeding of solutions with a range of viscosities was used to study the effect of viscosity on the rate of stomach emptying and intestinal transit. Increased viscosity was observed to slow gastro-intestinal transit and this provides one mechanism by which dietary fibre of the gel-forming type ray improve glucose tolerance.

Thesis (M.S.)--Baylor University, 2005. / Includes bibliographical references (p. 53-56).

Urban forests grow in stressful environments that can have negativerepercussions on tree energy reserves. The goal of this research was to evaluate theimpact of exogenously applied carbohydrates on growth and vitality of live oaks(Quercus virginiana P. Miller). An initial study focused on carbohydrate partitioningrevealed that annual mean glucose concentration in leaf tissues (49.55 mg·g-1 DW) wasalmost double that in twigs, trunks, or roots. Starch concentrations in roots and trunks(38.98 and 38.22 mg·g-1 DW of glucose, respectively) were higher during the dormantseason and approximately three times the concentrations found in other tissues. Aninvestigation of the effects of exogenous soil applications of glucose and starch on soilmicrobial activity revealed no significant differences using recoverable viable microbes.However, soil respiration was significantly increased (P<0.05) by glucose a week afterapplication, while higher starch concentrations (120 g·L-1) significantly increased(P<0.05) soil respiration after the fourth week. Although tree soil drenched withcarbohydrates in a different study showed significantly (P<0.05) greener leaf color, higher chlorophyll fluorescence, and increased soil respiration at higher concentrationsof starch (120 g·L-1), no significant differences were observed in photosynthesis or trunk,canopy, or root growth. Analysis of 13C signatures was unable to detect uptake ofexogenous carbohydrates. For trunk-injected trees with glucose and sucrose, trunkgrowth was significantly (P<0.05) increased by carbohydrate supplementation.Differences were also found in twig glucose content, root starch content, and chlorophyllfluorescence among overall concentration means. A study to compare field diagnostictools with carbohydrate laboratory analysis established that a portable blood glucosemeter can be used to measure glucose content in trees. However, ohmmeter,refractometer, chlorophyll fluorescence spectrometer, and iodine staining results did notcorrelate well with laboratory analysis of carbohydrate concentrations. Results fromthese studies reveal that soil applied carbohydrates can greatly increase soil microbialactivity, provide evidence that trunk-injected carbohydrates may improve growth andvitality of live oaks, and provide a new field diagnostic tool to increase the efficiency ofmeasuring carbohydrates in trees.

The transport of glucose across the plasma membrane is a fundamental mechanism to provide cells with its basic requirements for energy yielding processes. It is also vital for clearing glucose from blood into tissues, a process normally stimulated by the hormone insulin in mammals. The sympathetic nervous system, normally activated during stress, also regulates glucose transport. The sympathetic neurotransmitter noradrenaline, acts on the family of adrenergic receptors (ARs). An important subtype of the AR family is the β-AR, which is subdivided into the β1, β2, and β3-AR. Glucose is transported across the plasma membrane by the family of glucose transporters (GLUT1-12, and HMIT). In this thesis, I have investigated the β-AR regulation of GLUT1 and 4, and glucose uptake, in skeletal muscle cells and brown adipocytes in culture, model systems which correspond to metabolically active, sympathetically innervated and insulin-sensitive tissues. In brown adipocytes, activation of the β3-ARs induced the expression of GLUT1, resulting in a large increase of glucose uptake. In skeletal myotubes, we postulate there is a possible mechanism where β2-ARs can regulate the intrinsic activity of GLUT1. We found that insulin signaling, but not β-adrenergic signaling, mediated glucose uptake through class I phosphatidylinositol 3-kinase (PI3K). The β-adrenergic signaling to glucose uptake appeared to involve a PI3K related kinase (PIKK), in both skeletal myotubes and brown adipocytes. Furthermore, the increase of glucose uptake by β-ARs in brown adipocytes is partially mediated by AMP-activated protein kinase (AMPK). However, in an artificially constructed system, with cells expressing GLUT4 and β2-ARs, both insulin and β-adrenergic activation translocated GLUT4 and increased glucose uptake. These results show that β-adrenergic signaling increase glucose uptake by regulating glucose transporters through distinct pathways, in skeletal myotubes and brown adipocytes.

<p>Healthy cells require input from growth factor signaling pathways to maintain cell metabolism and survival. Growth factor deprivation induces a loss of glucose metabolism that contributes to cell death in this context, and we have previously shown that maintenance of glycolysis after growth factor deprivation suppresses the activation of p53 and the induction of the pro-apoptotic protein Puma to prevent cell death. However, it has remained unclear how cell metabolism regulates p53 activation and whether this increased glycolysis promotes cell survival in the face of additional types of cell stress. To examine these questions, we have utilized a system in which stable overexpression of the glucose transporter Glut1 and hexokinase 1 in hematopoietic cells drives growth-factor independent glycolysis. This system allows us to examine the effects of glucose metabolism in the absence of other signaling events activated downstream of growth factor receptors. Here, we demonstrate that elevated glucose metabolism, characteristic of cancer cells, can suppress PKC&#948;-dependent p53 activation to maintain cell survival after growth factor withdrawal. In contrast, DNA damage-induced p53 activation was PKC&#948;-independent and was not metabolically sensitive. Both stresses required p53 serine 18 phosphorylation for maximal activity but led to unique patterns of p53 target gene expression, demonstrating distinct activation and response pathways for p53 that were differentially regulated by metabolism.</p><p>Unlike the growth factor-dependence of normal cells, cancer cells can maintain growth factor-independent glycolysis and survival and often demonstrate dramatically increased rates of glucose uptake and glycolysis, in part to meet the metabolic demands associated with cell proliferation. Given the ability of elevated glucose metabolism to suppress p53 activity in the context of metabolic stress, we examined the effect of increased glucose uptake on leukemogenesis using a mAkt-driven model of leukemia and adoptive transfer experiments. We show here that elevated glucose uptake promoted leukemogenesis in vivo, perhaps through suppression of p53 transcriptional activity. During the process of leukemogenesis, cancerous cells can acquire growth factor independent control over metabolism and survival through expression of oncogenic kinases, such as BCR-Abl. While targeted kinase inhibition can promote cancer cell death, therapeutic resistance develops frequently and further mechanistic understanding regarding these therapies is needed. Kinase inhibition targets the necessary survival signals within cancerous cells and may activate similar cell death pathways to those initiated by growth factor deprivation. As we have demonstrated that loss of metabolism promotes cell death after growth factor withdrawal, we investigated whether cell metabolism played a role in the induction of apoptosis after treatment of BCR-Abl-expressing cells with the tyrosine kinase inhibitor imatinib. Consistent with oncogenic kinases acting to replace growth factors, treatment of BCR-Abl-expressing cells with imatinib led to reduced metabolism and p53- and Puma-dependent cell death. Accordingly, maintenance of glucose uptake inhibited p53 activation and promoted imatinib resistance, while inhibition of glycolysis enhanced imatinib sensitivity in BCR-Abl-expressing cells with wild type p53 but had little effect on p53 null cells. Together, these data demonstrate that distinct pathways regulate p53 after DNA damage and metabolic stress and that inhibition of glucose metabolism may enhance the efficacy of and overcome resistance to targeted molecular cancer therapies.</p> / Dissertation

Les glioblastomes sont, de par leur caractère invasif et infiltrant, des tumeurs cérébrales extrêmement résistantes aux thérapies classiques. Nous avons étudié les propriétés anti-cancérigènes de polyphénols extraits de produits naturels, notamment l'acide chlorogénique (CHL). Le CHL est un puissant inhibiteur fonctionnel du transporteur microsomale de glucose-6-phosphate (G6PT) responsable de l'étape limitante de la conversion du glucose-6-phosphate en glucose (Glc) et en phosphate inorganique par la glucose-6-phosphase (G6Pase) lors de la glyconéogenèse et de la glycogénolyse. En émettant l'hypothèse selon laquelle le système G6Pase se localise exclusivement dans les tissus glyconéogéniques tels que le foie et les reins, nos travaux ont alors pour objectif d'élucider le rôle de G6PT dans des tissus non producteurs de Glc tels les cellules gliales, et particulièrement dans la progression tumorale des glioblastomes. Au cours de notre étude, nous avons testé l'impact de l'inhibition de G6PT soit fonctionnelle par le CHL, soit génique par un siRNA spécifique, sur différents processus impliqués dans la survie et l'invasion tumorale tel que la migration cellulaire et la dégradation de la matrice extracellulaire (MEC) par les métalloprotéases (MMPs). Expérimentalement, les mesures des niveaux d'ARNm par RT-PCR, démontrent que l'expression génique de G6PT est plus élevée dans les glioblastomes U87, que dans toute autre lignée tumorale cérébrale testée. Le CHL inhibe la sécrétion de la MMP-2, et la migration cellulaire des U87, deux pré-requis pour l'invasion tumorale. La migration induite par G6PT recombinant suite à une transfection des cellules à l'aide du cDNA codant pour ce transporteur est aussi inhibée par le CHL. De plus, le CHL inhibe la migration cellulaire et la phosphorylation de ERK induite en réponse à la sphingosine-1-phosphate, un lysophospholipide abondant dans la MEC cérébrale. Nous démontrons, de plus, que le 2-deoxy-D-Glc et le 5-thio-Glc, deux analogues non métabolisables du Glc et menant à la déplétion de l'ATP intracellulaire, inhibent la sécrétion de MMP-2. Par ailleurs, l'inhibition de G6PT par un siRNA induit une mort cellulaire par apoptose détectée par cytométrie de flux. Une surexpression de la métalloprotéase membranaire de type 1 (MT1 -MMP) conduit à une diminution de l'expression génique de G6PT. De manière globale, nos résultats suggèrent que G6PT régulerait certaines fonctions invasives des cellules cancéreuses, et pourrait avoir une implication dans la signalisation intracellulaire de ces dernières, d'où son potentiel comme nouvelle cible pour les thérapies anti-cancer. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Glucose-6-phosphase, Acide chlorogénique, Glioblastomes.

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