Dissertations / Theses on the topic 'Glucose uptake'

Glucose uptake by the cellulclytic rumen anaerobe, Bacteroides succinogenes S85, was measured under conditions that maintained anaerobiosis and osmotic stability. This organism was found to possess a highly specific, active transport mechanism for glucose. Evidence for a phosphoenol-pyruvate:g1ucose phosphotransferase system was not detected. Compounds that inhibit electron transport systems (non-heme iron chelators, and sulfhydryl reagents) were effective inhibitors of glucose uptake. The strongest inhibitors were compounds (proton and metal ionophores) that interfere with maintenance of the proton motive force. Compounds which interfere with ATP synthesis also inhibited glucose uptake, but a role for ATP in energizing uptake could not be inferred from these results. Oxygen prevented glucose uptake (75% inhibition), reflecting possible active sulfhydryl centers (above) or autooxidation of electron transport components. The results suggest the fumarate reductase-coupled electron transport system of B. succinogenes can generate a proton motive force that is used to energize glucose uptake. Na+ and Li+. but not K+, stimulated glucose uptake and may partly account for the growth requirement of B. succinogenes for Na+. However, the data were insufficient to conclude that glucose uptake occurs by a Na+ symport mechanism. Spheroplasts of B. succinogenes transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose binding protein. A variety of sugars including the nonmetabolizable analog, [inversely proportional symbol]-methylglucoside. did not inhibit glucose uptake. Only cellobiose and 2-deoxyglucose were active and neither behaved as a competitive inhibitor. Metabolism of both sugars was probably responsible for the inhibition. Cellobiose-grcwn B. succinogenes showed a reduced ability to transport glucose compared to glucose-grown cells. This may indicate regulation of synthesis of the glucose carrier protein by cellobiose through a mechanism other than catabolite repression. Differences in the ability to transport glucose were detected between transition cells (transition from lag to log phase of growth) and log-phase cells. However, the differences were not due to different glucose transport mechanisms. Alterations in the structural integrity of the cell envelope, as reflected by osmotic- and cold-sensitivity features of transition and log cells, may have affected the glucose uptake abilities in these cell types.

The purpose of this investigation was to determine the relationship between calcium and glucose uptake following muscle contraction with the use of the calcium channel blocker dantrolene. In previous studies an exercise model has been used to investigate the role of calcium during post-exercise glucose uptake. This study utilized electrical stimulation. It has been shown that exercise-induced glucose uptake is calciummediated, but to date no one has shown that glucose transport induced by electrical stimulation is calcium-mediated. Twenty four male Sprague Dawley rats weighing 140 g were sacrificed and their epitrochlearis muscles were removed. Four treatment groups were established: control, muscle incubated in glucose (4mM); insulin, muscles incubated in glucose (4mM) and insulin (1000uU/ml); electrical stimulation, at 50 Hz for two five minute intervals separated by one minute rest periods; insulin (1000uU/ml) and electrical stimulation at 50 Hz for two five minute intervals separated by one minute intervals. Each group consisted of contain 8-10 muscle preparations. Glucose uptake was measured through the use of a double label of radioactive mannitol and 3-O-methylglucose and analyzed using liquid scintillation. This project followed a randomized group design. Treatments were measured with a one way ANOVA.
School of Physical Education

Insulin maintains glucose homeostasis through its binding of the insulin receptor and activation of the insulin signalling cascade in insulin sensitive tissues. Skeletal muscle is a major endocrine organ, and is responsible for the majority of post-prandial glucose disposal. The maintenance of glucose homeostasis is a delicate balance and impairments in glucose disposal can have significant physiological effects, resulting in the onset of metabolic diseases such as diabetes mellitus. Insulin stimulated glucose uptake involves a number of signalling proteins to enable uptake to occur. In order to understand the complexities associated with the insulin signalling cascade, cell culture models have provided a controlled and easily manipulated environment in which to investigate insulin stimulated glucose uptake in skeletal muscle. While the majority of these experiments have been conducted in conventional monolayer cultures, the growing field of three-dimensional tissue engineering provides an alternative environment in which skeletal muscle cells can be grown to investigate their physiological function. The purpose of this thesis was to investigate the use of different cell culture models for investigating the effects of acute and chronic insulin exposure on skeletal muscle. Initial investigations aimed to establish glucose uptake in tissue engineering skeletal muscle constructs using tritium labelled (H3) 2-deoxy-d-glucose. Monolayer cultures were used to developed base line conditions. In these cultures, concentrations greater than 0.5 μCi for 15 minutes of insulin stimulation suggested an initial assay window for investigating insulin stimulated glucose uptake. However, the duration of insulin stimulation was not effective in measuring uptake in tissue engineered skeletal muscle constructs based upon western blot experiments of Akt phosphorylation, therefore insulin stimulation in skeletal muscle tissue engineered constructs was increased to 30 minutes. Glucose uptake is mediated via specific glucose transporter protein, GLUT1 and GLUT4. Therefore, the transcriptional profile of these transporters was elucidated in monolayer culture and tissue engineered skeletal muscle constructs. Time course experiments showed an increase in GLUT4 transcription in tissue engineered and monolayer culture systems which is associated with an increase in the transcription of skeletal muscle development and myogenic genes. In two dimensional culture, skeletal muscle cells were exposed to insulin during differentiation and in post-mitotic skeletal muscle myotubes to investigating the potential effects upon metabolic genes and proteins involved in insulin signalling. Chronic exposure to insulin during skeletal muscle differentiation reduced insulin signalling and resulted in an increase in basal glucose uptake and ablated insulin stimulated glucose uptake. In contrast, post-mitotic skeletal muscle myotubes did not shown similar changes and were not as responsive to acute insulin exposure. Therefore future experiments exposed skeletal muscle to insulin during differentiation. Using the previous findings as a basis for experimentation, the effects of chronic and acute insulin exposure upon three dimensional skeletal muscle constructs were investigated. Fibrin and collagen constructs were grown for a total period of 14 days. Constructs were exposed to insulin during differentiation and acutely stimulated for 30 minutes at day 14. Although there was a mean increase in Akt protein phosphorylation in both types of tissue-engineered constructs, these changes were not significant following acute insulin stimulation. In addition, glucose uptake in fibrin skeletal muscle constructs increased as a result of acute insulin stimulation however was not significantly difference to unstimulated constructs. The work presented in this thesis provides initial experimental data of the use of different skeletal muscle cell culture models for investigating insulin signalling and glucose uptake. Further research should further characterise these in vitro models for investigating skeletal muscle metabolism.

I have investigated the potential role of Toll-Like Receptors (TLRs) in mediating adipose inflammation in obesity. TLRs are a family of pattern recognition receptors that play a key role in host defence and are also implicated in chronic inflammatory disorders. The finding that TLR4-deficient mice are protected against obesity-induced diabetes led me to hypothesise that TLR expression on adipocytes could play a role in obesity-induced adipose inflammation and metabolic dysfunction. The first chapter investigates the expression and function of TLRs in in vitro differentiated human subcutaneous adipocytes. I found that stimulation with ligands for TLR2, TLR3 and TLR4 but not the other TLRs, induces the expression of pro-inflammatory cytokines. I also explored the use of the TLR adapter molecules MyD88, Mal and TRIF by different TLRs. The second chapter examines whether TLR activation in adipocytes has an effect on glucose uptake. I established a 3H-2-deoxy-D-glucose (2DOG) uptake assay which led to an interesting yet unexpected observation: Stimulation with TLR3 and TLR4 ligands led to a decrease in insulin-stimulated glucose uptake but at the same time, insulin-independent glucose uptake was increased. I showed that these observations are at least partly due to altered expression of different glucose transporter (GLUT) isoforms. As the effects were seen only after prolonged TLR stimulation, I speculated that this was mediated via a secondary secreted factor. The third chapter is based on a cytokine and adipokine array, which I performed to identify cytokines that may be responsible for the effects described in Chapter 2. The secretion of several cytokines/chemokines with diverse pro-inflammatory functions was observed following stimulation with TLR3 and TLR4 ligands. The contribution of some of these factors to altered glucose handling was investigated. Whilst a contribution for ENA-78 was ruled out, I present evidence that IL-1 can contribute to this.

Both plasma glucose concentration and glucose uptake are deranged in insulin resistance. A high free fatty acid plasma level is a potential cause of insulin resistance, and therefore of type 2 diabetes mellitus animals and humans. The mechanism behind this is still unclear. The objectives of the present study were: (i) to research the effect of arachidonic acid (AA) as fatty acid representative, on glucose uptake into human isolated adipocytes, (ii) to investigate the uptake of AA into adipocyte membranes and nuclei, as a step to identify the mechanism whereby AA affects glucose uptake, and (iii) to verify the influence of insulin on AA uptake in adipocytes. The first objective was achieved by exposing adipocytes to AA and measuring the effect on deoxyglucose uptakt. To achieve the second objective, adipocytes were exposed to 14C-AA; radioactive uptake in membranes and nuclei was determined. The AA uptake into membranes was also determinate by membranes fatty acid profile using gas chromatography; the results of the two methods were compared. Finally, the third objective was achieved by exposing adipocytes to different concentrations of insulin and testing the effect by measuring arachidonic acid uptake by the entire cell. The results of this study shown that, acute (30 min) exposure of AA significantly stimulates glucose uptake by adipocytes (4.56 ± 0.6 nmole glucose /mg protein /min) compared to the control (3.12 ± 0.25 nmole glucose /mg protein /min). Secondly, 14C-AA was significantly taken up by the membranes between 20 and 30 minutes of exposure. The uptake into membranes was increased by 49.57 ± 29% and 123 ± 73% compared to the control 100% (1.77 ± 0.06 nmole AA /mg protein) respectively for 20 and 30 min exposure). AA significantly rose in the nuclei after 30 minutes (147 ± 19% increase) compared to the control 100% (2.25 ± 0.10 nmole AA /mg protein). The determination of AA uptake by gas chromatography analysis of the membrane fatty acid profile showed that the content of AA increased after 30 min exposure (0.57% AA of total membrane fatty acids) compared to the 10 min exposure (0.29% AA of total membrane fatty acid). Insulin was shown to stimulate 10 and 30 min AA uptake by adipocytes from a non-obese subject. The increases of AA uptake measured for 30 minutes were 20 ±8%, 21 ± 25% and 31 ± 4% compared to the control (0.58nmole AA / mg protein / min) respectively for the actions of 10nM, 20nM and 40 nM insulin. A similar tendency was observed when the AA uptake was measured for 10 min (81 ± 31% and 208 ± 36% respectively for the action of 10nM and 40nM insulin compared to the control 100% (0.06nmole AA/mg protein/min). In contrast to this finding, insulin depressed AA uptake by adipocytes from an obese subject (depression of 15 ± 5%, 14 ± 8% and 21 ± 5% respectively for 10nM, 20nM and 40nM insulin, compared to the control 100% (0.74 nmole AA/mg protein/min). In both situations the effect of insulin seemed dose dependent. The study demonstrated that AA acid positively modulates glucose uptake into adipocytes exposed for short periods (< 30 min). This was attributed to the probable this FA in the cell membrane, rather than its eventual effect on the DNA. The best method to measure membranes AA over short period of exposure when small amounts of adipocytes (2- 6 ml) are used was by radioactive means. It also suggested that insulin effect’s on AA acid uptake into adipocytes was dose dependent. This varies with the body mass index (BMI) of the patient, probably as a result of their cell’s insulin resistant state.
Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007.
Anatomy and Physiology
MSc
unrestricted

Pseudomonas aeruginosa è un patogeno opportunista che provoca una vasta gamma di infezioni nell’uomo. È noto che l’incidenza delle infezioni da P. aeruginosa è maggiore in persone che presentano elevati livelli di glucosio nel sangue, a causa della capacità del batterio di utilizzare il glucosio in eccesso come nutriente per la crescita. Pertanto, bloccare l’attività di proteine coinvolte nel trasporto o nell’utilizzazione del glucosio da parte del batterio potrebbe essere una buona strategia per lo sviluppo di farmaci anti-pseudomonas. Per valutare questa ipotesi, abbiamo costruito una serie di mutanti singoli e multipli difettivi nel trasporto del glucosio nel ceppo PAO1 di P. aeruginosa e li abbiamo analizzati per fenotipi correlati alla virulenza in saggi in vitro e in vivo, in due differenti modelli di infezione. In particolare, sono stati deleti i geni codificanti per i trasportatori della membrana interna Glt, GntP e Kgut, che mediano l’ingresso del glucosio e dei suoi derivati ossidati gluconato e 2-ketogluconato. Un triplo mutante privo dei suddetti trasportatori si è rivelato incapace di crescere su glucosio come unica fonte di carbonio (mutante GUN, Glucose Uptake Null). Più di 500 geni, che controllano sia funzioni metaboliche che la virulenza, sono espressi in modo differenziale nel mutante GUN rispetto al ceppo parentale. Coerentemente con i dati dell’analisi trascrittomica, i saggi di virulenza in vitro hanno mostrato che il mutante GUN si comporta diversamente dal ceppo parentale, avendo una ridotta capacità di formare biofilm e una maggiore secrezione di proteasi, piocianina e pioverdina. Inoltre, questo mutante ha una produzione alterata delle molecole segnale che attivano il sistema del quorum sensing. È interessante notare che il profilo trascrizionale e la maggior parte dei tratti fenotipici analizzati differiscono tra il mutante GUN e il ceppo parentale indipendentemente dalla presenza del glucosio, suggerendo che uno (o più) dei trasportatori deleti abbia una funzione addizionale non correlata al trasporto del glucosio. Infine, alcuni mutanti hanno mostrato un diverso grado di virulenza in saggi di infezione negli ospiti modello Caenorhabditis elegans e Galleria mellonella. In particolare, mentre in C. elegans la virulenza dei mutanti è risultata simile a quella del ceppo parentale PAO1, i mutanti deleti del gene kguT, che codifica per il trasportatore del 2-ketogluconato, sono meno virulenti di PAO1 in G. mellonella. L’attenuazione è particolarmente significativa per il doppio mutante ΔgntP ΔkguT e per il mutante GUN. Questo risultato suggerisce che l’efficacia antibatterica di composti che bloccano l’utilizzo del glucosio da parte di P. aeruginosa potrebbe presumibilmente variare a seconda della capacità del patogeno di adattarsi allo specifico contesto nutrizionale dell’ospite infettato. Una maggiore conoscenza delle interazioni metaboliche che avvengono tra patogeno ed ospite nel sito d’infezione diventa quindi sempre più necessaria per poter sviluppare terapie antibatteriche efficaci.
Pseudomonas aeruginosa is an opportunistic pathogen causing a wide range of infections in humans. Pathologies leading to hyperglycaemia have been associated with augmented risk of developing serious P. aeruginosa infections due to the ability of the bacterium to utilize glucose as carbon source for the growth. Therefore, preventing the import of glucose might be a good strategy to develop anti-pseudomonas drugs. To address this hypothesis, a collection of single and multiple glucose uptake defective mutants was generated in P. aeruginosa PAO1 and tested for virulence-related phenotypes in in vitro assays and in vivo, in two different infection models. In particular, we engineered mutants in genes encoding inner membrane (IM) proteins involved in the internalization of glucose and its oxidized derivatives gluconate and 2-ketogluconate, i.e. the Glt, GntP and KguT transporters. A triple mutant lacking these transporters was demonstrated to be completely unable to grow on glucose as sole carbon source (Glucose Uptake Null mutant, GUN). The transcriptomic analysis revealed a strong divergence in the GUN transcriptional profile relative to the parental strain, with more than 500 differentially expressed genes, controlling both metabolic functions and virulence traits. Consistent with the transcriptomic data, the GUN mutant showed a pleiotropic phenotype in the in vitro assays, with a reduction in biofilm formation and an increased secretion of proteases, pyocyanin and pyoverdine. Furthermore, the production of quorum sensing signal molecules was altered in this mutant. Interestingly, the gene expression profile and most phenotypic traits differ between GUN and the parental strain irrespective of the presence of glucose, suggesting a possible additional role for the deleted transporter(s). Finally, the in vivo assays demonstrated that while the virulence of all mutants in the Caenorhabditis elegans infection model was essentially comparable to that of the wild type strain, all mutants lacking kguT (i.e. the 2-ketogluconate transporter gene) and especially the double ΔgntP ΔkguT and GUN mutants, were attenuated in the Galleria mellonella model. This suggests that targeting glucose metabolism with specific drugs may alter P. aeruginosa pathogenicity depending on the ability of this pathogen to adapt to the specific nutritional context encountered at the site of infection. Therefore, a deeper knowledge of host-pathogen metabolic transactions is pivotal to develop effective antibacterial strategies based on hampering bacterial metabolic functions.

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.

The effect of Biosynthetic Human Proinsulin on glucose uptake by skeletal muscle was studied in the isolated perfused hindquarter of fasted rats. Animals were randomly assigned to the control group, insulin-perfused or proinsulin-perfused group. Glucose disappearance from the perfusate and muscle glycogen levels before and after 2 hours perfusion were measured. Perfusate glucose concentration showed the greatest decline in the insulin group, which was significantly lower (p < .01) than control from 60 to 120 min. Proinsulin perfusion resulted in a smaller and delayed decrease in perfusate glucose. The proinsulin perfusate glucose levels were significantly higher (p < .05) than the insulin glucose values during the second hour of perfusion. After the first hour of perfusion, insulin infusion resulted in higher rates of glucose uptake than control (p < .005) or proinsulin infusion (p < .05). The glucose uptake by muscles perfused with proinsulin was significantly different from control values only at the 2 hour time point (p < .05). Glycogen concentration following insulin infusion increased significantly in the oxidative muscles, i.e. soleus (p < .05) and red vastus (p < .002). These increases in glycogen were significantly different from the changes observed in muscles of control animals. The plantaris and white vastus muscles, which have fast twitch fibers, did not show a significant response to insulin. Proinsulin perfusion decreased glycogen levels regardless of the muscle type. This decline was significantly different from the glycogen changes in soleus (p < .025), plantaris (p < .001) and white vastus (p < .05) muscles of control animals. The proinsulin glycogen fall was also significantly different from the insulin induced response in soleus, plantaris and red vastus muscles (p < .001). These results show that proinsulin has 8.6 % of the biologic potency of insulin for glucose uptake in rat skeletal muscle. Insulin induced an increase in glycogen concentration in oxidative muscles, but proinsulin elicted a drop in glycogen level regardless of the muscle type.

Myo-[3H]-inositol accumulation in cultured bovine lens epithelial cells (BLECs) occurred by both high- and low affinity, Nat-dependent transport sites. High ambient glucose significantly inhibited myo-[ 3 H]-inositol uptake; the co-administration of sorbinil, an aldose reductase inhibitor, prevented the inhibitory effect on the low affinity transport site. A glucose-sensitive process for myo-[3 H]-inositol uptake on the high-affinity transport site was uncovered by Lineweaver-Burk analysis. Dixon plot analysis confirmed that the effect of glucose was due to competitive inhibition of the high-affinity myo-inositol transport site while the effect of sorbitol was due to competitive inhibition of the low-affinity myo-inositol transport site.

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

Elasmobranchs (sharks, skates, and rays) are a primarily carnivorous group of vertebrates that consume very few carbohydrates and have little reliance on glucose as an oxidative fuel, the one exception being the rectal gland. This has led to a dearth of information on glucose transport and metabolism in these fish, as well as the presumption of glucose intolerance. Given their location on the evolutionary tree however, understanding these aspects of their physiology could provide valuable insights into the evolution of glucose homeostasis in vertebrates. In this thesis, the presence of glucose transporters in an elasmobranch was determined and factors regulating their expression were investigated in the North Pacific spiny dogfish (Squalus suckleyi). In particular, the presence of a putative GLUT4 transporter, which was previously thought to have been lost in these fish, was established and its mRNA levels were shown to be upregulated by feeding (intestine, liver, and muscle), glucose injections (liver and muscle), and insulin injections (muscle). These findings, along with that of increases in muscle glycogen synthase mRNA levels and muscle and liver glycogen content, indicate a potentially conserved mechanism for glucose homeostasis in vertebrates, and argue against glucose intolerance in elasmobranchs. In contrast to the other tissues examined, there was a decrease in glut4 mRNA levels within the rectal gland in response to natural feeding, a factor known to activate the gland, suggesting mRNA storage for rapid protein synthesis upon activation. A similar trend was also shown for sglt1 in the rectal gland, and the ability of GLUT and SGLT inhibitors to prevent chloride secretion solidified the importance of glucose uptake for gland function. The exogenous factor of salinity was also investigated and high levels of glut mRNA were observed within the rectal glands of low salinity-acclimated fish relative to control and high salinity fish, reiterating the idea of mRNA storage when the gland is expected to be inactive. Taken together, the results of this thesis demonstrate that glucose is an important fuel in the dogfish (and likely other elasmobranchs) and that the dogfish is fully capable of regulating its storage and circulation, contrary to prior beliefs.

Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures.   The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.
QC 20110608

The resident microbes of human gastrointestinal tract cause both harmful and beneficial effects and these effects can be modulated by the administration of beneficial probiotic bacteria. Probiotics attribute several therapeutic and preventive beneficial effects, for both humans and animals. Despite the good effects of probiotic bacteria, the role of probiotic bacteria or their metabolites on the nutrient uptake by enterocytes is very less known. Most studies describe the genomic effects of probiotic bacteria on the transport properties. This thesis describes the short term (10 min or less) non-genomic effects of probiotic bacteria on the glucose uptake by human enterocytes like Caco-2 cells. The focus of the present study was to identify if metabolites of Lactobacilli sp. trigger a rapid non genomic regulation of glucose transporter proteins of enterocytes. The findings indicate that the regulatory molecules of bacterial metabolites can cause a rapid increase in glucose uptake by enterocytes.

To improve the efficacy of anticancer chemotherapy it is necessary to develop targeted treatments, which can be achieved by exploiting differences between normal and cancerous cells. The avid consumption of glucose by cancer cells compared with normal cells has been known for almost a century. The increased glucose uptake and its modified metabolism by cancerous cells, known as the Warburg effect, is recognised as a hallmark of malignant cells. A Warburg effect targeting group, rationally designed to facilitate uptake by GLUTs and be retained in cells due to phosphorylation by HK, has been synthesised. This targeting group can be conjugated to various metal complexes via an amide bond, with no deprotection reactions required after coupling, with the aim to enhance uptake and accumulation in cancer cells. The targeting vector has demonstrated inhibition of glucose phosphorylation by HK, indicating interaction with the enzyme and thereby providing the potential to facilitate an intracellular trapping mechanism for complexes it is conjugated to. Co(III)-tpa complexes have been shown to release bidentate ligands, presumably upon reduction to the more labile Co(II), in cells. To enhance delivery and uptake of anticancer agents through solid tumours a dual targeting strategy has been employed, combining Warburg effect targeting with bioreductive activation of Co(III) chaperone complexes, by conjugation of the targeting group with a tpa ligand. Uptake and distribution of the novel cancer targeted cobalt complex in cancer cell monolayers and spheroid tumour models has been investigated. Through accumulation studies, elemental mapping and fluorescent imaging the effectiveness of the Warburg effect targeting strategy has been evaluated. The novel targeting group shows promise, and distinct advantages over glucosamine, for exploiting the Warburg effect, with the targeting complex increasing delivery of a model cytotoxin to proliferating cells with enhanced glucose consumption.

Over the last 30 years data from manned spaceflight missions have indicated that microgravity affects a number of physiological systems including the skeletal muscles. The purpose of the experiments in this dissertation was to examine the influence of 14 days of simulated microgravity on insulin and exercise stimulated glucose utilization in rat hindlimb muscles. To accomplish this aim, male Sprague-Dawley rats (250-325 g) were suspended in a head down position (SUS) so that one or both hindlimbs were non-weight bearing. The results from hindlimb perfusion experiments indicated that glucose uptake rates for the entire hindquarter at both submaximally and maximally stimulating insulin concentrations were significantly higher (p ≤ 0.05) in the SUS rats (77% and 15%, respectively) than the cage control rats (CC), suggesting increases in insulin sensitivity and responsiveness. Insulin sensitivity for ¹⁴C glucose incorporation into glycogen was also increased for the soleus (SOL), plantaris (PL), and extensor digitorum longus (EDL) muscles in the SUS rats. When the suspended (SUS-E) and control (CC-E) rats were exposed to acute treadmill exercise at 80-90% of VO₂ max, hindlimb glucose uptake and its incorporation into glycogen in the absence of insulin were higher in the PL, EDL, and white gastrocnemius (GW) muscles from the SUS-E rats. However, hindlimb muscle responses to insulin appeared to be impaired after exercise for the SUS-E rats when compared to the CC-E rats, especially in the SOL muscle. To examine the influence of non-weight bearing per se on muscles during simulated microgravity, rats were suspended with the left hindlimb non-weight bearing (NWB) and the right hindlimb bearing 20% of pre-suspension body mass (WB). The results indicated that ³H 2-deoxyglucose uptake was significantly higher for the SOL, PL, EDL and GW muscles (21-80%), at a maximally stimulating insulin concentration, in both SUS-NWB and SUS-WB hindlimbs despite the prevention of SOL and PL muscle mass losses in the SUS-WB hindlimbs. Collectively, the results from this dissertation indicate that the suspension of the rat with hindlimbs non-weight bearing leads to enhanced muscle responses to insulin for glucose uptake and metabolism, and suggest that systemic influences may be involved. In addition, exercise induced glucose and glycogen utilization increase to a greater extent in the hindlimb muscles of suspended rats when compared to their controls; but, there was evidence that muscle responses to insulin were attenuated with exercise in the suspended animals.

The Effects of a glucocorticoid-antagonist on IGF1-stimulated glucose uptake in skeletal muscle of hindlimb suspended rats. Barnes B.R., T.C. Selix, D.C. Wright, and B.W. Craig. Ball State University, Muncie, IN.The purpose of this investigation was to determine the effects of a glucocorticoid-antagonist (RU486) on insulin-like-growth-factor-1 (IGF1)stimulated glucose transport following two weeks of hindlimb suspension (HS) on 100 gm male rats. After two weeks of HS and/or oral RU486 administration the animals were anesthetized, and the soleus (SOL) and extensor digitorum longus (EDL) muscles isolated and clamped at their resting length. Following an incubation series to prepare the muscle, the muscle was incubated in radioactive 3-O-methylglucose for 10 min. in the presence/absence of 75 ng/ml of IGF1, digested with 0.5 NaOH, and the amount of glucose transported measured. Two weeks of RU486 treatment significantly (P:5 0.05) elevated IGF1-stimulated glucose transport of SOL (0.576 ± 0.071 vs 1.405 ± 0.172), whereas the EDL was unaffected (2.728 0.258 vs 2.613 ± 0.182). The removal of glucocorticoids via RU486 administration significantly increased glucose uptake in HS exposed soleus muscles. The EDL was not affected by RU486 treatment.
School of Physical Education

Brown adipose tissue (BAT) is the main tissue involved in non-shivering heat production. A greater understanding of BAT could possibly lead to new ways of prevention and treatment of obesity and type 2 diabetes. The increasing prevalence of these conditions and the problems they cause society and individuals make the study of the subject important. An ongoing study performed at the Turku University Hospital uses images acquired using PET/MRI with 18F-FDG as the tracer. Scans are performed on sedentary and athlete subjects during normal room temperature and during cold stimulation. Sedentary subjects then undergo scanning during cold stimulation again after a six weeks long exercise training intervention. This degree project used images from this study. The objective of this degree project was to examine methods to automatically and objectively quantify parameters relevant for activation of BAT in combined PET/MRI data. A secondary goal was to create images showing glucose uptake changes in subjects from images taken at different times. Parameters were quantified in adipose tissue directly without registration (image matching), and for neck scans also after registration. Results for the first three subjects who have completed the study are presented. Larger registration errors were encountered near moving organs and in regions with less information. The creation of images showing changes in glucose uptake seem to be working well for the neck scans, and somewhat well for other sub-volumes. These images can be useful for identification of BAT. Examples of these images are shown in the report.

"Uniaxial Cyclic Stretch-Stimulated Glucose Transport Is Mediated by a Ca2+-Dependent Mechanism in Cultured Skeletal Muscle Cells" Pathobiology, v.74, n.3, pp.159-168を、博士論文として提出したもの。
名古屋大学博士学位論文 学位の種類:博士（リハビリテーション療法学）（課程）学位授与年月日:平成19年3月23日

Glucose and regulation of cell cycle in S. cerevisiae: analysis of mutants impaired in sugar uptake mechanisms Requisito fondamentale per la sopravvivenza di microrganismi a vita libera come il lievito S. cerevisiae è la capacità di regolare il proprio metabolismo e la progressione del ciclo cellulare in modo tale che la crescita sia rapida in presenza di abbondanti nutrienti e si arresti all’esaurirsi degli stessi. Perché questo sia possibile, nutrienti come il glucosio devono generare segnali che vengano recepiti ed elaborati dal complesso macchinario che governa il ciclo cellulare. S. cerevisiae possiede almeno tre meccanismi per rilevare variazioni dei livelli di glucosio nel mezzo di coltura: il pathway di Rgt2/Snf3, che controlla l’espressione dei trasportatori degli zuccheri esosi; il pathway cAMP/PKA, che regolando l’attività della protein-kinasi A promuove l’espressione di geni coinvolti nel metabolismo fermentativo e nella crescita cellulare e inibisce la trascrizione di geni coinvolti nella risposta agli stress; il glucose main repression pathway, che reprime l’espressione di geni coinvolti nella respirazione cellulare, nella gluconeogenesi e nell’utilizzo di fonti di carbonio alternative al glucosio. L’assunzione di glucosio nel citoplasma dall’ambiente esterno avviene attraverso i trasportatori codificati dalla famiglia di geni HXT (HeXose Transporter), che comprende almeno 20 membri: HXT1-17, RGT2, SNF3 e GAL2. Snf3 e Rgt2 sono incapaci di trasportare lo zucchero, ma agiscono piuttosto da sensori del livello di glucosio extracellulare: in particolare, Snf3 rileva basse concentrazioni dello zucchero inducendo l’espressione dei trasportatori ad alta affinità (codificati dai geni HXT2-HXT4), mentre Rgt2 rivela alte concentrazioni di glucosio promuovendo l’espressione dei trasportatori a bassa affinità (HXT1). Nessuno dei trasportatori è essenziale e solo la delezione di tutti i geni HXT (o almeno di quelli compresi tra 1-7 in alcuni background) rende la cellula di lievito incapace di crescere in presenza di glucosio come unica fonte di carbonio. L’espressione dei vari trasportatori è regolata a livello trascrizionale attraverso un complesso network che coinvolge tutti e tre pathway deputati al sensing del glucosio: come risultato, S. Cerevisiae è in grado di mantenere sempre un alto flusso glicolitico esprimendo il set di trasportatori più adatto alla quantità di glucosio disponibile. Le connessioni tra i pathway deputati al sensing del glucosio e gli elementi di regolazione del ciclo cellulare non sono completamente definite, anche perché risulta spesso difficile scindere il duplice ruolo dello zucchero come nutriente e come molecola segnale. Obbiettivo del presente progetto di ricerca è chiarire gli effetti di alterazioni nei meccanismi di sensing e (in modo particolare) di trasporto del glucosio sulla coordinazione tra crescita e divisione cellulare. In una prima fase dello studio sono stati presi in esame alcuni mutanti con delezioni nei geni HXT1-7, codificanti per i principali trasportatori degli zuccheri esosi: i dati presenti in letteratura certificano infatti come queste mutazioni siano sufficienti ad abolire sostanzialmente l’assunzione (uptake) cellulare del glucosio impedendo la crescita dei mutanti su tale fonte di carbonio. I parametri di crescita (tempo di duplicazione, indice di gemmazione, contenuto proteico e di DNA) di ciascuno dei ceppi sono stati determinati in due condizioni sperimentali: i) crescita esponenziale bilanciata in terreno csm/YNB addizionato di 2% etanolo o di una miscela 2% Etanolo + 2% glucosio, da cui è emerso come il glucosio possa esercitare un effetto sulle dimensioni celulari anche nei mutanti hxt (indipendente quindi dal suo ruolo come nutriente). Infatti, analogamente alle cellule wild-type, anche i ceppi con i trasportatori deleti mostrano dimensioni cellulari e contenuto proteico maggiore quando fatti crescere in glucosio+Etanolo, sebbene (diversamente dal ceppo wild type) la loro velocità di crescita sia simile a quella registrata in terreno con solo etanolo; ii) crescita durante shift-up nutrizionale etanolo => glucosio. All’aggiunta dello zucchero le cellule wild-type vanno incontro ad fase iniziale di adattamento (evidenziato dalla diminuzione transiente (10-15%) dell’indice di gemmazione), necessaria per reimpostare profilo trascrizionale, velocità di crescita e dimensioni cellulari e per il successivo passaggio ad un metabolismo energetico di tipo fermentativo. A differenza del ceppo wild type, dopo l’aggiunta di glucosio le cellule hxt(1-7) manifestano una drammatica e prolungata riduzione nell’indice di gemmazione e un forte rallentamento (arresto) nella progressione del ciclo cellulare. In seguito le cellule riprendono a dividersi con una velocità sostanzialmente identica a quella precedente lo shift, mentre volume cellulare e contenuto proteico medio aumentano sensibilmente: l’effetto del glucosio sulle dimensioni cellulari dei mutanti hxt(1-7) è tuttavia transiente e si esaurisce nell’arco di due/tre round di divisioni, quando le cellule tornano ad assumere le dimensioni tipiche della crescita su etanolo. I dati finora riportati sembrano quindi suggerire che, almeno inizialmente, gli effetti del glucosio sulle dimensioni cellulari dipendano dal sensing dello zucchero e non dal suo metabolismo. Tuttavia, sebbene il ceppo hxt(1-7) non sia in grado di crescere su glucosio come unica fonte, rimane comunque dotato di una capacità residua di trasporto dello zucchero, che sebbene insufficiente a sostenere il metabolismo glicolitico, potrebbe comunque assumere un’importanza decisiva per la regolazione delle dimensioni cellulari. Nel tentativo di scindere ancora più nettamente il duplice ruolo del glucosio come nutriente e come molecola segnale, in una successiva fase di studi sono stati utilizzati mutanti con delezioni in tutti i geni per i trasportatori del glucosio (hxt(1-17)), in cui ogni residuo trasporto dello zucchero risulta abolito. In aggiunta, si sono presi in esame una serie di mutanti con una capacità di uptake del glucosio progressivamente ridotta: nel dettaglio, la lista comprende (oltre ovviamente al ceppo wild type di riferimento): i) hxt(1-17)gal2, in cui il trasporto del glucosio è completamente abolito; ii) il ceppo hxt(1-17)) snf3, in cui l’inattivazione del sensore SNF3 rispristina una trascurabile capacità di trasporto del glucosio, insufficiente comunque a garantire la crescita in terreno liquido contenente glucosio come unica fonte: l’assunzione dello zucchero in questo caso sembrerebbe avvenire attraverso un trasportatore non ancora caratterizzato, la cui trascrizione risulta derepressa in assenza di SNF3; iii) il ceppo (hxt(1-17) gal2 HXT1, che esprime in modo costitutivo come unico trasportatore HXT1, un carrier a bassa affinità; iv) ) il ceppo (hxt(1-17) gal2 HXT7, che esprime in modo costitutivo come unico trasportatore HXT7, un carrier ad alta affinità; v) il ceppo snf3 rgt2, in cui l’uptake del glucosio è ridotto a causa dell’inattivazione del principale pathway che regola l’espressione dei maggiori trasportatori; vi) il triplo deleto hxk2 hxk1 glk1, che è in grado di trasportare glucosio nel citoplasma ma non è in grado di metabolizzarlo a causa dell’assenza di tutte e tre le chinasi che catalizzano il primo passaggio della glicolisi. I ceppi sopra elencati sono stati sottoposti alle analisi descritte in precedenza. Nel caso dei ceppi capaci di metabolizzare il glucosio, il tasso di crescita e le dimensioni cellulari su tale fonte sono generalmente correlate all’efficienza del sistema di trasporto dello zucchero nei vari mutanti: sembra esistere una relazione sostanzialmente lineare tra velocià di consumo del glucosio/velocità di crescita/dimensioni cellulari. Unica eccezione pare essere il ceppo snf3 rgt2, che manifesta dimensioni cellulari notevolmente più ridotte rispetto a quanto atteso sulla base del suo tasso di crescita: un risultato che sembrerebbe suggerire un ruolo diretto del pathway Snf3/Rgt3 nei meccanismi che regolano le dimensioni cellulari in risposta ai nutrienti. Diversamente da quanto emerso in precedenza, la crescita dei mutanti hxt(1-17) risulta fortemente inibita in terreni contenenti miscele di etanolo (o altra fonte non fermentabile) e glucosio, anche quando la concentrazione dello zucchero è a livelli sub-ottimali (0.05% anziché 2%). L’aggiunta di glucosio a cellule hxt(1-17) in crescita su etanolo (shift-up nutrizionale) determina l’arresto permanente del ciclo cellulare in G1 (cellule vitali, non gemmate con contenuto di DNA presintetico). Sembra quindi che la semplice presenza di glucosio nell’ambiente extracellulare - ma non il trasporto dello zucchero nel citoplasma - sia sufficiente ad impedire l’utilizzo di fonti di carbonio alternative presenti nel medium: ciò spiegherebbe la mancata crescita in terreni misti glucosio+etanolo da parte di cellule hxt(1-17), incapaci di effettuare l’uptake dello zucchero. Se tale ipotesi fosse corretta, inattivando contemporaneamente tutti i pathway deputati al sensing del glucosio dovrebbe essere possibile ripristinare la crescita di cellule hxt(1-17) in terreni contenenti miscele di glucosio ed etanolo. Al momento, si è appurato che l’inattivazione del ramo del cAMP/PKA pathway passante attraverso Gpr1/Gpa2 non è sufficiente a correggere il difetto di crescita del ceppo hxt(1-17)gal2 in fonte mista glucosio/etanolo. Al contrario, la semplice inattivazione di SNF3 (ma non di RGT2)sembra sostanzialmente azzerare l’effetto citostatico del glucosio sulla crescita del ceppo hxt(1-17) gal2 snf3. L’interpretazione di tale risultato è ovviamente complicata dal fatto che la delezione di SNF3 ripristina parzialmente il trasporto del gluccosio in un ceppo privo di tutti i trasportatori, sebbene, vale la pena ricordare, su scala estremamente ridotta e comunque insufficiente a sostenere la crescita, come confermato attraverso misurazioni dirette della velocità di consumo delo zucchero nel ceppo hxt(1-17)) snf3. Tuttavia, diversi dati in letteratura suggeriscono come il pathway Snf3/Rgt2 partecipi in qualche misura ai meccanismi della glucose repression, in particolare attrverso Mig2, un repressore trascrizionale che in presenza di glucosio collabora con Mig1 nel reprimere la trascrizione di geni richiesti per l’utilizo di fonti di carbonio alternative. La delezione di MIG2 non è tuttavia sufficiente a ripristinare la crescita su etanolo/glucosio del ceppo hxt(1-17)gal2: ulteriori indagini sono dunque necessarie per chiarire quale sia il ruolo giocato da SNF3 nell’intero processo. In aggiunta, il comportamento manifestato dal ceppo hxk2 hxk1 glk1 durante shift-up nutrizionale da etanolo a glucosio sembra ulteriormente confermare come in lievito lo zucchero sia in grado di regolare le dimensioni cellulari indipendentemente dal proprio metabolismo, almeno in una fase iniziale: le cellule hxk2 hxk1 glk1 in crescita su etanolo rispondono all’aggiunta di glucosio aumentando considerevolmente il proprio volume, in misura paragonabile a quanto si registra nel ceppo wild type; tuttavia, contrariamente al ceppo wild type, nel mutante hxk2 hxk1 glk1 l’aumento delle dimensioni celluari si accompagna ad un progressivo rallentamento della velocità di crescita, fino ad un totale arresto del ciclo di divisione cellulare che sopraggiunge a circa 12 ore dallo shift. Dopo una fase di lag piuttosto prolungata ed estremamente variabile, in cui le cellule, pur non dividendosi, si mantengono gemmate, si assiste alla rispresa del ciclo di divisione cellulare: le cellule tornano a dividersi lentamente utilizzando l’etanolo residuo nel terreno e nell’arco di due/tre generazioni assumono nuovamente le tipiche dimensioni ridotte associate alla crescita su fonte di carbonio non fermentabile. Ad ulteriore conferma di come l’effetto del glucosio sia solo temporaneo, dimensioni e contenuto proteico di cellule hxk2 hxk1 glk1 in crescita bilanciata su etanolo o su fonte mista etanolo/glucosio sono sostanzialmente identiche. Nonostante il sorprendente effetto citostativo dello zucchero, l’aumento delle dimensioni cellulari in risposta all’aggiunta di glucosio si manifesta anche nel ceppo privo di tutti i trasportatori (hxt(1-17) gal2)., sebbene in misura meno eclatante rispetto al triplo mutante hxk2 hxk1 glk1. Nell'insieme, tali risultati sembrano confermare come il glucosio sia in grado di modulare le dimensioni della cellula di lievito in maniera (almeno in parte) indipendente dal proprio ruolo come nutriente, funzionando in buona sostanza come un "ormone". Per chiarire le basi molecolari di tale fenomeno è necessario chiarire le connessioni tra i pathway deputati al sensing del glucosio e gli elementi di regolazione del ciclo di divisione cellulare in S. cerevisiae. A tal fine, si è ultimata la costruzione di una serie di mutanti esprimenti versioni “taggate” (HA-tag) di alcuni dei principali regolatori coinvolti nella transizione G1/S (nello specifico Cln3, Cln2, Far1, Sic1 e Clb5), così da facilitare l’analisi dei loro livelli di espressione e di localizzazione subcellulare. Gli studi in questo senso sono tuttavia in una fase ancora troppo preliminare per poter trarre conclusioni definitive. Da utlimo, si è cercato di valutare il contributo relativo di sensing, trasporto e metabolismo del glucosio alla regolazione trascrizionale del gene SUC2, uno dei marcatori più comunemente utilizzati per valutare il fenomeno della glucose repression in S. cerevisiae. SUC2 codifica per l’invertasi, un enzima chiave per l’utilizzo del disaccaride saccarosio e la sua espressione risulta completamente bloccata in presenza di alti livelli di glucosio mentre viene indotta da raffinosio o da bassi livelli di glucosio. I risultati ottenuti con i vari mutanti hanno evidenziato come in presenza di abbondante glucosio il livello basale di attività invertasica sia generalmente proporzionale alla velocità del flusso glicolitico, che dipende in larga misura dalla capacità di trasporto dello zucchero: nei ceppi aventi un sistema di uptake per il glucosio ad efficienza ridotta l’invertasi risulta parzialmente o addirittura competamente derepressa, come nel caso del mutante privo di tutti i trasportatori. Il ceppo snf3 rgt2 sfugge invece a questa regola, in quanto l’attività invertasica risulta sì parzialmente derepressa in presenza di alte concentrazioni di glucosio, ma non più inducibile da bassi livelli di glucosio o raffinosio. In aggiunta, l’inattivazione di SNF3 e di RGT2 abolisce completamente l’induzione dell’attività invertasica nel ceppo hxt(1-17)gal2 in presenza di glucosio. Nell’insieme, i dati appena descritti sembrano suggerire per il pathway Snf3/Rgt2 un ruolo decisamente più rilevante nella regolazione di SUC2 rispetto a quanto gli viene comunemente attribuito. Esperimenti futuri permetteranno di chiarire meglio la questione.

The STAC3 gene is a functionally undefined gene predicted to encode a protein containing two SH3 domains and one cysteine-rich domain. In this study, we determined the potential role of the STAC3 gene in proliferation and differentiation of bovine satellite cells. We isolated satellite cells from skeletal muscle of adult cattle and transfected them with STAC3 small interfering RNA (siRNA) or scrambled siRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. We assessed the differentiation status of bovine satellite cells by quantifying the expression levels of myogenin and myosin heavy chain genes, and by quantifying fusion index. STAC3 knockdown stimulated mRNA and protein expression of myogenin, and myosin heavy chain 3 and 7, and increased fusion index of bovine satellite cells. These data together suggest that STAC3 inhibits differentiation of bovine satellite cells into myotubes. To determine the underlying mechanism, we identified and validated AP1?1 as a STAC3-interacting protein by yeast two-hybrid screening and co-immunoprecipitation. In C2C12 cells, STAC3 knockdown decreased the expression level of AP1?1 protein. In bovine satellite cells, STAC3 knockdown increased the membrane localization of glucose transporter 4 (GLUT4) and glucose uptake. These data together suggest the following mechanism by which STAC3 inhibits differentiation of bovine satellite cells: STAC3 increases AP1?1 stability in cells; a high level of AP1?1 keeps GLUT4 from translocating to the plasma membrane; reduced membrane localization of GLUT4 impedes glucose uptake; and restricted glucose uptake inhibits differentiation of satellite cells into myotubes.
Master of Science

Fibroblast Growth Factor (FGF) 21 is a liver-derived endocrine factor with multiple metabolic effects on glucose and lipid homeostasis in animals. The adipose tissue has been proposed as a major target of FGF21, where it enhances glucose uptake and modulates lipolysis as well as thermogenesis. However, the molecular mechanisms underlying the pleiotropic effects of FGF21 in adipocytes and the physiological roles of FGF21 in regulating energy homeostasis remain poorly characterized. Therefore, the present study aimed to investigate: 1) the signal transduction pathway whereby FGF21 enhances glucose uptake in white adipocytes; 2) the role of FGF21 in lipolysis in both mouse and human white adipose tissues (WAT) and its underlying mechanisms involved; 3) the phenotypes of FGF21 knockout (KO) mice with respect to energy expenditure and adiposity under both standard chow and high fat diet. Key findings: 1. In vitro studies demonstrated that extracellular signal-regulated kinases (ERK1/2) play an obligatory role in mediating FGF21-induced upregulation of glucose transporter-1 (GLUT1) expression and hence elevation of glucose uptake in 3T3-L1 adipocytes. 2. Chromatin immunoprecipitation assay revealed that Serum Response Factor (SRF) and ETS-like protein-1 (Elk-1), the two transcription factors which are known as the downstream targets of ERK1/2, were recruited to the endogenous GLUT1 promoter in adipocytes. A conserved binding motif for these two transcription factors was also identified in the GLUT1 promoter responsive to FGF21 stimulation in 3T3-L1 adipocytes by site-directed mutagenesis and luciferase assay. 3. In WAT of diet-induced obese mice, FGF21-evoked downstream signaling events, including the phosphorylation of ERK1/2 and SRF/Elk-1, the upregulation of GLUT1, and the increased glucose uptake, were markedly blunted compared to lean controls, suggesting the existence of “FGF21 resistance” in obesity. 4. In vivo and ex vivo studies on fasted wild type and FGF21 KO mice demonstrated that FGF21 acutely suppressed basal and forskolin-stimulated lipolysis in WAT. 5. FGF21-inhibited lipolysis was mediated by Akt-dependent reduction of cyclic adenosine monophosphate (cAMP) levels in both mouse and human WAT. 6. FGF21 KO mice were resistant to diet- and aging-induced obesity, which was attributed to decreased fat mass. The increased lipolysis and fatty acid oxidation in FGF21 KO mice may explain in part the lean phenotype of FGF21 KO mice. Conclusions: These results collectively suggest FGF21 as a key modulator of glucose and lipid metabolism in WAT, by activation of ERK1/2 kinase and Akt respectively. FGF21 and its signaling components may represent potential targets for the future development of new strategies for treating obesity and its medical complications.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

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.

Final article is available at "wileyonlinelibrary.com" Takako Okamoto,Masaki Mandai,Noriomi Matsumura,Ken Yamaguchi,Hiroshi Kondoh,Yasuaki Amano,Tsukasa Baba,Junzo Hamanishi,Kaoru Abiko,Kenzo Kosaka,Susan K. Murphy,Seiichi Mori,Ikuo Konishi "Hepatocyte nuclear factor-1β (HNF-1β) promotes glucose uptake and glycolytic activity in ovarian clear cell carcinoma" Molecular Carcinogenesis 54:35–49 (2013)
Kyoto University (京都大学)
0048
新制・論文博士
博士(医学)
乙第12804号
論医博第2076号
新制||医||1001(附属図書館)
80848
京都大学大学院医学研究科医学専攻
(主査)教授 野田 亮, 教授 武藤 学, 教授 稲垣 暢也
学位規則第4条第2項該当

Glucose uptake occurs in skeletal muscle under basal conditions, and increases in response to stimuli such as insulin and exercise. Exercise is known to increase blood flow, and it appears that insulin has similar hemodynamic effects, including increased blood flow and capillary recruitment, which can modify the amount of glucose uptake occurring under each condition. Here we study factors affecting both basal and stimulated myocyte glucose uptake, with a particular focus on vasoactive agents. Insulin stimulates the release of endothelin-1 (ET-1), a potent vasoconstrictor, from endothelial cells in culture. As yet it is unknown whether ET-1 is a type A (causing nutritive perfusion) or a type B (non-nutritive) vasoconstrictor, so here we use the pump-perfused rat hindlimb to characterize the distribution effects of ET-1. We show that ET-1 causes a type A vasoconstriction, stimulating basal metabolism at low doses, while at high doses the distribution of flow changes to become non-nutritive, inhibitory to metabolism. As a general vasodilator prevents both metabolic and hemodynamic effects, the effects on metabolism are due to the redistribution of flow. These redistribution effects are confirmed by the ability of high dose ET-1 to decrease aerobic tension development in the contracting hindlimb, and by the ability of low dose ET-1 to increase the interstitial glucose concentration. Given this understanding of the effects of ET-1 alone, we can investigate the interactions between ET-1 and insulin. In the perfused rat hindlimb, insulin has not been observed to have any vasodilatory effect, whereas here for the first time insulin appears to have vasodilator-like actions against ET-1 mediated vasoconstriction. Also, the redistribution of flow by ET-1 does not appear to alter the metabolic effect of insulin to cause glucose uptake at either dose of ET-1 used. Nitric Oxide (NO) is thought to be the mechanism by which insulin causes vasodilation in muscle. A previous study has shown that methacholine (MC), by increasing NO, was able to augment insulin-mediated glucose uptake and capillary recruitment, while other NO donors were unable to do so. Here we show that, at the dose used to increase glucose uptake in the previous study, MC has only a vasodilatory effect, and no direct effect on glucose uptake, in the perfused rat hindlimb. At higher doses, an effect on glucose uptake can be observed. This means that the increase in capillary recruitment by MC was responsible for the elevated insulin-mediated glucose uptake, and there was no direct effect of MC on glucose uptake. A recent publication suggested that the `Na^+`-D-glucose cotransporter (SGLT1) was essential for insulin-mediated glucose uptake, although not required for basal glucose uptake. The implications of this detract from our proposed role of blood flow redistribution in insulin action. In attempting to reproduce these results in the perfused rat hindlimb we found that SGLT1 is not required for insulin-mediated glucose uptake, and confirmed this using a low sodium buffer, which would also inhibit the transporter. We conclude that SGLT1 is not required for insulin-mediated glucose uptake. Our results therefore suggest that complex interactions are involved in insulin action, some of which involve hemodynamic actions that are capable of altering insulin-mediated glucose uptake, and others in which insulin itself can limit the action of other vasomodulators, such as ET-1. It is apparent, however that SGLT1 in the endothelium may not be necessary for the metabolic effects of insulin, and that blood flow distribution, or capillary recruitment is therefore of great importance in delivering glucose to myocytes.

Glucose uptake occurs in skeletal muscle under basal conditions, and increases in response to stimuli such as insulin and exercise. Exercise is known to increase blood flow, and it appears that insulin has similar hemodynamic effects, including increased blood flow and capillary recruitment, which can modify the amount of glucose uptake occurring under each condition. Here we study factors affecting both basal and stimulated myocyte glucose uptake, with a particular focus on vasoactive agents. Insulin stimulates the release of endothelin-1 (ET-1), a potent vasoconstrictor, from endothelial cells in culture. As yet it is unknown whether ET-1 is a type A (causing nutritive perfusion) or a type B (non-nutritive) vasoconstrictor, so here we use the pump-perfused rat hindlimb to characterize the distribution effects of ET-1. We show that ET-1 causes a type A vasoconstriction, stimulating basal metabolism at low doses, while at high doses the distribution of flow changes to become non-nutritive, inhibitory to metabolism. As a general vasodilator prevents both metabolic and hemodynamic effects, the effects on metabolism are due to the redistribution of flow. These redistribution effects are confirmed by the ability of high dose ET-1 to decrease aerobic tension development in the contracting hindlimb, and by the ability of low dose ET-1 to increase the interstitial glucose concentration. Given this understanding of the effects of ET-1 alone, we can investigate the interactions between ET-1 and insulin. In the perfused rat hindlimb, insulin has not been observed to have any vasodilatory effect, whereas here for the first time insulin appears to have vasodilator-like actions against ET-1 mediated vasoconstriction. Also, the redistribution of flow by ET-1 does not appear to alter the metabolic effect of insulin to cause glucose uptake at either dose of ET-1 used. Nitric Oxide (NO) is thought to be the mechanism by which insulin causes vasodilation in muscle. A previous study has shown that methacholine (MC), by increasing NO, was able to augment insulin-mediated glucose uptake and capillary recruitment, while other NO donors were unable to do so. Here we show that, at the dose used to increase glucose uptake in the previous study, MC has only a vasodilatory effect, and no direct effect on glucose uptake, in the perfused rat hindlimb. At higher doses, an effect on glucose uptake can be observed. This means that the increase in capillary recruitment by MC was responsible for the elevated insulin-mediated glucose uptake, and there was no direct effect of MC on glucose uptake. A recent publication suggested that the Na+-D-glucose cotransporter (SGLT1) was essential for insulin-mediated glucose uptake, although not required for basal glucose uptake. The implications of this detract from our proposed role of blood flow redistribution in insulin action. In attempting to reproduce these results in the perfused rat hindlimb we found that SGLT1 is not required for insulin-mediated glucose uptake, and confirmed this using a low sodium buffer, which would also inhibit the transporter. We conclude that SGLT1 is not required for insulin-mediated glucose uptake. Our results therefore suggest that complex interactions are involved in insulin action, some of which involve hemodynamic actions that are capable of altering insulin-mediated glucose uptake, and others in which insulin itself can limit the action of other vasomodulators, such as ET-1. It is apparent, however that SGLT1 in the endothelium may not be necessary for the metabolic effects of insulin, and that blood flow distribution, or capillary recruitment is therefore of great importance in delivering glucose to myocytes.