Dissertations / Theses on the topic 'Monocarboxylate transporters (MCT)'

Chronic lymphocytic leukaemia (CLL) is a lymphoid malignancy which despite advances in the treatment options available is still incurable. Characterised by the gradual accumulation of CD5+ B cells, the paradigm that this is due to failed apoptosis has been challenged and a significant proliferative component has been identified. However, despite the crosstalk between pathways which regulate metabolism and proliferation the metabolic characteristics of these cells are not fully understood. Furthermore, there is a renewed interest in the field of cancer cell metabolism because of the Warburg effect, a hallmark of malignancy whereby cells preferentially switch to aerobic glycolysis and rapidly consume glucose. This has led to the development of new drugs such as AZD3965 an inhibitor of monocarboxylate transporter 1 (MCT1), which along with MCT4 mediates the export of lactate, a toxic bi-product of glycolysis, out of the cell. The aim of this project was to assess whether therapeutically targeting MCT -1 and -4 would be a viable approach for CLL. Chapter 2 of this thesis examines expression of MCT -1 and -4 as well as a specific chaperone protein needed for the surface expression of these proteins, CD147. This chapter confirms the presence of both MCT -1 and -4 and CD147 in normal B cells as well as demonstrating for the first time that these transporters are expressed in CLL cells using Western blotting and qRT-PCR to assess the MCTs and flow cytometry to measure CD147. The levels of both MCTs and CD147 are demonstrated to be significantly reduced in CLL cells in comparison normal B cells likely due to the adoption of a quiescent phenotype to aid cell survival. The following chapter investigates this further by assessing whether there are any changes in expression under the influence of microenvironmental stimuli, specifically CD40 ligand (CD40L). In this chapter it is demonstrated for the first time that MCT4 is upregulated in CLL cells in response to CD40L. Analysis of gene expression using a Fluidigm Biomark™ array suggests this is due to the induction of glycolysis and that CLL cells may promote fatty acid synthesis as well as instigating changes in the metabolism of the tumour stroma possibly to provide substrates. Finally, chapter 4 evaluates the sensitivity of CLL cell lines to AZD3965 using cell death and cell viability assays. Both MEC-1 and HG3 CLL cell lines are shown to be resistant to MCT1 inhibition using AZD3965 and silencing of MCT4 using siRNA cells also has no effect on the viability of MEC-1 cells. That MCT4 can compensate for MCT1 inhibition is shown by the transient expression of MCT4 in a Raji cell line where only MCT1 is expressed. Taken together, the data presented in this study indicates that while the inhibition of MCT1 is likely to be ineffective dual inhibition of both MCT -1 and -4 may be a viable strategy for the localised inhibition of CLL in the secondary tissues. Furthermore, MCT inhibition in this disease may have the potential to negate mechanisms of resistance and protection from oxidative stress mediated by CD40L.

Although it was once thought that neurons solely rely on glucose as a substrate for cellular energy production, it is now known that small monocarboxylate molecules, like pyruvate, lactate, and ketone bodies, are also utilized. Monocarboxylates are transported across plasma membranes via facilitated diffusion using a family of transport proteins known as monocarboxylate transporters (MCTs). Four MCTs (MCT1, MCT2, MCT3, and MCT4) are expressed within neural tissues. Expression of the MCTs has been tied to co-expression of a cell adhesion molecule belonging to the Basigin subset of the immunoglobulin superfamily (IgSF). Basigin gene products are known to interact with MCT1 and MCT4 in the mammalian neural retina and this association is essential to support the cellular energy needs of photoreceptors. A previous study indicated that Basigin gene products use hydrophobic amino acids within specific regions of the transmembrane domain to interact with MCT1. In the present study, it is hypothesized that the same amino acids within the transmembrane domain are used to interact with MCT4, but that no association exists with MCT2, which typically interacts with a different member of the IgSF subset. Therefore, the purpose of the present study was to assess the association between Basigin gene products and MCT4, and with MCT2. Recombinant proteins corresponding to the transmembrane domain of Basigin gene products were used in in vitro binding assays with endogenous MCT2 and MCT4 from mouse brain protein lysates. Contrary to the hypothesis, it was determined that the transmembrane domain of Basigin gene products binds to both MCT2 and MCT4 in vitro. Different amino acids within the transmembrane domain of Basigin gene products are used for each association and the pattern is different from that used in the association with MCT1. The data suggest that Basigin plays multiple roles in the nervous system.

Le catabolisme exacerbé du glucose et de la glutamine est actuellement reconnu comme une caractéristique des cellules cancéreuses, qui leur procure un avantage prolifératif via la production et l’accumulation de plusieurs métabolites au niveau du microenvironnement. Parmi ces métabolites, l’acide lactique représente une molécule de signalisation clé, favorisant la migration et les métastases. Mon projet de thèse s’inscrit dans le contexte d’une étude du métabolisme glycolytique associé aux cellules tumorales à division rapide. Durant ce projet, nous nous sommes intéressés à la caractérisation génétique et fonctionnelle des transporteurs MCT (MonoCarboxylate Transporters) 1 et 4, qui sont des symporteurs H+/lactate dont l’expression membranaire et la fonctionnalité requièrent la liaison avec une protéine chaperonne : CD147/BASIGINE (BSG). Afin de mieux explorer la physiologie des complexes MCT/BSG, et valider le ciblage de l’export d’acide lactique comme une nouvelle approche anti-cancer, nous avons développé une stratégie visant à invalider le gène BSG et/ou MCT4, en utilisant la technologie des Zinc Finger Nucleases (ZFN), dans des lignées cellulaires cancéreuses humaines de côlon, poumon et glioblastome. D’abord, nous avons démontré, que l’effet pro-tumoral majeur de BSG est lié à son action directe sur la stabilisation des MCTs au niveau des tumeurs glycolytiques et non pas à la production des metalloprotéases. Ensuite, nous avons démontré pour la première fois que l’inhibition concomitante de MCT1 et MCT4 est nécessaire pour induire une baisse significative de la tumorigénécité in vivo
Enhanced glucose and glutamine catabolism has become a recognized feature of cancer cells, leading to accumulation of metabolites in the tumour microenvironment, which offers growth advantages to tumours. Among these metabolites is emerging as a key signalling molecule that plays a pivotal role in cancer cell migration and metastasis. In this thesis, we focused on the genetic and functional characterization of monocarboxylate transporters (MCT) 1 and 4, which are H+/lactate symporters that require an interaction with an ancillary protein, CD147/BASIGIN (BSG), for their plasma membrane expression and function. To further explore the physiology of MCT/BSG complexes and validate the blockade of lactic acid export as an anti-cancer strategy, we designed experiments using Zinc Finger Nuclease mediated BSG and/or MCT4 gene knockouts in human colon adenocarcinoma, lung carcinoma and glioblastoma cell lines. First of all, we demonstrated that the major protumoural action of BSG is to control the energetics of glycolytic tumours via MCT1/4 activity and not to produce matrix metalloproteases. Second, we showed for the first time that combined inhibition of both MCT1 and MCT4 transporters is required to achieve a significant reduction in the tumour growth in vivo. Moreover, our findings reported that disruption of the BSG gene dramatically reduced the plasma membrane expression and lactate transport activity of both MCT1 and MCT4, leading to increased accumulation of intracellular pools of lactic and pyruvic acids, decreased intracellular pH and reduced rate of glycolysis

Les tissus adipeux thermogéniques beiges et bruns améliorent l'homéostasie énergétique et représentent des cibles thérapeutiques potentielles pour traiter les maladies métaboliques associées à l’obésité et au vieillissement. Malgré des décennies de recherche et le rôle très bien décrit de la signalisation noradrénergique, les mécanismes sous-jacents à leur plasticité, leur activation et leur fonction restent encore mal compris. Contrairement au tissu adipeux blanc qui stocke l’énergie pour la mettre à disposition de l'organisme, le tissu adipeux brun la dissipe sous forme de chaleur, et participe à la thermogénèse de non frisson. Cette spécificité métabolique est permise par les adipocytes bruns, aux fortes capacités oxydatives dues à leur richesse en mitochondries et à l’expression de la protéine découplante UCP1 (Uncoupling Protein 1). Les adipocytes beiges présentent des caractéristiques métaboliques similaires mais apparaissent dans des zones spécifiques de certains tissus adipeux blancs par le phénomène de brunissement, suite à une stimulation comme l’exposition au froid. Cependant, ces cellules apparaissent dans d’autres conditions de stress, ce qui suggère qu’elles puissent assurer d’autres fonctions que la thermogenèse. Les travaux de mon équipe ont montré que le lactate et les corps cétoniques, des métabolites produits lorsque les flux de substrats (glucose et acides gras respectivement) dépassent les capacités oxydatives et qui agissent comme des régulateurs du métabolisme redox au travers de dialogues inter-cellulaires et inter-tissulaires, sont de puissants inducteurs du brunissement. L’induction d’UCP1 par ces métabolites passe par un mécanisme dépendant du potentiel red/ox (augmentation du ratio NADH,H+/NAD+), et comme UCP1 permet de diminuer ce potentiel red/ox en accélérant le fonctionnement de la chaîne respiratoire, le brunissement apparaît comme un mécanisme adaptatif pour maintenir l’homéostasie red/ox
Brown and beige thermogenic adipose tissues improve energetic homeostasis and represent a potential therapeutic targets for the treatment of obesity and aging associated metabolic diseases. Besides decades of research and the very well-described role of noradrenergic signaling, the mechanisms underlying their plasticity, activation and function are still poorly understood. In contrast to white adipose tissue that stores energy to make it available to the organism, brown adipose tissue dissipates energy as heat, and is involved in non-shivering thermogenesis. This metabolic specificity is permitted by brown adipocytes, which exhibit strong oxidative capacities due to their high content in mitochondria and the expression of the uncoupling protein 1 (UCP1). Beige adipocytes have similar metabolic characteristics but appear in specific regions of certain white adipose tissues by the browning phenomenon, following stimulation such as cold exposure. However, these cells appear in other stress situations, suggesting that they may have other functions than thermogenesis. My team's work has previously shown that lactate and ketone bodies, metabolites produced when substrate fluxes (glucose and fatty acids respectively) exceed oxidative capacities and act as regulators of redox metabolism through inter-cellular and inter-organ dialogues, are powerful inducers of browning. The induction of UCP1 by these metabolites is due to a redox mechanism (increase in NADH,H+/NAD+ ratio), and because UCP1 reduces this redox pressure by accelerating the respiratory chain, browning thus appears as an adaptive mechanism to maintain redox homeostasis. Because the underlying molecular mechanisms were poorly understood, my thesis objective was to characterize the expression of lactate transporters in adipocytes and to understand their role in their plasticity and metabolic activity. The fine mapping of the subcutaneous inguinal adipose tissue in mice, using laser microdissection experiments, gene expression measurement and confocal imaging, revealed i) a strong positive correlation between the expression of the lactate transporter Mct1 (monocarboxylate transporter 1) and that of Ucp1 and (ii) the appearance of UCP1 following cold exposure restricted to the subpopulation of adipocytes expressing MCT1 and pre-existing at thermoneutrality. These results highlight the MCT1 protein as a marker of dormant beige adipocytes, able of be activated during cold exposure. This finding is reinforced by the absence of the MCT1 protein in perigonadic adipose tissue which is resistant to browning, and its strong expression in classical brown adipocytes. While MCT1 is necessary for lactate-induced UCP1 expression, we showed that it was not involved in the Ucp1 regulation by adrenergic signaling. However, lactate oxidation and isotopic profiling experiments showed that MCT1 was essential for the metabolic activity of beige adipocytes, by controlling lactate export and import. Lactate export by MCT1 is necessary for glucose consumption, especially during ß3 adrenergic agonist stimulation, by maintaining the redox NADH,H+/NAD+ ratio which is fundamental for the control of glycolysis. MCT1-dependent lactate import feeds the oxidative metabolism and kreb cycle of these cells. A genetically engineered mouse model showed that inducible MCT1 loss of function in adipocytes impact glycemia during cold exposure, confirming the crucial role of MCT1 and lactate fluxes in the control of glucose metabolism in brown/beige adipose tissues. The proposed mechanisms highlight the fundamental role of MCT1 in beige adipocytes biology and could be extrapolated to brown adipocytes

The current understanding of lactate metabolism in fish is based almost entirely on interpretation of concentration measurements that cannot be used to infer changes in flux. Moreover, the transporters regulating these fluxes have never been characterized in rainbow trout. My goals were: (1) to quantify lactate fluxes in rainbow trout under normoxic resting conditions, during acute hypoxia, and exercise by continuous infusion of [U-14C] lactate; (2) to determine lactate uptake capacity of trout tissues by infusing exogenous lactate in fish rest and during graded exercise, and (3) to clone monocarboxylate transporters (MCTs) and determine the effects of exhausting exercise on their expression. Such information could prove important to understand the mechanisms underlying the classic “lactate retention” seen in trout white muscle after intense exercise. In normoxic resting fish, the rates of appearance (Ra) and disappearance (Rd) of lactate were always matched (~18 to 13 µmol kg-1 min-1), thereby maintaining a low baseline blood lactate concentration (~0.8 mM). In hypoxic fish, Ra lactate increased from baseline to 36.5 µmol kg-1 min-1, and was accompanied by an unexpected 52% increase in Rd reaching 30.3 µmol kg-1 min-1, accounting for a rise in blood lactate to 8.9 mM. In exercising fish, lactate flux was stimulated > 2.4 body lengths per second (BL s-1). As the fish reached critical swimming speed (Ucrit), Ra lactate was more stimulated (+67% to 40.4 μmol kg-1 min-1) than Rd (+41% to 34.7 μmol kg-1 min-1), causing an increase in blood lactate to 5.1mM. Fish infused with exogenous lactate stimulated Rd lactate by 300% (14 to 56 μmol kg-1 min-1) during graded exercise, whereas the Rd in resting fish increased by only 90% (21 to 40 µmol kg-1 min-1). Four MCT isoforms were partially cloned and characterized in rainbow trout: MCT1b was the most abundant in heart, and red muscle, but poorly expressed in gill and brain where MCT1a and MCT2 were prevalent. MCT4 was more expressed in the heart. Transcript levels of MCT2 (+260%; brain), MCT1a (+90%; heart) and MCT1b (+50%; heart) were stimulated by exhausting exercise. This study shows that: (i) the increase in Rd lactate plays a strategic role in reducing the lactate load imposed on the circulation. Without this response, blood lactate accumulation would double; (ii) a high capacity for lactate disposal in rainbow trout tissues is elicited by the increased blood-to-tissue lactate gradient when extra lactate is administered; and (iii) rainbow trout may be unable to release large lactate loads rapidly from white muscle after exhausting exercise (lactate retention) because they poorly express MCT4 in white muscle and fail to upregulate its expression during exercise.

It has been shown that tumour cells are capable of switching to glycolytic metabolism for the production of ATP even in the presence of oxygen, this is known as aerobic glycolysis or the 'Warburg effect'. The glycolytic phenotype has been associated with tumour aggressiveness and poor outcome in several cancer types. This makes the area of cancer metabolism an attractive area for the potential identification of new therapeutic targets. One key component, required for cells to maintain the glycolytic phenotype, is the presence of monocarboxylate transporters that are capable of exporting lactate. These transporters are vital for the maintenance of the intracellular pH of cells under these conditions. This study was centred around the hypothesis that altering expression of MCTs would impact on the metabolism of tumour cells and, therefore, other key characteristics of cells relating to metastatic capabilities and survival following treatment. For the purpose of this work, prostate cancer cell lines were transfected with lentiviral particles targeting overexpression of MCT1 or MCT4, or knockdown of MCT4. Following transfection, cellular metabolic profiles were assessed under normoxic and hypoxic conditions and the metastatic phenotype of each cell line was investigated. Additionally, the effect of MCT expression on response to chemotherapy and radiation therapy was explored, and a siRNA metabolome screen was performed to identify combinations of targets that may produce synthetic lethality in prostate cancer cell lines. It was shown that changes in the expression of MCT1 or MCT4 did not cause significant changes in the metastatic phenotypes of the prostate cancer cell lines investigated. Some differences were observed in the metabolic pathways used by these prostate cancer cells following alterations in MCT expression. For example, overexpression of MCT1 in DU145 cells resulted in an increase in intracellular lactate. Additionally, MCT4 knockdown in PC3 cells was able to reduce OXPHOS under reduced oxygen. MCT1 overexpression was able to sensitise androgen-independent prostate cancer cells to treatment with chemotherapy and radiation therapy. Furthermore, combinations of siRNA treatments were identified that may be capable of producing synthetic lethality. In summary, findings in this study indicated that targeting MCT1 and MCT4 expression could offer therapeutic benefit in prostate cancer. However, it was also highlighted that the roles of these transporters are specific to cancer type, and even cell line.

Basigin and Embigin are members of the immunoglobulin superfamily that function as cell adhesion molecules. Studies of Basigin null mice revealed reproductive sterility, increased pain sensitivity, and blindness. It is thought that the mechanism causing blindness involves misexpression of monocarboxylate transporter 1 (MCT1) in the absence of Basigin. It is known that the transmembrane domain of Basigin interacts with MCT1. In the absence of Basigin, MCT1 does not localize to the plasma membrane of expressing cells and photoreceptor function is disrupted. Studies of the Basigin null mouse brain suggest that MCT1 is properly expressed, which suggests a separate mechanism causes the increased pain sensitivity in these animals, and also that a different protein directs MCT1 to the plasma membrane of expressing cells in mouse brain. Embigin is known to interact with MCT2 in neurons and with MCT1 in erythrocytes. It is not known, however, if Embigin normally interacts with MCT1 in the mouse brain or if Embigin acts to compensate for the lack of Basigin in the Basigin null animals. Therefore, the purpose of this study was to determine if Embigin normally interacts with MCT1, 2, or 4 in the mouse brain and if so, whether the interaction is similar to that between Basigin and MCT1. Expression of Basigin, Embigin, MCT1, MCT2, and MCT4 in mouse brain was assessed via immunoblotting and immunohistochemical analyses. In addition, recombinant protein probes corresponding to the Embigin transmembrane domain were generated for ELISA binding assays using endogenous mouse brain MCTs. It was determined that the proteins in question are rather ubiquitously expressed throughout the mouse brain, and that the cell adhesion molecules Basigin and Embigin may be co-expressed in the same cells as the MCT2 and MCT4 transporter proteins. In addition, it was determined that the Embigin transmembrane domain does not interact with the MCTs. The data therefore suggest that MCTs do not require Basigin or Embigin for plasma membrane expression in mouse brain.

Upon activation, T cells shift towards a metabolic program characterized by increased glucose metabolism in order to sustain proliferation and effector function. Surprisingly, while resting T lymphocytes degrade glucose aerobically to CO2, proliferating T cells metabolize glucose almostentirely to lactate in the presence of oxygen through aerobic glycolysis (the Warburg effect). This metabolic switch comprises the upregulation of glycolytic enzymes and glucose transporters to the cell membrane, leading to an increase of glycolytic flux and the concomitant production of lactate. Despite many decades of research, we still do not fully understand the mechanisms that make proliferating T cells choose glycolysis rather than oxidation of glucose to produce energy. Since activated T lymphocytes depend on a glycolytic metabolism, they must release lactate, which inthese cells is facilitated by the proton-linked monocarboxylate transporter MCT1. The transporter is part of a protein family of 14 members among which MCT1–4 facilitate the passive transmembrane transport of monocarboxylates such as lactate, pyruvate and ketone bodies. The observation that pharmacological MCT1 inhibition has shown anti-proliferative effect on T cells suggests that lactate transport is essential to T cell expansion triggered after antigen recognition. The aim of our research is to investigate the importance of MCT1-dependent regulation in T cellmetabolism. Following TCR stimulation, MCT1 was expressed early in T cells unlike MCT4 whose significant expression was detected at later time point. To investigate the role played by MCT1 in the early steps of T cell activation, we generated a transgenic mouse model where conditional deletion of the MCT1 gene was achieved specifically in T cells. Phenotype and T cell distribution in thymus and peripheral organs were normal in MCT1fl/fl CD4Cre mice. However, lack of MCT1 expression decreased the proliferative capacity of in vitro activated CD4+ or CD8+ T cells without altering their viability. We observed that the IL-2 production was also affected by the lack of MCT1 expression, in line with decreased proliferative ability. Moreover, in vivo, T cell expansion that followed antigenic stimulation as well as T cell-mediated immune response to infection were deficient in MCT1fl/flCD4Cre mice. Our data indicate that this situation resulted from a cellular energy shortage caused by reduced glycolytic activity soon after activation. Moreover, energy crisis was amplified by the necessity to use ATP-consuming mechanisms for excluding H+ protons from the cytosol of activated MCT1-deficient T cells. Thus, in T cells, early MCT1 expression after activation ensures an energy saving mechanism for regulating cytoplasm acidification. Our observations also indicate that a high glycolytic flux is required in dividing T cells to maintain pH homeostasis.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

La fermentation des fibres alimentaires par la microflore colique aboutit à la production d'acide gras à chaines courtes comme l'acétate, le propionate et le butyrate. Ce dernier joue un rôle majeur dans le maintien de l'intégrité de la muqueuse colique. Il constitue la première source d'énergie de l'épithelium colique et a de nombreux effets physiologiques comme la régulation de la proliferation cellulaire, la différenciation et l'apoptose. L'utilisation du butyrate est diminuée dans de nombreuses situations pathologiques comme les maladies inflammatoires chroniques de l'intestin et les cancers colorectaux. Le transporteur des monocarboxylates 1 (MCT1=SLC16A1) transporte le butyrate à travers la membrane apicale des colonocytes. Une diminution de l'expression de MCT1, qui réduit la disponibilité intracellulaire du butyrate diminue non seulement son oxidation mais aussi ses effets régulateurs. Dans cette étude, nous montrons tout d'abord que MCT1 est un bon marqueur de l'exposition de la muqueuse colique au butyrate observé. Dans un deuxième temps, nous démontrons que le défaut d'oxydation du butyrate observé dans les maladies inflammatoires de l'intestin est la conséquence d'une diminution du transport du butyrate, lui même associé à une diminution de l'expression de MCT1 dans la muqueuse colique
Dietary fibers are digestible food ingredients that reach the colon and are then fermented by colonic bacteria, resulting mainly in the formation of short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate. Especially butyrate plays an important part in maintaining the health and integrity of the colonic mucosa. It is the primary energy source for the colonic epithelium and has most important physiological effect, including regulation of cell proliferation, differentiation and apoptosis. Butyrate oxidation was decrease in pathological situations like chronic inflammatory bowel diseases or colorectoral cancer. The monocarboxylate transporter 1 (MCT1 = SLC16A1) transports butyrate across the apical membrane of human colonocytes. Thus, a decrease in MCT1 expression, which reduces the intra-cellular availability of butyrate could affect not only its oxidation, but also its cell regulatory effects. In this study, we investigated that MCT1 can be identified as a good marker of butyrate exposition in colon epithelial cells and we demonstrate that the defect in butyrate oxidation reported in inflammatory bowel diseases is the consequence of a decrease in butyrate transport into the colonocyte, itself related to a reduced expression of the butyrate intestinal transporter MCT1

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O transportador de monocarboxilato isoforma 1 (MCT1), presente na membrana das hemácias, e sua proteína auxiliar CD147 têm como função transportar H+ e lactato do plasma para dentro das hemácias, mantendo assim, a homeostase ácido-base e retardando a acidose sistêmica e fadiga muscular. Dessa forma, o objetivo desse estudo foi comparar as quantidades das proteínas MCT1 e CD147 em hemácias de cavalos Árabes e Quartos de Milha com diferentes níveis de desempenho atlético. Além disso, objetivou-se buscar por polimorfismos para os genes MCT1, CD147, DMRT3 e PDK4, a fim de checar associações entre os polimorfismos e o desempenho nas raças. Cavalos Árabes e Quartos de Milha foram divididos em dois grupos de acordo com o desempenho em provas de enduro e provas de corridas, respectivamente. A quantidade de MCT1 e CD147 na membrana plasmática das hemácias foi determinada por western blotting com unidades arbitrárias de densidade óptica (OD) e anticorpos reagentes à espécie humana anti-MCT1 e anti-CD147. Os dados para as quantidades de proteínas foram analisados pelo PROC MIXED do SAS. O modelo incluiu a idade como covariável e os efeitos fixos de sexo, raça e grupo de desempenho dentro de raça. As correlações foram analisadas pelo teste de Pearson pelo procedimento PROC CORR. P-valores <0,01 foram considerados estatisticamente significantes. Os polimorfismos dos genes foram analisados por sequenciamento (MCT1 e CD147), PCR-RFLP (DMRT3) e ARMS-PCR (PDK4). Os pacotes estatísticos Genetics, Lattice e GenABEL foram utilizados para comparar as frequências dos grupos de desempenho no software R, com o teste exato de Fisher a 5% de significância. As proteínas MCT1 e CD147 foram encontradas nas hemácias de todos os animais. A quantidade de MCT1 foi significativamente (p<0,0001) maior em Quartos de Milha (2,99 ± 0,35 OD) do que em Árabes (1,04 ± 0,08 OD). Quartos de Milha (3,23 ± 0,38 OD) também apresentaram maior conteúdo de CD147 do que Árabes (0,88 ± 0,06 OD). Não houve diferença estatística nas quantidades de proteínas para os grupos de desempenho de ambas as raças. Correlação positiva foi encontrada entre as quantidades de MCT1 e CD147 (r=0,95; p<0,0001). O Alelo A dos polimorfismos Lys457Gln:1573A>C do gene MCT1 e Ile51Val:168A>G do gene CD147 estavam fixados em ambas as raças. Um novo polimorfismo (AY457175.1:c1498G>A) foi encontrado na sequência do gene MCT1. Para o DMRT3, todos os animais apresentaram o alelo C fixado para o polimorfismo. Árabes mostraram maior frequência para o alelo G do que Quartos de Milha (p<0,01) para o polimorfismo no gene PDK4. Entretanto, não houve diferença entre os grupos de desempenho para as duas raças. Dessa forma, conclui-se que Quartos de Milha têm maiores quantidades de MCT1 e CD147 do que Árabes. Não foi possível determinar a influência dos polimorfismos nos genes MCT1, CD147 e DMRT3 no desempenho atlético das duas raças visto que seus alelos estavam fixados. Além disso, houve diferença significativa nas frequências do polimorfismo no gene PDK4 entre Árabes e Quartos de Milha, mas não houve diferença entre os grupos de desempenho.
Monocarboxylate transporter isoform 1 (MCT1), present in the red blood cell membranes and its ancillary protein CD147 have as function transport H+ and lactate ions from the plasma into the red blood cells, thereby maintaining acid/base homeostasis and retarding systemic acidosis and muscular fatigue. Thereby, the aim of this study was to compare the amount of MCT1 and CD147 proteins in the red blood cells of Arabian and Quarter Horses with different levels of athletic ability. Furthermore, we investigated polymorphisms for MCT1, CD147, DMRT3, and PDK4 genes in Arabian and Quarter Horses in order to check associations between the polymorphisms and the performance in these breeds. Arabian horses were divided into two groups according to their performance in endurance competition and Quarter Horses were separated by its performance in races, determined by Speed Index. The amount of MCT1 and CD147 proteins in the plasma membrane of red blood cells was determined by western blotting analysis with arbitrary optical density units (OD), using a human specific anti-MCT1 and anti- CD147 antibody. Data for the amounts of proteins were analyzed using the PROC MIXED procedure of SAS software. The model for the analysis included the effects of sex, breed and performance group within breed as fixed effect and age as covariate. The correlations were analyzed by Pearson correlation test using the PROC CORR procedure of SAS software. P values <0.01 were considered statistically significant. Polymorphisms of the genes were analyzed by sequencing (MCT1 and CD147), PCR-RFLP (DMRT3) and ARMS-PCR (PDK4) techniques. The statistical packages Genetics, Lattice and GenABEL were used to compare the frequencies of the groups using the software R, with the Fisher's exact test being performed with significance level of 5%. MCT1 and CD147 proteins were found in the red blood cell membranes of all studied animals. The amount of MCT1 was significantly (p<0.0001) higher in Quarter Horses (2.99 ± 0.35 OD) than in Arabians (1.04 ± 0.08 OD). Quarter Horses (3.23 ± 0.38 OD) also showed bigger contents of CD147 than Arabians (0.88 ± 0.06 OD). There was not statistical difference in the amounts of MCT1 and CD147 between the performance groups of both breeds. Positive correlation was found between the amounts of MCT1 and CD147 (r=0.95; p<0.0001). The A allele from the polymorphisms Lys457Gln:1573A>C of MCT1 and Ile51Val:168A>G of CD147 gene, were fixed in both breeds. A new polymorphism (AY457175.1:c1498G>A) was found in the MCT1 gene sequence. For DMRT3 mutation, all the animals shown to have the C allele fixed for the polymorphism. Arabians showed significant greater frequency of the G allele than Quarter Horses (p<0.01) for the PDK4 polymorphism. However, there was not difference between the groups of performance for both breeds. In summary, it follows that the Quarter Horses have greater amount of MCT1 and CD147 proteins than Arabian. It was not possible to determine the influence of polymorphisms in MCT1, CD147 and DMRT3 genes in the athletic performance of these breeds since they had alleles fixed. There was a significant difference in the frequencies of the PDK4 polymorphism between Arabians and Quarter Horses, but there was not difference between the performance groups.
FAPESP: 2012/24193-0
FAPESP: 2012/20697-9

L'hypothalamus est fortement impliqué dans la régulation nerveuse de l'homéostasie énergétique. Il existe dans cette structure des neurones spécialisés (gluco-sensibles) qui détectent notamment l'hyperglycémie puis déclenchent des réponses adaptées comme le maintien de la glycémie, en stimulant la sécrétion d'insuline ou encore le rassasiement. Les astrocytes sont suspectés de participer à la détection neuronale du glucose. Dans l'ensemble du cerveau, il existe un couplage métabolique entre astrocytes et neurones. Le lactate, issu de la métabolisation du glucose par les astrocytes, est transporté par les neurones par des transporteurs aux monocarboxylates (MCTs). De plus, il a récemment été montré que les jonctions gap (GJ), à l'origine de la formation de réseaux au sein des astrocytes sont indispensables au passage du glucose de la circulation sanguine vers les neurones en activité. Ces GJ astrocytaires sont formées majoritairement de connexines 43 et 30 (Cxs).Mon travail de thèse s'est orienté suivant deux axes, qui ont visé à étudier le rôle des astrocytes dans la détection hypothalamique du glucose et du lactate. Dans un premier temps, nous avons montré que le lactate, comme le glucose, est détecté au niveau central et induit une sécrétion d'insuline. Dans un modèle de rat hyperglycémique pendant 48h (qui présente aussi une hyperlactatémie), nous avons montré que la détection du glucose et du lactate est altérée. Ces modifications ne sont pas dues à une variation de l'expression protéique des MCTs astrocytaires ou neuronale de l'hypothalamus.Dans un deuxième temps, nous nous sommes intéressés au rôle des Cxs astrocytaires. La Cx43 est très exprimée autour des micro-vaisseaux sanguins de l'hypothalamus médio-basal (MBH), un site présentant de nombreux neurones gluco-sensibles. L'expression de la Cx30 est plus diffuse dans cette structure. Nous montrons également que l'expression protéique des Cxs astrocytaires varie très rapidement suite à des modifications du statut métabolique (jeûne, réalimentation, hyperglycémie). Afin d'évaluer l'implication de la Cx43 astrocytaire (majoritaire) dans la détection hypothalamique du glucose, nous avons inhibé son expression dans le MBH, in vivo, en injectant des siRNA permettant d'inhiber la synthèse de cette protéine. L'inhibition de la Cx43 (30% à 72h) induit une diminution de la prise alimentaire sans modification du poids, de la glycémie et de l'insulinémie comparée aux témoins. Suite à l'injection carotidienne de glucose (censée mimer une hyperglycémie), la sécrétion d'insuline est fortement inhibée chez les animaux siCx43. De même, l'effet satiétogène du glucose semble inhibé chez ces animaux lors de la réalimentation après un jeûne.Ces résultats montrent pour la première fois, de façon intégrée, l'importance des connexines, et probablement des réseaux astrocytaires, lors de la détection hypothalamique du glucose. Ces nouvelles données renforcent l'importance du rôle métabolique des astrocytes lors de fonctions neuronales précises

Der Monocarboxylattransporter 8 (Mct8) ist ein spezifischer Schilddrüsenhormon (SDH)-Transporter. MCT8-Mutationen führen zu einer psychomotorischen Retardierung in Kombination mit abnormalen SDH-Serumkonzentrationen. Das konstitutiv Mct8-defiziente Mausmodell repliziert den endokrinologischen, jedoch nicht den humanen neurologischen Phänotyp. Um die Hypothese eines stark beeinträchtigten T3-Transportes speziell in Neuronen als Ursache zu untersuchen, wurde das Neuron-spezifische Mct8-defiziente Mausmodell (CamK-Cre;Mct8fl/fl) generiert. Neben einer funktionalen, Mct8-exprimierenden Blut-Hirn-Schranke liegt eine funktionale Hypophysen-Hypothalamus-Schilddrüsen Achse vor. NMR-Analysen des zerebralen Energiestoffwechsels von CamK-Cre;Mct8fl/fl-Mäusen zeigen nach [1-13C] Glukoseinfusion verringerte Laktatintensitäten sowie eine reduzierte Laktatdehydrogenase-Aktivität. Zudem sind Astrozyten-spezifische Transporter und Enzyme des Neurotransmitterstoffwechsels und deren Biosynthese in ihrer Genexpression reduziert. Somit führt der neuronale Mct8-Verlust zu einem verlangsamten zerebralen Metabolismus sowie einer reduzierten neuronalen Aktivität. Die Rolle von Mct8 im Energiestoffwechsel wurde außerdem in primären Mct8-defizienten Astrozyten- und Neuronkulturen mittels Seahorse Flux Analyzer untersucht. In Mct8-defizienten Neuronen kommt es zu einer verringerten SDH-Aufnahme, was in einer verringerten Expression von OXPHOS-relevanten Proteinen sowie in einer verringerten Sauerstoffverbrauchsrate resultiert. Somit stützen die in vitro Daten die des CamK-Cre;Mct8fl/fl-Mausmodelles bezüglich einer reduzierten neuronalen Aktivität sowie eines verlangsamten zerebralen Stoffwechsels. Zusammenfassend zeigen die Ergebnisse, dass grundlegende Mechanismen des zerebralen Stoffwechsels bei neuronaler Mct8-Defizienz beeinträchtigt sind und die Rolle von Mct8 mit Hilfe weiterer konditioneller Mausmodelle (Astrozyten-spezifisch) und primären Ko-Kulturmodellen untersucht werden muss.
The monocarboxylate transporter 8 (Mct8) is the most specific thyroid hormone (TH) transporter. Mutations lead to a severe form of psychomotor retardation in combination with abnormal TH concentrations in sera. The global Mct8-deficient mouse model was intensively studied and it replicates the endocrine, but not the human neurological phenotype. To test the hypothesis, that a disturbed uptake of T3 especially into neurons is responsible for the phenotype, we generated a neuron-specific Mct8-deficient mouse model (CamK-Cre;Mct8fl/fl). CamK-Cre;Mct8fl/fl mice exhibit a functional Mct8-expressing blood-brain-barrier and a functional hypothalamus pituitary thyroid axis. NMR analyses of the cerebral energy metabolism of CamK-Cre;Mct8fl/fl mice after [1-13C] glucose injection revealed less enrichment of lactate and a reduced lactate dehydrogenase activity. Moreover, especially astrocyte-specific expressed transporter and enzymes of neurotransmitter metabolism and their biosynthesis are significantly reduced in comparison to control mice. These results point to a decelerated cerebral metabolism as well as a reduced neuronal activity caused by the neuronal loss of Mct8. In addition, we studied the impact of Mct8 on the energy metabolism in primary wildtype and Mct8-deficient astrocyte and neuron cultures by use of the Seahorse Flux Analyzer. Mct8-deficient neurons show a reduced uptake of TH, which results in a reduced expression of OXPHOS relevant proteins as well as a reduced oxygen consumption rate. Therefore, the in vitro raised data provide the observed changes of the CamK-Cre;Mct8fl/fl mice regarding a reduced synaptic activity as well as a reduced cerebral metabolism. Taken together, the data clearly shows that basic mechanisms of the cerebral metabolism are hampered in neuronal Mct8 deficiency. The role of Mct8 in this context needs further analyses with the help of conditional mouse models (astrocyte-specific) and primary co-culture models.

Monocarboxylate transporters (MCTs) have been shown to be important in regulating metabolism during hypoxia in mammals. However, the role of MCTs in hypoxic survival in lower vertebrates is currently unclear. The goal of this study was to investigate the coordination of MCTs along with other metabolic proteins during hypoxia. Therefore, we subjected zebrafish (Danio rerio) to 1.5 mg L-1 O2 over 48 and 96-hr and measured tissue-specific transcriptional changes of MCTs (1, 2 and 4), lactate dehydrogenase A (LDHa), citrate synthase (CS), and other metabolic proteins using real-time RT-PCR. There were no changes in mRNA in muscle at 48 and 96-hr. When data from both time points were pooled in brain, a significant increase was found in MCT4 (+102%) and LDHa (+28%) mRNA indicating a preference towards glycolysis. In gills, there were increases in LDHa at 48-hr (+101%) and MCT1 (+24%) mRNA from pooled data suggesting that both anaerobic and aerobic metabolism is being utilized. Heart had the greatest changes in transcriptional levels compared to other tissues. At 48-hr, increases were found in MCT1 (+117%), MCT4 (+86%), LDHa (+197%), and pooled data showed an increase in CS (+18%) mRNA. These results indicate that the influx and efflux of lactate are both employed as strategies in cardiac tissue during hypoxia. This study has shown that fish utilize tissue-specific regulation of MCTs along with other metabolic genes during hypoxia.
Thesis (Master, Biology) -- Queen's University, 2009-08-24 13:44:06.114

Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.

Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.

The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.

Dissertation

Monocarboxylate transporters (MCTs) are bi-directional symporters that couple proton movement to a variety of glycolytic metabolites, including lactate and pyruvate. As such, MCTs not only play a crucial role in pH regulation, but could also function in the distribution of substrates between organelles, cells and tissues. This function may be critically important during hypoxia, when tissues depend on glycolytic flux for energy. We have examined the role of MCTs during hypoxia in an estuarine teleost, the common killifish, Fundulus heteroclitus, that routinely encounters environmental hypoxia. We cloned and sequenced a killifish MCT that resembled the MCT-2 isoform of mammals, and examined its expression pattern and regulation during hypoxia using real-time quantitative PCR. Killifish MCT mRNA levels were highest in the intestine (~2x) and lowest in the heart (~0.2x) as compared to the expression in the liver. Exposure to hypoxia (0.5 mg L-1) resulted in a significant increase in plasma lactate and glucose within 4 hours. However pyruvate and glycogen concentration in white muscle did not change significantly during this time. We observed no change in the mRNA levels of this MCT isoform in any tissue during 16h of hypoxia. This could indicate the regulation of this MCT at another level, a secondary role in hypoxia, or a role for this isoform unrelated to hypoxia tolerance.
Thesis (Master, Biology) -- Queen's University, 2009-09-02 15:54:00.643

Mastitis is often bacterial in origin. Lipoteichoic acid (LTA) and lipopolysaccharide (LPS), endotoxins from gram-positive and gram-negative bacteria, respectively, are potent inducers of mammary gland inflammation. Inflammation can alter expression of transporters responsible for transport of substrates important in synthesis of milk constituents and cellular metabolic energy. Since, gram-positive and gram-negative bacterial infections cause a different clinical course of mastitis, I investigated whether LTA and LPS differentially alter proton-coupled (MCT1) and sodium-coupled monocarboxylate transporter (SMCT1, SMCT2) expression and functional outcomes of altered expression. Human mammary epithelial cells (MCF-12A) were incubated with 1 microgram/mL LPS or LTA for 6, 12 and 24 hours and mRNA expression of TNF-alpha, IL-1β, IL-6, MCT1, SMCT1, and SMCT2 were measured using Quantitative RT-PCR. LPS decreased SMCT1, but increased SMCT2 expression after 6 h, while LTA increased MCT1 expression at 6 h, followed by gradual decrease in expression until 24 h. To know whether such differential changes in transporter expression by LPS and LTA could cause changes in cellular energy production, I quantified creatine (Cr) and high-energy phosphate substrates (CrP, ATP, ADP, AMP) and oxygen consumption rates using HPLC and Hansatech oxygen electrode, respectively. At 12 h, LPS increased concentrations of Cr, CrP, ATP and ADP, whereas LTA caused changes in CrP and ADP concentrations relative to control. Both LPS and LTA decreased oxygen consumption rates after 12 h. Furthermore, to know whether changes in transporter expression lead to differences in substrate availability, I performed uptake studies for carnitine using radiolabelled tritium L-carnitine. LPS and LTA challenge did not affect the affinity, but caused a 2-3-fold increase in maximal activity (Vmax) of carnitine transport. Although increases in Vmax were not significant, the increase in Vmax after 12 h exposure by LPS and LTA corresponds to changes in mRNA expression of the OCTN2 transporter (previously reported in the laboratory). In conclusion, LPS and LTA differentially alter mRNA expression of transporters, which leads to changes in cellular energy levels and oxygen consumption rates and possibly to changes in the functional activity of transporters. Whether such differences contribute to the different clinical course of mastitis warrants further investigation.

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012
Desde a descoberta do “Efeito de Warburg”, a primeira alteração bioquímica qualitativa descoberta em células cancerígenas que tem por base a alteração do mecanismo preferencial de produção de ATP, que o metabolismo tumoral tem vindo progressivamente a ser considerado como uma das mais importantes características dos tumores. Esta característica foi efectivamente considerada como uma das principais características tumorais emergentes (Hanahan e Weinberg, 2011). Células não tumorais fazem a produção de ATP através da fosforilação oxidativa, enquanto algumas células tumorais parecem optar por um mecanismo alternativo de produção de ATP, a glicólise anaeróbia (Berg et al., 2006). Ao optar por esta via alternativa as células cancerígenas estão efectivamente a escolher uma via que produz menos moléculas de ATP por mole de glucose. Assim, vários trabalhos têm-se debruçado sobre os motivos pelos quais as células tumorais optam por esta via e hoje em dia pensa-se que a produção de lactato leva a uma acidificação do meio circundante que pode ser benéfico para as células tumorais uma vez que está ligado em alguns cancros a um comportamento mais invasivo e metastático (Postovit et al., 2002; He et al., 2004). Foi também proposto que o efeito de Warburg nas células tumorais pode permitir às células a produção de maior quantidade de amino ácidos, lípidos, ácidos gordos e nucleótidos ao permitir que a glucose seja utilizada por percursores da síntese destas molélulas (Vander Heiden et al., 2009). O lactato é produto da glicólise, sendo que em mamíferos o lactato proveniente de tecidos glicolíticos sofre gluconeogénese no fígado através do ciclo de Cori (Berg et al., 2006). O lactato foi proposto como sendo uma molécula sinalizadora (Hashimoto et al. 2007). O alastramento do lactato em tecidos está geralmente associado a hipóxia (baixos níveis de oxigénio) e áreas de necrose (Shcroeder et al., 2005 ). Os transportadores de monocarboxilados (MCTs) medeiam o transporte de moléculas com um grupo carboxílico (monocarboxilados) através das membranas celulares. Em células não cancerígenas eles são responsáveis pela manutenção do pH intracelular através do efluxo de lactato (Dimmer et al., 2000). Vários monocarboxilados usam estes transportadores tais como piruvato e lactato, demonstrando a sua importância na comunicação intercelular (Poole and Halestrap, 1993). Os diferentes MCTs parecem ser definidos pela sua ligação ao substrato e especificidade, provavelmente através do seu C-terminal que é menos conservado que o N-terminal (Halestrap and Meredith, 2004). Até à data foram identificados 14 MCTs em mamíferos sendo que o MCT1, MCT2, MCT3 e MCT4 já foram avaliados como melhorando o transporte de monocarboxilados ligado a H+ entre os locais de secreção e remoção (Halestrap and Price, 1999). Pensa-se que os MCTs 1 e 4 estão associados a um aumento da agressividade tumoral, sendo que necessitam de uma molécula auxiliar, CD147 (também conhecido por basigin), para a sua expressão funcional correcta e para o transporte de lactato (Kirk et al., 2000). As lactato desidrogenases (LDHs) catalizam a conversão que se dá de piruvato a lactato (LDHA) e vice versa (LDHB). A LDH tem cinco isozimas diferentes, que ao associar dois péptidos (M e H) codificados por dois genes (A e B, respectivamente) dão origem a tetrâmeros (Koen and Goodman, 1969). Existe também uma outra forma tetramérica, LDH-C4, codificada por um outro gene (LDHC) presente apenas nos testículos maduros e esperma (Markert et al., 1975). Pensa-se que a expressão programada de LDH ocorre durante o desenvolvimento, no estadio de diferenciação das células germinativas (Thomas et al., 1990). Leucemias são cancros do sangue e são classificadas de acordo com a sua linhagem na hematopoiese, pelo que podem ser divididos de acordo com as duas principais linhagens sanguíneas: mielóides e linfóides. A leucemia mielóide aguda é um neoplasmo maligno clonal das células hematopoiéticas progenitoras da medula óssea em que estes blastos perdem a capacidade de se diferenciarem normalmente e de responder a reguladores normais da proliferação (Passegué et al., 2003). O que acontece na leucemia mielóide aguda é que as células sanguíneas anormais sobrepopulam a medula óssea, substituindo as células sanguíneas normais e interrompendo a hematopoiese normal (Goldsby et al., 2002). As leucemias são classificadas pela Federação Francesa- Americana-Britânica (FAB) de acordo com vários critérios como a morfologia, citoquímica e fenótipo imunológico, definindo assim 8 principais subtipos de leucemia baseados no tipo de linhagem e no grau de diferenciação dos blastos (Head et al., 1985). A acidose láctica é definida por níveis elevados de lactato e está geralmente associada a exercício físico e respiração de baixas taxas de oxigénio mas também tem sido descrita em doenças e cancros, especialmente malignâncias hematológicas como leucemias e linfomas (Friedenberg et al. 2007). Em tumores os elevados níveis de lactato derivados da glicólise e que rapidamente aumentam o nível intracelular de lactato e que são exportados para a corrente sanguínea são a causa da acidose láctica. A acidose láctica em malignâncias hematológicas está associada a um prognóstico da doença extremamente mau, tendo sido recentemente descrito um caso de morte fulminante por acidose láctica (Sillos et al., 2001; Terpe et al., 2012) O factor de crescimento VEGF é descrito como tendo um papel na função celular de tumores (proliferação, sobrevivência, migração e invasão) e no homing de progenitores da medula óssea (Ellis and Hicklin, 2008). Em células HL60 foi demonstrado que o loop autócrino interno e externo do VEGF com um dos seus receptores (VEGFR2) está associado à regulação da sobrevivência em leucemia (Santos et al., 2004). Este trabalho visa elucidar o papel do metabolismo do lactato em leucemia mielóide aguda pelo que foram propostos alguns objectivos específicos, como a avaliação da modelação da expressão de MCT1, MCT4, LDHA e LDHB pelo lactato, a avaliação da incorporação de carbonos do lactato em outros compostos e a avaliação do papel do VEGF na expressão de MCT1, MCT4, LDHA e LDHB. Concluímos que os níveis elevados de MCT1 detectados à altura do diagnóstico de pacientes podem estar relacionados com os níveis de blastos leucémicos. Os ensaios com a linha celular de leucemia mielóide aguda HL60 revelaram que os níveis de expressão de proteína de MCT1 e LDHA parecem ser modulados pelo lactato, enquanto os níveis de expressão proteica de MCT4 parecem a estar a ser também modulados pelo VEGF. Os nossos resultados sugerem a existência de um loop funcional de influxo/efluxo de NaLac mediado pelo MCT1 e MCT4, sendo mantido devido à acção catalítica da LDHA e LDHB. A exposição crónica das células HL60 a lactato parece conferir às células capacidade de adaptação ao ambiente mais ácido, através do aumento da expressão de MCT1 e LDHA. A análise do ensaio de NMR revelou que os carbonos do lactato-C13 marcado estão a ser incorporados em diversos constituintes como amino ácidos e acetato. Assim, como conclusão final do trabalho propomos o MCT1 e/ou MCT4 como possíveis alvos terapêuticos uma vez que os resultados obtidos sugerem fazerem parte de um loop funcional de influxo/efluxo de lactato que pode estar envolvido na manutenção da acidose láctica intracelular.
Tumour cells opt for alternative pathways for ATP production, a pathway generally used in hypoxia but that tumour cells use even in the presence of oxygen. This metabolic switch is known as the Warburg effect. The Warburg effect has been described as having a protective role for tumour cells by increasing acidosis which is associated with higher levels of invasion and metastasis. Lactate (NaLac) has been described as a signalling molecule and its role in tumour metabolism has recently been the object of several studies. Monocarboxylate transporters are membrane transporters of monocarboxylates, such as lactate and pyruvate, both of which are implicated in the Warburg effect. They have a major role in NaLac transport, namely of MCT1 and MCT4, which transport NaLac into and from the cells,, also regulating the pH in the cells and serving as intercellular communicators. Lactate dehydrogenases are enzymes responsible for the reactions occurring between pyruvate and lactate, where LDHA catalyses the reaction of pyruvate to NaLac and LDHB the opposite reaction. LDHs are thereby essential in sustaining glycolysis. Lactic acidosis has been linked to haematological malignancies such as leukaemias, where it is caused by the intracellular increase in NaLac due to glycolysis. This work aims to elucidate the role of NaLac metabolism in acute myeloid leukaemia. As such we evaluated the modulation of MCT1, MCT4, LDHA and LDHB expression by NaLac, but also the incorporation of carbons from lactate in other compounds and evaluated the role of VEGF in MCT1, MCT4, LDHA and LDHB expression. Overall, NaLac and VEGF modulate MCT1, MCT4, LDHA and LDHB expression at least at the protein synthesis level. A functional loop of NaLac influx/efflux mediated by MCT1 and MCT4 that is also maintained due to the catalytic action of LDHA and LDHB is suggested by our results. In our model, the role of NaLac as a carbon source was shown. Nevertheless, the role of NaLac as a signalling molecule should be addressed in future studies. We conclude that MCT1 and/or MCT4 may be a suitable therapeutic approach as our results suggest they are part of NaLac functional influx/efflux loop that could be involved in maintaining intracellular lactic acidosis.

Dissertação de mestrado em Genética Molecular
Colorectal cancer (CRC) is one of most commonly diagnosed cancer worldwide. A normal human intestine harbours hundreds of different bacterial species which play several roles in human health, such as protection against pathogens, immune system maturation, degradation of toxic substances, digestion of complex carbohydrates and production of short-chain fatty acids (SCFAs). SCFAs, specifically acetate, propionate and butyrate are produced by propionibacteria and constitute a major source of energy for colonocytes. Previous reports from our group showed that acetate inhibits CRC cell proliferation, induces apoptosis, promotes lysosomal membrane permeabilization, increases CRC cell glycolytic phenotype and regulate its own uptake by increasing the expression of monocarboxylate transporters (MCTs). However, the signalling pathways associated to the phenotypic changes induced by acetate have not been characterized. In order to clarify this issue, here we aimed at evaluate the involvement of acetate in the expression levels of KRAS/BRAF oncogene signalling pathways molecules known to be important in CRC cells survival namely PI3K/AKT and MAPK pathways. We also aimed at understanding the role of KRAS and BRAF oncogenes in the regulation of glycolytic metabolism and uncover the role of MCTs in the regulation of the KRAS/BRAF signalling pathways in CRC cells exposed to acetate. Our data suggest that acetate treatment is able to modulate the expression levels of some signalling molecules namely phosphorylated cRAF and ERK, in a time and dose-dependent manner. Moreover, preliminary results herein presented show that acetate may activate some feedback mechanism to maintain the uptake of SCFAs when there is downregulation of the MCT-1 expression levels. To the best of our knowledge this is the first work studying the interplay between acetate and two important hallmarks of cancer, namely oncogene signalling activation and metabolism reprogramming in CRC. This study might help in the discovery of new approaches in prevention/therapy of CRC.
O cancro colorretal (CCR) é um dos tipos de cancro mais comumente diagnosticado em todo o mundo. Um intestino humano normal abriga centenas de espécies bacterianas diferentes que desempenham diversos papéis na saúde humana, como proteção contra agentes patogénicos, maturação do sistema imunológico, degradação de substâncias tóxicas, digestão de carbohidratos complexos e produção de ácidos gordos de cadeia curta (AGCC). Os AGCC, especificamente o acetato, propionato e butirato são produzidos pela propionibacteria e constituem uma importante fonte de energia para os colonócitos. Recentemente, foi demonstrado pelo nosso grupo que o acetato inibe a proliferação de células de CCR, induz apoptose e promove a permeabilização da membrana lisossomal, aumenta o fenótipo glicolítico destas células e regula a sua própria absorção aumentando a expressão de transportadores de monocarboxilatos (MCTs). No entanto, as vias de sinalização associadas às alterações fenotípicas induzidas pelo acetato não foram ainda devidamente caracterizadas. Para clarificar esta questão, nesta tese avaliámos o envolvimento do acetato nos níveis de expressão de moléculas das vias de sinalização dos oncogenes KRAS/BRAF, importantes na sobrevivência das células de CCR, nomeadamente as vias da PI3K/AKT e das MAPK. De seguida, tentamos compreender o papel dos oncogenes KRAS e BRAF na regulação do metabolismo glicolítico e entender o papel dos MCTs na regulação das mesmas vias de sinalização em células de CCR expostas ao acetato. Os nossos resultados sugerem que o tratamento com acetato é capaz de modular os níveis de expressão de algumas moléculas de sinalização, nomeadamente a cRAF e a ERK fosforilada, de uma maneira dependente da dose e do tempo. Além disso, os resultados preliminares aqui apresentados demonstram que o acetato poderá ativar algum mecanismo de feedback que mantenha a entrada de AGCC quando existe uma redução dos níveis de expressão de MCT-1. Este constitui o primeiro trabalho no qual se estuda a interação entre o acetato e duas importantes características do cancro, nomeadamente a ativação da via de sinalização oncogénica e a reprogramação do metabolismo energético no CCR. Assim, este estudo poderá ajudar na descoberta de novas abordagens na prevenção/terapia do CCR.