Dissertationen zum Thema „Pyruvate mitochondrial“

Biomedical Sciences
Ph.D.
Mitochondria shape cytosolic Ca2+ (cCa2+) transients. Ca2+ entry into the mitochondria is driven by the highly negative mitochondrial membrane potential and through a highly selective channel, the Mitochondrial Calcium Uniporter (MCU). Mitochondrial Ca2+ (mCa2+) is utilized by the matrix dehydrogenases for maintaining cellular bioenergetics. The TCA cycle-derived NADH and FADH2 are mCa2+ dependent thus, feed into the electron transport chain (ETC) to generate ATP. Either loss of mCa2+ or metabolite uptake by the mitochondria results in a bioenergetic crisis and mitochondrial dysfunction. Reciprocally, sudden elevation of cCa2+ under conditions of stroke or ischemia/reperfusion injury (I/R) drives excessive mCa2+ overload that in turn leads to the opening of a large channel, the mitochondrial permeability transition pore (PTP) that triggers necrotic cell death. Thus, Ca2+ and metabolite equilibrium is essential to maintain a healthy mitochondrial pool. Our laboratory has previously showed that loss of mCa2+ uptake leads to decreased ATP generation and cell survival through autophagy. Although metabolite scarcity also results in similar reduction in ATP generation, the molecular mechanisms by which metabolites control mitochondrial ion homeostasis remain elusive. Deprivation of glucose or supplementation of mitochondrial pyruvate carrier (MPC) transport blocker UK5099 and or carnitine-dependent fatty acid blocker etomoxir triggered an increase in the expression of MICU1, a regulator of the mitochondrial calcium uniporter (MCU) but not the MCU core subunit. Consistently, either RNAi-mediated deletion of MPC isoforms or dominant negative human mutant MPC1 R97W showed significant induction of MICU1 protein abundance and inhibition of MCU-mediated mCa2+ uptake. Moreover, TCA cycle substrate-dependent MICU1 expression is under the control of EGR1 transcriptional regulation. Reciprocally, the MICU1 dependent inhibition of mCa2+ uptake exhibited lower NADH production and oxygen consumption and ATP production. The reduction of mitochondrial pyruvate by MPC knockdown is linked to higher production of mitochondrial ROS and elevated autophagy markers. These studies reveal an unexpected regulation of MCU-mediated mCa2+ flux machinery involving major TCA cycle substrate availability and possibly MICU1 to control cellular switch between glycolysis and oxidative phosphorylation. While mCa2+ is required for energy generation, sustained elevation of mCa2+ results in mitochondrial swelling and necrotic death. Hence, it was thought that preventing mCa2+ overload can be protective under conditions of elevated cCa2+. Contrary to this, mice knocked-out for MCU, that demonstrated no mCa2+ uptake and hence no mitochondrial swelling, however failed protect cells from I/R- mediated cell death. MCU-/- animals showed a similar infarct size comparable to that of control animals, suggesting that prevention of MCU-mediated mCa2+ overload alone is not sufficient to protect cells from Ca2+ -induced necrosis. The absence of mCa2+ entry revealed an elevation in the upstream cCa2+ transients in hepatocytes from MCUDHEP. Ultra-structural analysis of liver sections from MCU-/- (MCUDHEP) and MCUfl/fl animals revealed stark contrast in the shape of mitochondria: MCUfl/fl liver sections showed long and filamentous mitochondria (spaghetti-like) while MCUDHEP mitochondria were short and circular (donut-like). Furthermore, challenging MCUfl/fl and MCUDHEP hepatocytes with ionomycin caused a marked increase in cCa2+ and a simultaneous change in mitochondrial shape (from spaghetti to donut), a phenomenon we termed mitochondrial shape transition (MiST) that was independent of mitochondrial swelling. The cCa2+-mediated MiST is induced by an evolutionarily conserved mitochondrial surface EF-hand domain containing Miro1. Glutamate and Ca2+ -stress driven cCa2+ mobilization cause MiST in neurons that is suppressed by expression of Miro1 EF1 mutants. Miro1-dependent MiST is essential for autophagosome formation that is attenuated in cells harboring Miro1 EF1 mutants. Remarkably, loss of cCa2+ sensitization by Miro1 prevented MiST and mitigated autophagy. These results demonstrate that an interplay of ions and metabolites function in concert to regulate mitochondrial shape that in turn dictates the diverse mitochondrial processes from ATP generation to determining mechanisms of cell death.
Temple University--Theses

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix, thereby linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient c.C289T and c.T236A MPC1 alleles disrupt MPC function. MPC1 c.C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and full-length point mutant (R97W). MPC1 c.T236A encodes a full-length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from human patient MPC1 mutations and inform fundamental MPC structure-function relationships. These results also demonstrate an efficient molecular genetic system using the mouse C2C12 cell line to mechanistically investigate human inborn errors in pyruvate metabolism.

Le pyruvate est rapidement decarboxyle par les mitochondries des tumeurs d'ehrlich pour former de l'acetoine qui, si ajoutee aux mitochondries, est rapidement utilisee pour former de petites quantites d'ethanol et de citrate. Elle a aussi ete detectee dans les mitochondries as 30-d mais pas dans celles du foie de rat controle. Elle resulte de la condensation d'acetaldehyde active et d'acetaldehyde par le complexe pyruvate deshydrogenase tumoral (pdh). Elle controle le metabolisme du pyruvate par l'inhibition competitive du complexe pdh, ainsi que l'oxydation du succinate qu'elle inhibe. La forte accumulation de cholesterol dans les membranes mitochondriales entraine une diminution de la fuite passive des protons. Cette derniere, avec la presence insolite d'isozymes de la creatine kinase et la presence d'une hexokinase liee a la membrane externe de la mitochondrie alimentant la glycolyse a partir d'atp mitochondrial, contribuent a distribuer efficacement les molecules energetiques pour les besoins cellulaires comme la division. Une anomalie de la restriction de l'adn mitochondrial as 30-d accompagne ce metabolisme deviant: un des deux sites de restriction par l'enzyme xba i a disparu. Les molecules normales et mutees coexistent avec une heteroplasmie d'environ 50%

Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Metabolic cycles are a fundamental element of cellular and organismal function. Among the most critical in higher organisms is the Cori Cycle, the systemic cycling between lactate and glucose. Here, skeletal muscle-specific Mitochondrial Pyruvate Carrier (MPC) deletion in mice increased muscle glucose uptake but diverted pyruvate into the circulation as lactate, driving increased Cori Cycling and energy expenditure. Loss of muscle MPC activity evoked adaptive glutaminolysis, increased fatty acid oxidation, and resulted in a striking resistance to gains in fat mass with age with perfect sparing of muscle mass and strength. Furthermore, chronic and acute muscle MPC deletion accelerated fat mass loss on a normal diet after high fat diet-induced obesity. Our results illuminate the role of the skeletal muscle MPC as a central node for whole-body carbohydrate, fat, and amino acid metabolism. They highlight the potential utility of decreasing muscle pyruvate oxidation to ameliorate obesity and type 2 diabetes.

This thesis will be organized into two chapters discussing the role of hypoxia in the human placenta. The goal of this thesis is to characterize pyruvate kinase M2, mammalian target of rapamycin, mitochondrial function, and cell invasion in hypoxic conditions in the trophoblast. Understanding the mechanisms of placental metabolism can lead to further treatments for placental diseases. Chapter one covers the background of intrauterine growth restriction, hypoxia, placental metabolism, and pyruvate kinase M2 (PKM2). Little is currently understood about the role of the mitochondria in placental diseases. Expression of PKM2, trophoblast cell invasion, and mitochondrial function is shown to be inhibited by hypoxia. PKM2 inhibition decreases trophoblast cell invasion and nuclear expression of PKM2, but increases mitochondrial function. Studying how hypoxia affects the placenta during placental diseases can help clarify the mechanisms by which these diseases occur. Chapter two further characterizes the background of intrauterine growth restriction and hypoxia. It also covers the background of mammalian target of rapamycin. The objective of this chapter was to assess activated mTOR in the trophoblast in hypoxia. Decreased placental and fetal weights, as well as trophoblast cell invasion were observed in hypoxia. A decrease in the activation of mTOR was also found in the hypoxic placenta. This study could provide insight into the physiological relevance of the pathways and could be targeted to help alleviate placental diseases.

Le cancer du sein triple négatif (TNBC) est un sous-type particulièrement agressif du cancer du sein, traité principalement par chimiothérapie. Cependant, environ 50% des patients connaissent une rechute avec métastases dans les 3 à 5 ans suivant le traitement. Afin de mieux comprendre l'évasion post-chimiothérapie et la formation de métastases des cellules cancéreuses TNBC, nous avons établi des modèles de cellules TNBC en traitant les cellules SUM159-PT et MDA-MB-231 avec de l'épirubicine, du cyclophosphamide et du paclitaxel, simulant des protocoles cliniques. Nous nous sommes initialement concentrés sur l'adaptation mitochondriale de ces cellules persistantes. Les cellules MDA-MB-231 ont montré une sensibilité réduite à la chimiothérapie, associée à une phosphorylation oxydative accrue et à des intermédiaires du cycle de l'acide tricarboxylique modifiés. En revanche, les cellules SUM159-PT ont conservé leur sensibilité. Le ciblage du métabolisme mitochondrial du pyruvate avec le UK-5099 a resensibilisé les cellules persistantes MDA-MB-231 aux agents thérapeutiques. Les cellules persistantes ont montré une migration, une invasion et une survie accrues en culture en suspension, les cellules SUM159-PT présentant une adhésion accrue aux cellules endothéliales. Des études de xénogreffe in vivo ont confirmé ces observations, mettant l'accent sur une croissance cellulaire accrue et une colonisation métastatique dans des organes vitaux, en particulier le cerveau. Le tropisme accru pour le cerveau pourrait s'expliquer par le fait que les cellules TNBC persistantes présentaient une capacité accrue de traverser la barrière hémato-encéphalique, d'envahir le parenchyme cérébral et de croître dans une matrice 3D similaire au cerveau. Dans une deuxième phase de notre étude, nous avons étudié les mécanismes moléculaires facilitant la formation de métastases cérébrales de ces cellules persistantes. L'analyse protéomique a identifié des protéines surexprimées, notamment le COL1A1, fréquemment élevé chez les patients atteints de TNBC. Une augmentation de COL1A1 était corrélée à un mauvais pronostic et à une augmentation de la formation de métastase. L'inhibition de COL1A1 a réduit le potentiel métastatique à la fois in vitro et in vivo, soulignant son potentiel en tant que cible thérapeutique pour prévenir les métastases cérébrales après un traitement par chimiothérapie.L'ensemble de ces résultats offre un aperçu des mécanismes d'adaptation mis en place par les cellules cancéreuses en réponse à la chimiothérapie, et suggère que cibler le métabolisme du pyruvate mitochondrial pourrait contribuer à surmonter les adaptations mitochondriales des cellules de cancer du sein triple négatif. De plus, nos résultats mettent en lumière la manière dont une chimiothérapie combinée et séquentielle peut accroître le potentiel métastatique des cellules TNBC, en particulier vers le cerveau. Nous avons identifié la protéine COL1A1 comme un élément clé favorisant les différentes étapes de formation des métastases cérébrales dans les cellules TNBC résistantes à la chimiothérapie. Des recherches complémentaires sont nécessaires pour élucider les mécanismes détaillés de la surexpression de COL1A1.En utilisant le même schéma thérapeutique, nous avons mis en œuvre un traitement court de 48h, combiné et séquentiel pour évaluer les modifications protéomique précoces dans les vésicules extracellulaires libérées par les cellules TNBC persistantes. Avec cette approche, on a également exploré l'impact de la chimiothérapie sur les facteurs angiocrines des cellules endothéliales, suggérant le rôle du sécrétome induit par la chimiothérapie dans la facilitation des métastases post-chimiothérapie. Bien que ce volet de notre étude en soit à un stade préliminaire, les résultats encouragent à approfondir davantage l'investigation expérimentale
Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype, primarily treated with chemotherapy. However, approximately 50% of patients experience relapse with metastasis within 3 to 5 years post-treatment. To gain insight into the post-chemotherapy escape and metastasis formation of TNBC cancer cells, we established TNBC cell models by treating SUM159-PT and MDA-MB-231 cells with epirubicin, cyclophosphamide, and paclitaxel. simulating clinical protocols. We initially focused on the mitochondrial adaptation of these chemo-persistent cells. MDA-MB-231 cells showed reduced chemosensitivity, associated with increased oxidative phosphorylation and altered tricarboxylic acid cycle intermediates. In contrast, SUM159-PT cells retained sensitivity. Targeting mitochondrial pyruvate metabolism with UK-5099 re-sensitized persistent cells to therapeutic agents, suggesting a potential strategy to overcome mitochondrial adaptation. Persistent cells exhibited increased migration, invasion, survival in suspension culture, with SUM159-PT cells displaying increased adhesion to endothelial cells. In vivo xenograft studies confirmed these observations, emphasizing increased cell growth and metastatic colonization in vital organs, particularly the brain. The enhanced trophism for brain could be explained by the fact that persistent TNBC cells exhibited increased abilities to transmigrate through BBB, to invade the brain parenchyma and to grow in a brain-like 3D matrix. In a second phase of our study, we investigated the molecular mechanisms facilitating brain metastasis of these persistent cells. proteomic analysis identified upregulated proteins, notably COL1A1, frequently elevated in TNBC patients. Increased COL1A1 correlated with poor prognosis and enhanced metastasis. Inhibition of COL1A1 reduced metastatic potential both in vitro and in vivo, highlighting its potential as a therapeutic target in preventing brain metastasis post chemotherapy treatment.Collectively, these findings provide insight into the adaptive mechanisms employed by cancer cells in response to chemotherapy, and suggest that targeting mitochondrial pyruvate metabolism may help to overcome the mitochondrial adaptations in TNBC cells. Furthermore, our data illuminate how combined and sequential chemotherapy may increase the metastatic potential of TNBC cells, particularly towards the brain. We have pinpointed COL1A1 as a key factor promoting various stages of brain metastasis formation in chemotherapy-resistant TNBC cells. Additional research is required to elucidate the detailed mechanisms behind COL1A1 overexpression.Using the identical drug regimen, we implemented a short, combined, and sequential treatment to replicate initial proteomic alterations in extracellular vesicles released by persistent TNBC cells. This approach also explored the impact of chemotherapy on angiocrine factors from endothelial cells, suggesting the role of the chemo-induced secretome in evading treatment and facilitating metastasis post-chemotherapy. Although this aspect of our study is currently in its early phases, the findings underscore the necessity for further experimental validation

Eurycoma longifolia (Tongkat Ali
TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report 
regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods This study 
encompasses two parts (part 1: on spermatozoa
part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups, 
washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 &mu
g/ml) for 1 hour at 37°
C. A sample without addition of TA served as control. After incubation with TA, 
the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen 
species (ROS
dihydroethidium test
DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (&Delta
&psi
m) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells 
incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 &mu
g/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were 
evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant 
dose-dependent trends were found 
for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 &mu
g/ml, yet, no significance could be 
observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor &Delta
&psi
m.

L'objectif de ce travail est d'explorer en détail le métabolisme de lignées astrocytaires dérivant de la moelle épinière d'embryons de rat. La respiration mitochondriale, certains paramètres de la glycolyse et l'activité de l'oncogène Akt sont étudiés dans les lignées en passages précoces (EP) et dans les lignées en passages tardifs (LP), qui se sont spontanément transformées après avoir été maintenues pendant plus de 35 passages dans le milieu de culture ne contenant pas de pyruvate. Dans les LP, en comparaison avec les EP, il existe une diminution de la glycolyse, une réduction du nombre de mitochondries par cellule et un déficit de la respiration portant sur les complexes I et II+III de la chaîne respiratoire mitochondriale. Le traitement des LP par le pyruvate, pendant 20 passages supplémentaires, ne modifie pas l'état de transformation des cellules. Alors que ce traitement rétablit la glycolyse, aucun effet bénéfique n'est constaté sur le déficit respiratoire. Le traitement des EP par du pyruvate, jusqu'à ce qu'elles soient par définition en passages tardifs, prévient la transformation spontanée. Alors que les EP traitées par le pyruvate ont un déficit modéré de la respiration, elles renferment 2 fois plus de mitochondries par cellule que les EP non traitées. L'augmentation du nombre de mitochondries compense le déficit modéré de la respiration afin de maintenir les capacités d'oxydation dans ces lignées tardives qui ne sont pas transformées. Dans l'ensemble des lignées, l'activité de l'oncogène Akt varie dans le même sens que la glycolyse et que la respiration et est directement stimulée par le pyruvate dans les lignées traitées. En conclusion, ce travail ouvre de nouvelles perspectives quant à la compréhension des mécanismes possiblement associés à la transformation astrocytaire et met en évidence de nouvelles voies thérapeutiques potentielles afin d'améliorer le contrôle des proliférations astrocytaires.

Le cancer de la plèvre est grave car très résistant à la chimiothérapie. Nous avons testé un nouveau traitement basé sur la glycolyse particulière des cellules cancéreuses aboutissant à la formation d’acide lactique, fait décrit par Otto Warburg, deux fois prix Nobel, qui considérait ce phénomène du à un défaut de la respiration cellulaire comme étant à l’origine du cancer. Nous avons étudié l’effet du 2-Deoxyglucose, du 3-Bromopyruvate et du Citrate, sur 2 lignées cellulaires de mésothéliome humain en culture, puis sur un modèle de carcinose péritonéale obtenu chez la souris nude. Les deux lignées ont réagi d’une façon presque inversée à ces composés, soit en ralentissant leur prolifération, soit en mourant par un mécanisme apoptotique (voie mitochondriale) ou par empoisonnement nécrotique. In vivo, le 3-BrPA et le citrate ont entraîné un gain de survie très significatif (p<0. 0001), le cisplatine, drogue de référence, n’ayant pas d’effet. Nous avons tenté de mieux comprendre la biochimie particulière de la cellule cancéreuse et de sa mitochondrie, et de commencer à la croiser avec les mécanismes de résistance à l’apoptose. En définitive, la cellule cancéreuse et la tumeur sont de véritables parasites pour l’organisme, détournant l’énergie pour proliférer. Par le biais de stratégies anti-énergétiques nouvelles, il semble donc possible de ralentir ou stopper la prolifération tumorale et peut-être d’aider à rendre efficace d’autres stratégies (siRNA anti-famille Bcl-2 ou dirigés contre des enzymes clés ou limitants de la glycolyse ou de la lipolyse…), ou d’induire la mort par apoptose ou par empoisonnement, tandis que des thérapies de « support » peuvent être imaginées pour aider l’organisme à mieux lutter contre le cancer-parasite. Nous concluons par un schéma explicatif du métabolisme et l’amorce d’une nouvelle théorie de la cancérisation, dans laquelle comme le pensait Warburg, le défaut de la respiration cellulaire pourrait bien être la cause originelle du cancer, se situant en amont des altérations géniques
Pleural cancer is a poor prognosis disease because it is resistant to chemotherapy. We tested a new anti-cancer approach based on the fact that cancer cells have an impaired metabolism of the glucose, leading to the secretion of lactic acid, as first described by Otto Warburg, two times honoured by Nobel price, considered this fact as the origin of cancer. We studied the effect of 2-Deoxyglucose, of 3-Bromopyruvate and of citrate, in vitro on 2 human mesothelioma cell lines, and in nude mice developing peritoneal carcinomatosis. The two cell lines reacted in a nearly opposite way to these molecules, either by a slow-down of the proliferation, or by cell death via the mitochondrial apoptosis pathway, or by a poisoning-necrosis death. In vivo, 3-BrPA and citrate increased the survival very significantly (p<0. 0001), whereas cisplatin, the current reference drogue has no effect. We tried to understand the particular biochemistry of the transformed cell and its mitochondria, and begun to decipher the mechanisms of resistance to apoptosis. Cancer cells steal the energy of the organism for its own profit and proliferation, whereas the organism is unable to survive. Using novel anti-energetic strategies, we think it is nowadays possible, either to slow down the proliferation and to facilitate action of other strategies (siRNA directed against members of the Bcl-2 family or against key or regulators enzymes of the glycolysis or lipolysis…), or to provoke death either by the apoptotic pathway or by necrosis-poisoning mechanisms. Supportive energetic strategies could be developed also to improve the general condition of patients, and the fight against cancer disease. We conclude by presenting an explicative schema of the metabolism of cancer cell, and by suggesting a new theory of the origin of cancer, that like Warburg thought, could be due to a defect of the cell mitochondrial respiration, an event occurring before the multiple genetics abnormalities described afterwards

1. La d-3-hydroxybutyrate deshydrogenase (bdh) est une enzyme specifiquement activable par la phosphatidylcholine (pc). Par western blot, dot blot, immunoprecipitation, test elisa en competition, et coupures proteolytiques, nous avons teste la specificite de 7 anticorps monoclonaux (mac) pour la bdh, la reactivite croisee des mac pour l'enzyme des 2 especes rat et buf; nous avons determine le type d'epitope (sequentiel ou conformationnel) et localise certains dans la sequence primaire (dont un a l'extremite c-terminale), et par rapport a la surface ou a l'interieur de la proteine native. Nous avons montre que pc dans l'environnement phospholipidique de la bdh modifie le profil proteolytique de l'enzyme et notamment protege l'extremite c-terminale. Celle-ci semble etre indispensable a l'insertion de l'apoenzyme dans la bicouche mais non indispensable a la stabilite du complexe bdh-phospholipides preforme avant coupure. 2. Nous avons identifie, par western blot sur 34 serums, les differents antigenes mitochondriaux de la cirrhose biliaire primitive, maladie autoimmune caracterisee par des auto-anticorps anti-mitochondries. La reactivite des ac auto-immuns sur les sous-unites du complexe multienzymatique pyruvate deshydrogenase purifie a montre que 3 de ses sous-unites sont antigenes. Aucune correlation n'a pu etre trouvee entre les differents profils reconnus par les serums et le sexe des patients, les classes et les sous-classes des ac, l'existence ou non d'un traitement particulier, la greffe d'un foie sain

Indiana University-Purdue University Indianapolis (IUPUI)
The goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.

碩士
國立陽明大學
生化暨分子生物研究所
103
Sirtuins catalyze the deacylation of lysine residues of target proteins and regulate a wide range of cellular functions, mitochondrial biogenesis and energetic metabolism. Recent studies showed that the mitochondrial sirtuins, Sirt3 and Sirt5, could regulate the activities of key metabolic enzymes of intermediary metabolism. Deacetylation catalyzed by Sirt3 has been reported to be involved in the regulation of β-oxidation of fatty acids, TCA cycle, and urea cycle. In previous studies, we demonstrated that an increase of ROS elicited by pathogenic mitochondrial DNA (mtDNA) mutations is involved in the impairment of bioenergetic function and other cellular function in patients with mitochondrial disease. We showed that mitochondrial dysfunction could down-regulate the expression of sirtuins. In this study, human cybrids harboring A8344G mutated mtDNA (MERRF cybrids) were used as a cell model to explore the role of sirtuins-mediated protein acetylation in mitochondrial disease. The results showed a decrease of Sirt3 expression and increase in the levels of protein acetylation in MERRF cybrids. A large number of acylated lysine residues were identified in metabolic enzymes, especially the pyruvate dehydrogenase complex (PDHC), which had been reported to be functionally regulated by PTM. Recently, acetylation of PDHC was shown to inhibit its enzymatic activity by enhancing pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation. The enzyme activity levels of PDHC in the MERRF cybrids were found to be significantly lower than that of the wild-type cybrid. Moreover, MERRF cybrids had higher levels of phosphorylated PDHC despite decreased level of PDK1 phosphorylation. I used immunoprecipitation to capture acetyl-lysine and found that the acetylation of PDHC was increased in mutant cybrids compared with the wild-type cybrid. By using an antibody to pull down the PDHC, we found that the PDHE1α and pyruvate dehydrogenase phosphatase 1 (PDP1) interact with Sirt3 in MERRF cybrids. I found that the PDH activity was increased in mutant cybrids overexpressing Sirt3 relative to control. I also discovered that the levels of oxygen consumption rate and mitochondrial membrane potential were increased in cybrids overexpressing Sirt3. These results suggest that impairment of Sirt3-mediated lysine deacetylation may lead to the decrease of PDH activity and thus compromise oxidative metabolism in human cells with A8344G mutation of mtDNA. Taken together, these observations have provided new information for a better understanding of how protein modifications can modulate the PDH activity and mitochondrial function in human cells. Such findings can help us understand the function and targets of mitochondrial sirtuins, and advance the development of novel strategies to treat mitochondrial diseases caused by mtDNA mutations.

Dissertação de mestrado em Biologia Celular e Molecular apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
A doença de Huntington (HD, do inglês „Huntington’s disease’) é uma doença neurodegenerativa autossómica dominante, caracterizada por perda selectiva dos neurónios estriatais. É marcada por movimentos corporais involuntários (coreia), discurso “arrastado”, dificuldades em engolir, distúrbios emocionais e demência. A HD é causada por uma mutação (expansão de CAGs) no gene IT15 (ou gene HD), que codifica para a proteína huntingtina (Htt). Nos doentes de HD, a Htt mutante (mHtt) apresenta um elevado número de poliglutaminas no seu terminal amínico. De entre vários mecanismos celulares, a mHtt interfere com o sistema ubiquitina-proteassoma, mecanismos apoptóticos, autofagia, transcrição e homeostase mitocondrial. Recentemente foi demonstrado pelo nosso grupo que a actividade e expressão da enzima piruvato desidrogenase (PDH) estão diminuídas em cíbridos citoplasmáticos de HD, resultando em alterações bioenergéticas nessas células. Actualmente, não existe um tratamento para esta doença devastadora. No entanto, evidências anteriores sugerem que a insulina e o insulin-like growth factor-1 (IGF-1) podem actuar como potenciais agentes protectores na HD. Assim, colocámos a hipótese de que ambos os factores neurotróficos poderiam prevenir ou atenuar as alterações bioenergéticas após expressão da mHtt completa (full-length). Para além disso, os inibidores das desacetilases de histonas (iHDACs) podem também ser usados para reverter a disfunção associada à HD, melhorando o processo transcricional alterado após expressão de mHtt. Assim, neste trabalho utilizámos uma linha de células estriatais derivadas de ratinhos (murganhos) knock-in homozigóticos que expressam mHtt com 111 glutaminas (STHdhQ111/Q111) versus células estriatais obtidas de ratinhos wild-type (que expressam Htt com 7 glutaminas) tratadas com insulina, IGF-1, e os iHDACs tricostatina A (TSA), butirato de sódio (SB) e 4-fenilbutirato (PB). Por fim, de modo a estudar o papel da mitocôndria em células humanas que expressam mHtt produziram-se células rho-zero a partir de linfoblastos humanos.Os nossos resultados mostraram que, em comparação com células wild-type, as células estriatais STHdhQ111/Q111 apresentaram maior produção de espécies reactivas de oxigénio (ROS), maior fosforilação/inactivação da PDH e diminuição da actividade da PDH e dos níveis da sua subunidade E1alpha, o que poderá contribuir para a diminuição da viabilidade celular (por aumento da apoptose e necrose). Para além disso, as células STHdhQ111/Q111 exibiram algumas alterações bioenergéticas, incluindo a diminuição dos rácios ATP/ADP e lactato/piruvato, da actividade da enzima lactato desidrogenase (LDH) e um aumento dos níveis de fosfocreatina (PCr) e piruvato. Por outro lado, a exposição das células ao dicloroacetato, um activador indirecto da PDH, diminuiu a produção de ROS em células STHdhQ111/Q111. A pré-incubação com insulina e IGF-1 não afectou significativamente as alterações bioenergéticas acima referidas. Contudo, observámos um efeito promissor de IGF-1 (1 nM) no restabelecimento da actividade da LDH, assim como uma ligeira diminuição dos níveis de piruvato. Adicionalmente, mostrámos que os iHDACs podem ter efeitos protectores na HD, diminuindo a morte celular por apoptose nas células STHdhQ111/Q111 e, consequentemente, aumentando o número de células viáveis. Ainda, a exposição a SB (500 μM) diminuiu a fosforilação da PDH e aumentou os níveis da sua subunidade E1alpha nas células STHdhQ111/Q111. Por fim, produzimos células rho-zero após tratamento com baixas concentrações de brometo de etídio, durante 27 dias. Porém, o processo de criação de células rho-zero pareceu ser dependente do tipo de linha celular. Assim, serão necessários estudos futuros de forma a definir um protocolo exacto para a criação de células rho-zero a partir de linhas celulares linfoblastóides. A maior limitação parece ser a rapidez com que se observa a repopulação mitocondrial, o que poderá trazer algumas dificuldades à manutenção do estadio rho-zero. Em conclusão, os dados apresentados neste estudo descrevem alguns défices mitocondriais presentes nas células STHdhQ111/Q111, enfatizando a PDH como um importante alvo terapêutico. Adicionalmente, demonstrámos que, no modelo celular testado, os iHDACs (em particular o SB), podem prevenir estes défices de forma mais eficaz comparativamente aos efeitos mediados pela insulina ou IGF-1. Assim, os iHDAC representam uma estratégia terapêutica promissora na HD.
Huntington‟s disease (HD) is an autosomal dominant neurodegenerative disease, characterized by selective neuronal loss of striatal and cortical neurons. It is marked by involuntary body movements (e.g. chorea), slurred speech, difficulties in swallowing, emotional disturbances and dementia. HD is caused by a mutation (CAG expansion) in the IT15 (or HD) gene, which encodes the protein huntingtin (Htt). In HD patients, mutant Htt (mHtt) presents an increased number of polyglutamines in its N-terminal. Among other pathways, mHtt interferes with the ubiquitin-proteasome system (UPS), apoptotic mechanisms, autophagy, transcription and mitochondrial homeostasis. Recent studies from our group have shown that pyruvate dehydrogenase (PDH) activity and expression were decreased in HD cytoplasmic hybrids (cybrids), resulting in changes in mitochondrial bioenergetics in these cells. Currently, there is no effective treatment for this devastating disease. However, previous evidence suggests that insulin, insulin-like growth factor-1 (IGF-1) and also histone deacetylase inhibitors (iHDACs) may offer potential therapeutic protection against HD. Thus, the aim of this work was to analyze the levels of several energy metabolic parameters and to evaluate the protective effect of both IGF-1 and insulin in these parameters; and, also, to investigate the influence of iHDACs (trichostatin A (TSA), sodium butyrate (SB) and 4-phenylbutyrate (PB)), on PDH protein expression and regulation. For this purpose, we used striatal STHdhQ111/Q111 cell line derived from HD knock-in mice expressing mHtt with 111 glutamines versus striatal cells from wild-type mice (expressing Htt with 7 glutamines) treated with insulin, IGF-1 and the iHDACs. In addition, in order to study the role of mitochondrial dysfunction in HD, we produced rhozero cells from HD patients‟ and control lymphoblasts. Our results showed that, in comparison with wild type cells, striatal STHdhQ111/Q111 cells produced high levels of reactive oxygen species (ROS), increased PDH phosphorylation/inactivation, and decreased PDH activity and E1alpha protein levels, which could contribute for lower cell viability, as a result of increased apoptosis and necrosis. Also, STHdhQ111/Q111 cells showed a decrease in ATP/ADP ratio and lactate dehydrogenase (LDH) activity and increased phosphocreatine (PCr) levels. Moreover,pyruvate levels were increased in mutant cells whereas lactate levels were unchanged. Insulin (0.1 nM) and IGF-1 (1 nM) treatment significantly decreased PCr levels, whereas IGF-1 (1 nM) also decreased pyruvate levels and LDH activity in mutant cells, when compared to wild-type cells. Also, exposure to dicloroacetate, an indirect activator of PDH, decreased ROS production in STHdhQ111/Q111 cells. Additionally, we showed that iHDACs can have protective effects in HD, by decreasing cell death by apoptosis in STHdhQ111/Q111 cells, thereby increasing the number of viable cells. In the presence of SB (500 μM), PDH E1alpha subunit levels increased and PDH phosphorylation decreased in STHdhQ111/Q111 cells. Also, we were able to produce rho-zero lymphoblast cells using ethidium bromide, for 27 days. Nevertheless, the production of rho-zero cells seemed to be dependent on the cell line. Thus, further studies will be needed in order to define a precise protocol to create a rhozero status from lymphoblastoid cell lines. The main limitation seems to be a rapid mitochondrial repopulation, which presents some concerns to the maintenance of rho-zero lines. Data presented in this study describe some mitochondrial deficits present in HD striatal cells, emphasizing PDH as an interesting therapeutic target. Also, we show that, in this cell model, iHDACs (and particularly SB) more efficiently prevented the observed mitochondrial deficits compared to both insulin or IGF-1. Therefore, iHDACs represent a promising therapeutic strategy in HD.
Huntington‟s disease, pyruvate dehydrogenase, HDAC inhibitors, insulin, rhozero cells.

Tese de doutoramento em Ciências da Saúde, ramo Ciências Biomédicas apresentada à Faculdade de Medicina da Universidade de Coimbra
Huntington’s disease (HD) is a neurodegenerative disorder that gradually affects memory, cognitive skills and normal movements of the affected individuals and for which no disease modifying treatments exist. The disease is caused by an expanded CAG trinucleotide repeat (of variable length) in the HTT gene that encodes the huntingtin protein (HTT). Mutant HTT (mHTT) exhibits an abnormal elongated polyglutamine stretch that confers a toxic gain of function and predisposes the protein to fragmentation and aggregation, resulting in neuronal dysfunction and selective death in striatum and cortex. There is strong evidence that transcriptional deregulation and altered mitochondrial function occurs early and acts causally in HD pathogenesis; importantly, these events may be related to altered acetylation of proteins. Therefore, pharmacological strategies that interfere with both nuclear and mitochondrial protein acetylation may be therapeutically useful to hinder neuronal dysfunction in the course of HD pathology. In the present work, distinct lysine deacetylases (a heterogeneous group of proteins that remove acetyl groups from histones and other proteins regulating their structure and function), were pharmacologically or genetically targeted with the main purpose of counteracting mitochondrial and metabolic deficits in in vitro and in vivo models expressing human full-length mHTT. We have previously shown that mitochondrial dysfunction in HD persists at the level of pyruvate dehydrogenase (PDH), which functionally links glycolysis to oxidative phosphorylation. In Chapter 3 we report that decreased PDH activity in HD striatal cells expressing 111 glutamines (STHdhQ111/Q111) occurs through enhanced PDH kinases (PDKs) and reduced PDH phosphatase 1 protein expression, which trigger inhibitory phosphorylation of catalytic PDH E1α subunit in three different serine sites (Ser293, Ser300 and Ser232). Classes I and IIa histone deacetylase inhibitors (HDACi), sodium butyrate (SB) and phenylbutyrate, increased histone H3 acetylation and enhanced metabolism and mitochondrial respiration, similar to PDH activator dichloroacetate, suggesting that HDACi may favor activation of PDH complex. Concordantly, SB significantly decreased the expression of PDK2 and PDK3 in STHdhQ111/Q111 cells, which led to decreased PDH E1α phosphorylation in all serine sites. This effect occurs since HDACi SB stimulates HIF-1α degradation, a transcription factor that actively suppresses metabolism by directly transactivating genes encoding for PDKs. Therefore, partial depletion of PDK3 enhanced mitochondrial respiration and ATP synthesis in both wild-type and mutant STHdh cells, similarly to the effects achieved with SB treatment. YAC128 transgenic HD mice also exhibited decreased PDH E1α phosphorylation and PDK1-3 expression after SB treatment, which positively influenced central and peripheral metabolism. Further studies were conducted using modulators of class III lysine deacetylases, classically known as sirtuins. SIRT1, the best studied member of the sirtuins family, has been shown to exert neuroprotective roles in several models of neurodegeneration, boosting the interest in the development of SIRT1 activators. However, recent reports on SIRT1 inhibitors have also shown protective effects, casting doubt regarding previous findings. To better understand this paradox we tested resveratrol (RESV, a SIRT1 activator) and nicotinamide (NAM, a SIRT1 inhibitor) in in vitro models and in an animal model expressing mHTT (Chapter 4). We found that RESV enhanced lysine deacetylation activity and recovered abnormal mitochondrial phenotype of lymphoblasts from affected HD patients and cortical and striatal primary neurons isolated from YAC128 mice, which was associated with increased mitochondrial transcription and biogenesis. NAM had no effects on mitochondrial biogenesis, however increased NAD+ levels following NAM treatment may explain the positive impact on mitochondrial function in vitro. In symptomatic YAC128 mice, RESV completely abrogated deficits in motor learning and coordination, two well-established features of this transgenic HD model, and increased transcription of mitochondrial-encoded electron transport chain genes. In turn, NAM supplementation in vivo proved to be deleterious. The positive effects achieved with the SIRT-activating compound RESV led us to specifically target SIRT3 (Chapter 5), the major mitochondrial deacetylase that has received much attention for its role in oxidative metabolism and aging. We found increased SIRT3 levels and activity in HD lymphoblasts and in STHdhQ111/Q111 cells. Considering its potential neuroprotective role in HD, we overexpressed (OE) SIRT3, which was shown to co-localize more in mitochondria from HD striatal cells than in wild-type counterparts. Cortical tissue from YAC128 mice also exhibited lower acetylation of superoxide dismutase 2, a recognized target of SIRT3, suggesting increased SIRT3 activity. When OE in STHdh, SIRT3 enhanced lysine deacetylation and mitochondrial transmembrane potential. SIRT3 OE also reverted the loss in mitochondrial-related transcription factors, PGC-1α and TFAM, induced by mHTT. Still, increased mitochondrial content was not observed. Ultimately, STHdhQ111/Q111-SIRT3 OE cells showed lower susceptibility to apoptotic cell death. Overall, this study provides novel insights into the molecular deficits underlying mitochondrial dysfunction in HD and explores promising drugs that effectively modulate acetylation landscape, further impacting on mitochondrial and mitochondrial-related transcription proteins activity. Finally, increased mitochondrial activity and transcription partially control HD-related motor disturbances and neuronal death.
A Doença de Huntington (DH) é uma doença neurodegenerativa que afeta gradualmente as capacidade cognitivas e motoras dos indivíduos afetados, e para a qual não existe tratamento neuroprotetor ou cura. A DH é causada por uma expansão de trinucleótidos CAG (de tamanho variável) no gene HTT que codifica para a proteína huntingtina (HTT). A HTT mutante (mHTT) possui uma expansão anormal de poliglutaminas que lhe confere uma função tóxica, predispondo-a para a fragmentação e agregação, resultando em disfunção e morte seletiva de neurónios estriatais e corticais. Fortes evidências sugerem que alterações na regulação da transcrição e na função mitocondrial ocorrem em estádios iniciais da doença e são fatores causais para a patogénese da DH; estes eventos poderão estar relacionados com alterações na acetilação de proteínas. Assim, estratégias farmacológicas que interfiram com a acetilação de proteínas nucleares e mitocondriais poderão ser profícuas no combate à disfunção neuronal no decurso da DH. Neste trabalho diferentes desacetilases de lisinas (um grupo heterogéneo de proteínas que remove grupos acetil de histonas ou outras proteínas, regulando a sua estrutura e função) foram farmacologicamente ou geneticamente moduladas com o objetivo de neutralizar os défices mitocondriais e metabólicos em modelos que expressam a mHTT. Anteriormente o nosso grupo de investigação mostrou que a disfunção mitocondrial ocorre ao nível da piruvato desidrogenase (PDH), um complexo enzimático que faz a ligação entre a glicólise e a fosforilação oxidativa. No Capítulo 3 desta tese mostrámos que a diminuição da atividade da PDH em células estriatais que expressam 111 glutaminas (STHdhQ111/Q111) ocorre devido ao aumento das cinases da PDH (PDKs) e à diminuição da fosfatase 1 da PDH, desencadeando a fosforilação (inibitória) da subunidade catalítica PDH E1α em três resíduos de serina (Ser293, Ser300 e Ser232). Inibidores das classes I e IIa das desacetilases de histonas (HDACi), os compostos butirato de sódio (BS) e fenilbutirato, aumentaram a acetilação da histona H3 e melhoraram o metabolismo e a respiração mitocondrial, de forma semelhante ao observado com dicloroacetato, um reconhecido ativador da PDH, sugerindo que os HDACi poderão favorecer a atividade da PDH. Em concordância, o SB diminuiu a expressão da PDK2 e PDK3 nas células STHdhQ111/Q111, o que levou a uma diminuição da fosforilação da PDH E1α em todos os resíduos de serina. Este efeito mediado pelo SB parece ter resultado da estimulação da degradação do HIF-1α, um fator de transcrição que inibe o metabolismo celular através da transativação de genes que codificam para as PDKs. De forma semelhante ao SB, a redução parcial da expressão da PDK3 aumentou a respiração mitocondrial e a síntese de ATP em ambas as células STHdh wild-type e mutante. No murganho transgénico YAC128 verificou-se uma diminuição na fosforilação da PDH E1α e na expressão das PDK1-3 após tratamento com SB, influenciando positivamente o metabolismo energético. Nos estudos que se seguiram foram utilizados moduladores de desacetilases de lisinas de classe III, conhecidas como sirtuinas. A SIRT1, o membro da família mais estudado, tem vindo a mostrar um papel neuroprotetor em vários modelos de neurodegenerescência, aumentando o interesse no desenvolvimento de ativadores da SIRT1. No entanto, em estudos mais recentes os inibidores da SIRT1 também mostraram resultados protetores, colocando em dúvida as observações anteriores. Para compreender melhor este paradoxo, testámos o efeito do resveratrol (RESV, ativador da SIRT1) e da nicotinamida (NAM, inibidor da SIRT1) em modelos in vitro e num animal modelo que expressa a mHTT (Capítulo 4). O RESV aumentou a atividade desacetilase e reverteu a disfunção mitocondrial quando testado em linfoblastos de doentes de Huntington e em neurónios corticais e estriatais isolados de embriões do murganho YAC128; estas observações foram associadas ao aumento da transcrição e biogénese mitocondrial. O tratamento com NAM não teve qualquer efeito na biogénese mitocondrial; no entanto, o aumento dos níveis de NAD+ poderão justificar o impacto positivo observado na função mitocondrial testada in vitro. Quando administrado numa fase sintomática do murganho YAC128, o RESV anulou os défices de aprendizagem e coordenação motora, duas características deste animal transgénico, e aumentou a transcrição de genes da cadeia respiratória mitocondrial codificados pelo DNA mitocondrial. Por sua vez, a administração de NAM teve um efeito deletério in vivo. Os efeitos positivos obtidos com o ativador da SIRT1, resveratrol, levou-nos a modular especificamente a SIRT3 (Capítulo 5), uma desacetilase mitocondrial que tem recebido especial atenção pelo seu papel no metabolismo oxidativo e no processo de envelhecimento. Em linfoblastos DH e em células STHdhQ111/Q111 observámos um aumento dos níveis proteícos, de RNAm e da atividade da SIRT3. Considerando o potencial terapêutico da SIRT3 na DH, sobre-expressámos (SE) esta proteína em células estriatais e verificámos que se co-localiza preferencialmente nas mitocôndrias das células estriatais mutantes, comparativamente às células wild-type. O córtex do murganho YAC128 também exibiu menor acetilação da proteína superóxido dismutase 2, um alvo da SIRT3, sugerindo uma maior atividade desta sirtuína no contexto da DH. Quando SE em células STHdh, a SIRT3 aumentou a desacetilação e o potencial de membrana mitocondrial. A SE da SIRT3 também reverteu a diminuição dos fatores de transcrição associados à mitocôndria, PGC-1α e TFAM, induzida pela mHtt. Contudo, não se observou um aumento da massa mitocondrial. Por fim, as células STHdhQ111/Q111 que com SIRT3 SE apresentaram uma menor suscetibilidade à morte celular por apoptose. Em conclusão, este estudo fornece novas perspetivas sobre os défices moleculares subjacentes à disfunção mitocondrial na DH, e explora estratégias farmacológicas promissoras que modificam a acetilação e que têm posterior impacto na atividade da mitocôndria e nos fatores de transcrição associados a este organelo. Adicionalmente, o aumento da função e transcrição mitocondrial influenciam os distúrbios motores e a morte neuronal associada à DH. Palavras chave: Doença de Huntington, mitocôndria, piruvato desidrogenase, desacetilases de lisinas, neuroproteção.
Neuroscience prize 2013, supported by Santa Casa da Misericórdia de Lisboa

Dissertação de Mestrado em Saúde Ocupacional apresentada à Faculdade de Medicina
Os citostáticos usados no tratamento de neoplasias e doenças crónicas são identificados como perigosos para profissionais de saúde laboralmente expostos.Eles atuam a nível celular, mas existe pouca informação sobre o seu efeito na disfunção mitocondrial, que pode ser avaliada, entre outras, pela determinação de piruvato (AP) e lactato (AL) e rácio AL/AP. O objetivo do estudo era perceber se existem diferenças entre os valores de AL, AP e rácio AL/AP entre as trabalhadoras expostas a citostáticos e o grupo controlo.Realizou-se um estudo transversal exploratório onde se observou o posto de trabalho, aplicou a história clínica-laboral, exame físico e parâmetros analíticos incluídos AL e AP nas trabalhadoras expostas laboralmente a citostáticos: grupo 1(enfermeiras), grupo 2 (farmacêuticas) e grupo 3 (técnicas de farmácia) e o grupo controlo, da mesma instituição.Foram referidos alguns sintomas no grupo de exposição; 64% do grupo 1 não usa equipamentos de protecção individual adequados. Analiticamente observou-se que os valores de AP têm média superior nos grupos expostos e existem diferenças significativas nos valores de AL (F3, 31 = 5.184, p=0.005). Houve diferenças significativas nos valores de neutrófilos, %: F3,30 =3.203, p=0.037; nos basófilos, %: F3,30 = 5.156, p=0.005; nos basófilos em contagem: F3,30 = 5.156, p=0.005 e nos monócitos, em contagem: F3,30 =3.041, p=0.044.Os protocolos de boas praticas podem ser melhorados de acordo às recomendações internacionais. A vigilancia de saúde das trabalhadoras expostas poderia incluir os valores de AL e AP. Mais estudos podem ser úteis.Palavras-chave: citostáticos, ácido pirúvico, ácido láctico, disfunção mitocondrial.
Cytostatics drugs, used in cancer and chronic diseases treatment, have been recognized as hazardous to healthcare workers (HCWs) in occupational settings.They act on the cellular system but there is little information about mitochondrial dysfunction, which can be measured, among others, by pyruvate (AP), lactate (AL) and ratio AL/AP. The objective is understanding if there is a difference in pyruvate, lactate and ratio AL/AP values between cytostatic occupational exposure and nonexposure group.An exploratory transversal study was conducted with workplace observation, applied an occupational and clinical histories, medical examination and underwent clinical analyses included AL and AP profiles of cytostatic occupational exposure women workers: group 1: nurses; group 2: pharmacists and group 3: pharmacy technicians and a control group of the same health institution.Some symptoms have been reported by exposure group and 64% of group 1 did not use an adequate individual protection equipment (IPE).Statistically significant differences were found for AL profiles (F3, 31 = 5.184, p =0.005); neutrophils % (F3,30 =3.203, p=0.037), basophils % F3,30 = 5.156, p=0.005, basophils number F3,30 = 5.156, p=0.005 and monocytes % F3,30 =3.041, p=0.044.Good-practice protocols can be improved in terms of international recommendations. Lactate and Pyruvate values may be helpful indicators for health surveillance. Further studies could be useful.Key words: cytostatic, pyruvate; lactate; mitochondrial dysfunction.
Outro - A dissertação foi financiada pelo ICBAS – Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Departamento de Estudos de Populações pela Diretora do Laboratório de Ecotoxicologia e Ecologia (ECOTOX), a Professora Catedrática Lúcia Guilhermino. A oferta consistiu no fornecimento dos kits de piruvato e lactato necessários à realização do estudo.

Le diabète est une maladie métabolique qui se caractérise par une résistance à l’insuline des tissus périphériques et par une incapacité des cellules β pancréatiques à sécréter les niveaux d’insuline appropriés afin de compenser pour cette résistance. Pour mieux comprendre les mécanismes déficients dans les cellules β des patients diabétiques, il est nécessaire de comprendre et de définir les mécanismes impliqués dans le contrôle de la sécrétion d’insuline en réponse au glucose. Dans les cellules β pancréatiques, le métabolisme du glucose conduit à la production de facteurs de couplage métabolique, comme l’ATP, nécessaires à la régulation de l’exocytose des vésicules d’insuline. Le mécanisme par lequel la production de l’ATP par le métabolisme oxydatif du glucose déclenche l’exocytose des vésicules d’insuline est bien décrit dans la littérature. Cependant, il ne peut à lui seul réguler adéquatement la sécrétion d’insuline. Le malonyl-CoA et le NADPH sont deux autres facteurs de couplage métaboliques qui ont été suggérés afin de relier le métabolisme du glucose à la régulation de la sécrétion d’insuline. Les mécanismes impliqués demeurent cependant à être caractérisés. Le but de la présente thèse était de déterminer l’implication des navettes du pyruvate, découlant du métabolisme mitochondrial du glucose, dans la régulation de la sécrétion d’insuline. Dans les cellules β, les navettes du pyruvate découlent de la combinaison des processus d’anaplérose et de cataplérose et permettent la transduction des signaux métaboliques provenant du métabolisme du glucose. Dans une première étude, nous nous sommes intéressés au rôle de la navette pyruvate/citrate dans la régulation de la sécrétion d’insuline en réponse au glucose, puisque cette navette conduit à la production dans le cytoplasme de deux facteurs de couplage métabolique, soit le malonyl-CoA et le NADPH. De plus, la navette pyruvate/citrate favorise le flux métabolique à travers la glycolyse en réoxydation le NADH. Une étude effectuée précédemment dans notre laboratoire avait suggéré la présence de cette navette dans les cellules β pancréatique. Afin de tester notre hypothèse, nous avons ciblé trois étapes de cette navette dans la lignée cellulaire β pancréatique INS 832/13, soit la sortie du citrate de la mitochondrie et l’activité de l’ATP-citrate lyase (ACL) et l’enzyme malique (MEc), deux enzymes clés de la navette pyruvate/citrate. L’inhibition de chacune de ces étapes par l’utilisation d’un inhibiteur pharmacologique ou de la technologie des ARN interférant a corrélé avec une réduction significative de la sécrétion d’insuline en réponse au glucose. Les résultats obtenus suggèrent que la navette pyruvate/citrate joue un rôle critique dans la régulation de la sécrétion d’insuline en réponse au glucose. Parallèlement à notre étude, deux autres groupes de recherche ont suggéré que les navettes pyruvate/malate et pyruvate/isocitrate/α-cétoglutarate étaient aussi importantes pour la sécrétion d’insuline en réponse au glucose. Ainsi, trois navettes découlant du métabolisme mitochondrial du glucose pourraient être impliquées dans le contrôle de la sécrétion d’insuline. Le point commun de ces trois navettes est la production dans le cytoplasme du NADPH, un facteur de couplage métabolique possiblement très important pour la sécrétion d’insuline. Dans les navettes pyruvate/malate et pyruvate/citrate, le NADPH est formé par MEc, alors que l’isocitrate déshydrogénase (IDHc) est responsable de la production du NADPH dans la navette pyruvate/isocitrate/α-cétoglutarate. Dans notre première étude, nous avions démontré l’importance de l’expression de ME pour la sécrétion adéquate d’insuline en réponse au glucose. Dans notre deuxième étude, nous avons testé l’implication de IDHc dans les mécanismes de régulation de la sécrétion d’insuline en réponse au glucose. La diminution de l’expression de IDHc dans les INS 832/13 a stimulé la sécrétion d’insuline en réponse au glucose par un mécanisme indépendant de la production de l’ATP par le métabolisme oxydatif du glucose. Ce résultat a ensuite été confirmé dans les cellules dispersées des îlots pancréatiques de rat. Nous avons aussi observé dans notre modèle que l’incorporation du glucose en acides gras était augmentée, suggérant que la diminution de l’activité de IDHc favorise la redirection du métabolisme de l’isocitrate à travers la navette pyruvate/citrate. Un mécanisme de compensation à travers la navette pyruvate/citrate pourrait ainsi expliquer la stimulation de la sécrétion d’insuline observée en réponse à la diminution de l’expression de IDHc. Les travaux effectués dans cette deuxième étude remettent en question l’implication de l’activité de IDHc, et de la navette pyruvate/isocitrate/α-cétoglutarate, dans la transduction des signaux métaboliques reliant le métabolisme du glucose à la sécrétion d’insuline. La navette pyruvate/citrate est la seule des navettes du pyruvate à conduire à la production du malonyl-CoA dans le cytoplasme des cellules β. Le malonyl-CoA régule le métabolisme des acides gras en inhibant la carnitine palmitoyl transférase 1, l’enzyme limitante dans l’oxydation des acides gras. Ainsi, l’élévation des niveaux de malonyl-CoA en réponse au glucose entraîne une redirection du métabolisme des acides gras vers les processus d’estérification puis de lipolyse. Plus précisément, les acides gras sont métabolisés à travers le cycle des triglycérides/acides gras libres (qui combinent les voies métaboliques d’estérification et de lipolyse), afin de produire des molécules lipidiques signalétiques nécessaires à la modulation de la sécrétion d’insuline. Des études effectuées précédemment dans notre laboratoire ont démontré que l’activité lipolytique de HSL (de l’anglais hormone-sensitive lipase) était importante, mais non suffisante, pour la régulation de la sécrétion d’insuline. Dans une étude complémentaire, nous nous sommes intéressés au rôle d’une autre lipase, soit ATGL (de l’anglais adipose triglyceride lipase), dans la régulation de la sécrétion d’insuline en réponse au glucose et aux acides gras. Nous avons démontré que ATGL est exprimé dans les cellules β pancréatiques et que son activité contribue significativement à la lipolyse. Une réduction de son expression dans les cellules INS 832/13 par RNA interférant ou son absence dans les îlots pancréatiques de souris déficientes en ATGL a conduit à une réduction de la sécrétion d’insuline en réponse au glucose en présence ou en absence d’acides gras. Ces résultats appuient l’hypothèse que la lipolyse est une composante importante de la régulation de la sécrétion d’insuline dans les cellules β pancréatiques. En conclusion, les résultats obtenus dans cette thèse suggèrent que la navette pyruvate/citrate est importante pour la régulation de la sécrétion d’insuline en réponse au glucose. Ce mécanisme impliquerait la production du NADPH et du malonyl-CoA dans le cytoplasme en fonction du métabolisme du glucose. Cependant, nos travaux remettent en question l’implication de la navette pyruvate/isocitrate/α-cétoglutarate dans la régulation de la sécrétion d’insuline. Le rôle exact de IDHc dans ce processus demeure cependant à être déterminé. Finalement, nos travaux ont aussi démontré un rôle pour ATGL et la lipolyse dans les mécanismes de couplage métabolique régulant la sécrétion d’insuline.
Diabetes is a metabolic disorder characterized by a combination of insulin resistance in peripheral tissues with an inappropriate amount of insulin secreted by the pancreatic β-cells to overcome this insulin resistance. In order to help find a cure for diabetic patients, we need to elucidate the mechanisms underlying the proper control of insulin secretion in response to glucose. In pancreatic β-cells, glucose metabolism leads to the production of metabolic coupling factors, like ATP, implicated in the regulation of insulin vesicle exocytosis. The mechanism linking ATP production by the oxidative metabolism of glucose to the triggering of insulin release that involves Ca2+ and metabolically sensitive K+ channels is relatively well known. Other mechanisms are also involved in the regulation of insulin secretion in response to glucose and other nutrients, such as fatty acids and some amino acids. Malonyl-CoA and NADPH are two metabolic coupling factors that have been suggested to be implicated in the transduction of metabolic signaling coming from glucose metabolism to control the release of insulin granules. However, the mechanisms implicated remained to be defined. The goal of the present thesis was to further our understanding of the role of the pyruvate shuttles, derived from mitochondrial glucose metabolism, in the regulation of insulin secretion. In pancreatic β-cells, pyruvate shuttles are produced by the combination of anaplerosis and cataplerosis processes and are thought to link glucose metabolism to the regulation of insulin secretion by the production metabolic coupling factors. In our first study, we wished to determine the role of the pyruvate/citrate shuttle in the regulation of glucose-induced insulin secretion. The pyruvate/citrate shuttle leads to the production in the cytoplasm of both malonyl-CoA and NADPH and also stimulates the metabolic flux through the glycolysis by re-oxidating NADH. A previous study done in the group of Dr Prentki has suggested the feasibility of the pyruvate/citrate shuttle in pancreatic β-cells. To investigate our hypothesis, we inhibited three different steps of this shuttle in INS 832/13 cells, a pancreatic β-cell line. Specifically, we repressed, using pharmacological inhibitors or RNA interference technology, the mitochondrial citrate export to the cytoplasm and the expression of malic enzyme (MEc) and ATP-citrate lyase (ACL), two key enzymes implicated in the pyruvate/citrate shuttle. The inhibition of each of those steps resulted in a reduction of glucose-induced insulin secretion. Our results underscore the importance of the pyruvate/citrate shuttle in the pancreatic β-cell signaling and the regulation of insulin secretion in response to glucose. Other research groups are also interested in studying the implication of pyruvate cycling processes in the regulation of insulin exocytosis. They suggested a role for the pyruvate/malate and the pyruvate/isocitrate/α-ketoglutarate shuttles. Therefore, three different shuttles derived from the mitochondrial glucose metabolism could be implicated in the regulation of glucose-induced insulin release. All those three shuttles can produce NADPH in the cytoplasm. In the pyruvate/malate and the pyruvate/citrate shuttles, the NADPH is formed by cytosolic malic enzyme (MEc), whereas in the pyruvate/isocitrate/α-ketoglutarate, NADPH is produced by cytosolic isocitrate dehydrogenease (IDHc). In our first study, we established the importance of MEc expression in the regulation of insulin secretion. In our second study, we wanted to investigate the importance of IDHc expression in glucose-induced insulin secretion. The reduction of IDHc expression in INS 832/13 cells stimulated insulin release in response to glucose by a mechanism independent of ATP production coming from glucose oxidative metabolism. This stimulation was also observed in isolated rat pancreatic cells. IDHc knockdown cells showed elevated glucose incorporation into fatty acids, suggesting that isocitrate metabolism could be redirected into the pyruvate/citrate shuttle in these cells. Taken together, these results suggest that IDHc is not essential for glucose-induced insulin secretion and that a compensatory mechanism, probably involving the pyruvate/citrate shuttle, explains the enhanced insulin secretion in IDHc knockdown cells . The pyruvate/citrate shuttle is the only pyruvate shuttle that is linked to the production of malonyl-CoA. Malonyl-CoA is a known inhibitor of carnitine palmitoyl transferase 1, the rate-limiting step in fatty acid oxidation. Therefore, the raising level of malonyl-CoA in response to glucose redirects the metabolism of fatty acids into the triglycerides/free fatty acids cycle which combine esterification and lipolysis processes. Previous studies done in the laboratory of Dr Prentki supported the concept that lipolysis of endogenous lipid stores is an important process for the appropriate regulation of insulin secretion. A first lipase, hormone-sensitive lipase (HSL), has been identified in pancreatic β-cells. HSL expression is important, but not sufficient, for the β-cell lipolysis activity. In a complementary study, we have investigated the role of another lipase, adipose triglyceride lipase (ATGL), in the regulation of insulin secretion in response to glucose and to fatty acids. We first demonstrated the expression and the activity of ATGL in pancreatic β-cells. Reducing ATGL expression using shRNA in INS 832/13 cells caused a reduction in insulin secretion in response to glucose and to fatty acids. Pancreatic islets from ATGL null mice also showed defect in insulin release in response to glucose and to fatty acids. The results demonstrate the importance of ATGL and intracellular lipid signaling in the regulation of insulin secretion. In conclusion, the work presented in this thesis suggests a role for the pyruvate/citrate shuttle in the regulation of insulin secretion in response to glucose. This mechanism possibly implicates the production of NADPH and malonyl-CoA in the cytoplasm. The results also points to a re-evaluation of the role of IDHc in glucose-induced insulin secretion. The precise role of IDHc in pancreatic β-cells needs to be determined. Finally, the data have also documented a role of lipolysis and ATGL in the coupling mechanisms of insulin secretion in response to both fuel and non-fuel stimuli.

Sink limitations in plants reduce the potential for photosynthesis and yield, particularly under conditions that favour enhanced source activity such as elevated CO2 (EC). Dark respiration, considered catabolic, has rarely been exploited to enhance sink activity in plants. Arabidopsis thaliana L. lines with partially-suppressed mitochondrial pyruvate dehydrogenase (mtPDH) kinase (mtPDHK), a negative post-translational regulator of the mtPDH complex, was shown previously to have both elevated mtPDH complex activity and increased seed weight and oil content at ambient CO2 (AC), suggesting an enhancement of sink activity. The mtPDH links glycolysis with the tricarboxylic acid (TCA) cycle. It was hypothesized that Arabidopsis having suppressed mtPDHK will display their greatest plant productivity at EC through a combined enhancement of source and sink activities. Control and transgenic Arabidopsis having either constitutive or seed-specific expression of antisense mtPDHK were grown at either AC or EC. Expression of mtPDHK and mtPDH complex activity in rosette leaves and reproductive tissues were measured, which required the development of an assay to quantify mtPDH activity. Vegetative and reproductive growth over time, seed oil parameters, and leaf net C exchange were also quantified. A parabolic relationship was found between mtPDHK expression and mtPDH activity, reflecting a role for mtPDH in balancing photosynthetic and respiratory processes. A number of growth and seed oil parameters were improved in transgenic lines, particularly at EC; many of these parameters showed a significant linear or quadratic correlation with mtPDHK expression and mtPDH activity. The proportion of very long chain fatty acids was increased in transgenic lines. Leaf net C exchange was enhanced at AC and EC, and particularly in lines showing repression of mtPDHK. The greatest enhancement in total seed and oil productivity was found for the constitutive lines 104 and 31 at EC (up to 2.8 times). These two lines exhibited a significant increase in inflorescence size, an increase in leaf water use efficiency, the lowest rate of mtPDH complex inactivation by ATP, and an intermediary enhancement of mtPDH complex activity in seeds. Thus, it is concluded that the mtPDH plays a key role in regulating sink and source activities in plants.
Natural Sciences and Engineering Research Council (NSERC) through the Green Crop Networks Research Network; Ontario Graduate Scholarship; Syngenta Graduate Scholarship; Ball Farm Services and Agrico Canada Ltd. Scholarship; Mrs. Fred Ball Scholarship; Arthur D. Latornell Scholarship; Hoskins Scholarship; Robb Travel Grant; Registrars and the Deans Scholarship and travel awards and bursaries from the University of Guelph, and the Ontario Agricultural College.

Multiple myeloma (MM) is an incurable B-cell malignancy and the second most common haematological cancer in the world. MM patients easily develop innate and acquired chemotherapy resistance due to the dynamic gene mutations. The current standard MM treatment is not promising as patients go through cycles of remission and relapse and eventually treatment failure. Therefore, new treatments and management approaches are needed. MM displays a glycolytic phenotype (Warburg effect) that contributes to cancer development, survival, and drug resistance. Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase (PDK) inhibitor that can reverse the glycolytic phenotype. DCA is an inexpensive and bioavailable drug that has been used to treat mitochondrial malfunctions in humans for decades. Studies have shown DCA to have metabolic modulatory and cytotoxicity effects when used at clinically unachievable concentrations (>10 mM) in various cancer types including MM, but there is a lack of evidence of the on-target and anti-cancer effects of DCA when it is used at clinically achievable concentrations. DCA inhibits its own metabolism through inactivation of its only known metabolising enzyme, GSTZ1. It has been hypothesised that GSTZ1 polymorphisms alter a patient’s ability to metabolise DCA, thus it has been proposed that a personalized DCA dose regimen based on GSTZ1 genotype be applied for clinical use. This study examined the on-target and anti-cancer effects of DCA at mechanistically relevant concentrations under clinically relevant conditions in MM cell lines, and investigated the factors contributing to the variable sensitivity to DCA. The clinical implication of DCA was examined in a phase-2 clinical trial in MM patients and the pharmacokinetics and pharmacogenetics of DCA were evaluated. Results of this study showed that DCA at mechanistically relevant concentrations inhibited glycolysis and cell proliferation but did not induce apoptosis in MM cell lines. DCA can act on-target by reducing phosphorylated pyruvate dehydrogenase (pPDH) when used at concentrations achieved in our clinical trial. The effect of DCA in reducing pPDH was cumulative in vitro over time. This study demonstrated that MM cells displayed heterogeneous metabolic profiles. The degree of dependence on glycolysis was a key contributing factor in the sensitivity of MM cells to DCA. Moreover, the growth inhibition effect of DCA required glucose and an active glycolysis pathway. Under hypoxic conditions that mimic the bone marrow (BM) microenvironment (BMM), DCA can induce apoptosis in a non-glycolytic cell line that has the greatest glycolytic reserve and the highest increase in the targets of DCA, PDK1 and PDK3. Thus, DCA can have greater growth inhibition effect and even cytotoxic effects in hypoxic cancers. The clinical use of DCA in cancer had been tested in four clinical trials in solid tumours, with results showing that DCA was well tolerated but there was no direct efficacy information. The clinical use of DCA in haematological cancer patients was investigated through our world-first trial of DCA in MM patients. The results demonstrated that DCA was quickly absorbed and maintained at mechanistically relevant concentrations for inhibition of PDK2. MM patients tolerated DCA well despite having baseline neuropathy. The main side effect of DCA was neuropathy but this was reversible. The disease burden was reduced, and a response was achieved on day 28 in 33% of patients, but this effect was not maintained. A GSTZ1 promoter polymorphism correlated with one patient’s elevated DCA serum levels and side effects, and it may be the driving variant in determining the serum levels of DCA in long-term use. This trial suggested that the DCA dosing regimen needs to be increased in order for it to inhibit the targets PDK1 and PDK3 in cancer patients in future trials. The combination of DCA with common chemotherapy drugs, such as dexamethasone (DEX) and lenalidomide (LEN), significantly decreased the total viable cells numbers compared to when DEX or LEN was used as a single agent. This indicates that DCA is not interfering with the conventional chemotherapy agents. Thus, DCA has the potential to be used as a low-toxicity addition to conventional chemotherapy for the treatment of MM. This study provides DCA dosing guidance and opens windows for future clinical trials in cancers that display a glycolytic phenotype.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018
Homeostasis of metabolic networks and respective regulatory processes is affected by nutrient availability and also by enzymatic, genetic and epigenetic mechanisms. Acetylation of lysine residues in proteins, including histones, depend of the combined activity of acetyltransferases and deacetylases (KDAC) that involve NAD+ and acetyl-Coenzyme A (CoA). The latter is a critical metabolite that modulates protein acetylation beyond its role in key cellular processes such as mitochondrial energy metabolism. Mitochondrial dysfunction is one important mechanism that may underlie potential drug-induced pharmacological or toxicological effects. Previous studies of our group have already shown that adverse liver toxicity and microvesicular steatosis induced by the antiepileptic drug valproic acid (VPA), may be associated with specific mitochondrial effects, including: i) inhibition of carnitine palmitoyltransferase 1 (CPT1A) activity; ii) inhibition of DLDH, the E3 subunit of 2-oxoacid dehydrogenase complex; iii) impairment of mitochondrial pyruvate carrier; iv) and deregulation of hepatic NAD+ levels. This work focused on this KDAC inhibitor, linking the direct or indirect effects of epigenetic modulation to specific targets of mitochondrial energy metabolism, CPT1A and pyruvate dehydrogenase complex (PDH complex). To better elucidate the implications of acetyl-CoA availability in the acetylation-mediated regulation of hepatic key enzymes, the experimental work aimed to study VPA-induced effects in vivo on PDH complex. The respective activity was measured through a radiochemical assay based on the conversion rate of [14C]-pyruvate to 14CO2 using rat liver tissues obtained from animal studies. In addition, we analyzed PDH complex subunits (E1-α, E2, E3-bp) or Cpt1 protein expression and overall proteins acetylation status in rat liver through western-blot analyses. Pdha1 gene expression was analysed through RT-qPCR and fatty acids and TCA cycle metabolites were analysed in rat urine samples through GC-MS. In this report, results suggest for the first time a VPA-induced inhibition on PDH complex activity. It was observed that VPA-associated samples showed a lower enzymatic activity, a decrease on subunits expression and a reduction in Pdha1 gene expression. A global protein lysine hyperacetylation was observed in liver lysates associated with repeated administration of VPA. Differential analysis of acetylated proteins revealed the highest variation for the 43 kDa band that may hypothetically indicate E1-α subunit among other possible proteins. Analysis of Cpt1 expression showed no significant differences, suggesting that valproyl-CoA-induced inhibition on Cpt1 activity, previously reported, seems independent of protein expression. Results of metabolites profiling analyses are in agreement with our published reports relating VPA-induced inhibitory effects on FAO and TCA cycle with targeted-metabolomic studies in plasma or liver suggesting potential crosstalks with L-lysine degradation pathway. Overall, it is shown that VPA may play a key role as a disruptor of hepatic acetyl-CoA levels, with cumulative effects on acetyl-CoA producing processes, such as PDH-mediated reaction and FAO pathway. This mechanism may contribute to elucidate potential hepatotoxic effects caused by treatment with this drug and also to elucidate the anticancer properties of VPA, linking the signaling metabolite acetyl-CoA with mitochondrial energy metabolism and epigenetic effects.

Two Arabidopsis lines, 10’4 and 3’1, with partial-repression, constitutively of mitochondrial pyruvate dehydrogenase kinase that could alter dark respiration (Rd) were grown on rockwool to reduce off-gassing from peat that interfered with assessment of Rd. At the rosette stage, Rd and photosynthesis (Pn) at high CO2 were greater than at ambient CO2, and Rd was greater for whole-plant than single leaf measurements due to the contribution of non-laminar tissues. However, whole-plant and leaf Rd and Pn were similar on a leaf-area-basis comparing mutants with controls. Whole-plant, analyses during reproductive stage showed that although, the inflorescence contributed as much as 90% of daily C-gain when the rosette leaves senesced, canopy-Pn on a surface-area-basis at each CO2 level remained similar to those at the rosette stage. At each CO2 level, the transgenic and control lines were similar indicating that the mutation resulted in no direct or indirect effect of Rd or Pn.
Ontario Graduate Scholarship, Green Crop Net Work

碩士
臺北醫學大學
細胞及分子生物研究所
95
Insulin is a major anabolic hormone that stimulates synthesis of protein, lipid and glycogen in liver, adipose tissue and muscles. Two main signal transduction pathways downstream of insulin receptor are the phosphatidylinositol 3 kinase/ PDK-1/ Akt pathway and the MAP kinase pathway. How insulin might regulate pyruvate dehydrogenase (PDH) activity in mitochondria is not completely known. Our laboratory has previously found that mitochondrial GSK3b was associated with PDH E3 subunit as a complex. In the present study, we demonstrated that?nAkt was translocated to mitochondria upon insulin stimulation, and the mitochondrial Akt was in its phosphorylated and active state. Activation of Akt is known to phosphorylate and inhibit its downstream enzyme, GSK3b?nat Ser9. Inhibitory phosphorylation of GSK3b maintains PDH at its non-phosphorylated and active state. Consistently, treatment of Hep G2 cells with insulin increased phosphorylation of mitochondrial GSK-3b, which was associated with an increase of PDH activity. Activation of GSK3b by doxorubicin suppressed the PDH activity, and this effect was reversed by pretreatment of cells with TDZD-8, a GSK3b-specific inhibitor?|?n Furthermore, treatment of Hep G2 cells with doxorubicin increased phosphorylation of PDH E3 binding-protein as revealed by 2D-immunoblotting, and the inhibition of GSK3b?n?nby TDZD-8 abolished this phosphorylation. Taken together, our results suggest that translocation of Akt to mitochondria and subsequent GSK3b?nphosphorylation may regulate PDH activity in mitochondria by phosphorylating the PDH E2 or E3 binding-protein?|?n These findings might provide clues to understand the mechanism by which insulin regulates mitochondrial pyruvate dehydrogenase activity through GSK3b?|