Thèses sur le sujet « Citrate metabolism »

OBJETIVOS: O aumento da expectativa de vida tem gerado um envelhecimento populacional global, fazendo com que a proporção de indivíduos com mais de 60 anos de idade cresça mais rapidamente do que as demais faixas etárias. A incidência de litíase urinária em indivíduos idosos tem aumentado nos últimos anos, principalmente em nações industrializadas. Aspectos particulares do envelhecimento orgânico aumentam a morbidade associada à litíase urinária em idosos, tornando a prevenção e o tratamento clínico ainda mais relevantes nessa faixa etária. Nosso objetivo é analisar a avaliação metabólica de homens idosos portadores de cálculos renais. MATERIAIS E MÉTODOS: Realizamos um estudo clínico tipo caso-controle. Os critérios de inclusão foram: indivíduos do sexo masculino com mais de 60 anos de idade, com antecedente de cólica renal ou diagnóstico incidental de litíase urinária após os 60 anos (grupo caso); no grupo controle foram incluídos pacientes da mesma faixa etária sem antecedente de cólica renal ou diagnóstico incidental de litíase renal. Todos os participantes foram submetidos à anamnese e aqueles selecionados realizaram perfil metabólico para diagnóstico de litíase urinária: dosagem sérica de cálcio total, cálcio ionizado, ácido úrico, fósforo, glicemia, uréia, creatinina e paratormônio (PTH); coleta de urina para urocultura e pH urinário, e amostras de urina de 24 horas para quantificação do volume e dosagem de cálcio, citrato, creatinina, ácido úrico e sódio. Foram também submetidos à radiografia simples de abdome e ultrassonografia do aparelho urinário. Os indivíduos do grupo caso realizaram dois perfis metabólicos completos de urina de 24 horas, enquanto os do grupo controle somente um perfil. Os resultados foram submetidos à análise estatística. RESULTADOS: Cento e dez indivíduos foram convocados e, após aplicação dos critérios de inclusão, 70 foram selecionados. Cinquenta e um indivíduos concluíram a investigação clínica, sendo 25 no grupo caso e 26 no controle. Cinquenta e seis por cento dos pacientes do grupo caso apresentaram hipocitratúria comparados a 15,4% do grupo controle (p=0,002). A determinação de sódio em urina de 24 horas também apresentou diferença estatisticamente significativa entre casos e controles: 64% versus 30,8%, respectivamente (p=0,017). Estes resultados foram submetidos à análise de regressão logística univariada e múltipla, respectivamente, e mantiveram seus níveis de significância. CONCLUSÃO: A hipocitratúria e a hipernatriúria são os principais distúrbios metabólicos apresentados por indivíduos idosos do sexo masculino portadores de cálculo urinário
PURPOSES: Rise in life expectancy has caused a global populational ageing and people older than 60-years have increased more than any other age group. The incidence of urinary lithiasis in aging people has increased during the last years, mainly in developed nations. Some aspects concerning organic ageing increase morbidity related to urolithiasis in older individuals making prevention and medical management of urinary stones relevant in this age group. Our objective is to evaluate metabolic parameters in men older than 60 years with urinary stones. MATERIALS AND METHODS: A case-control study was performed. Inclusion criteria were: men older than 60- years old with renal pain episodes or incidental diagnosis of urinary lithiasis beginning after 60-years old (case arm). Control group was constituted by patients older than 60 years without renal colic past or diagnosis of urolithiasis. Patients were recruited from a database from the Urologic Clinic at University of São Paulo Medical School Hospital. Each individual was submitted to anamnesis and those selected underwent a metabolic evaluation for urinary stones: serum dosages of total and ionized calcium, uric acid, phosphorus, glucose, urea, creatinine and parathyroid hormone (PTH); urine culture and urinary pH. Twenty four hour urine samples were obtained for volume quantification and for calcium, citrate, creatinine, uric acid and sodium dosages. An abdominal x-ray and ultrasonography were performed in all patients. Case arm patients underwent two complete metabolic urinary investigations while control arm individuals to only one. All results were submitted to statistical analysis. RESULTS: One hundred and ten individuals were called up for initial evaluation and 70 were selected. Fifty-one individuals concluded the whole clinical investigation: 25 in the case arm and 26 in the control arm. Hypocitraturia was present in 56% of case arm patients and 15,4% of the control arm patients (p=0,002). Hypernatriuria in 24-hour urine samples was found in 64% of case arm patients and in 30,8% of control arm patients (p=0,017). These results were submitted to univariate and multiple logistic regressions and maintained their levels of significancy. CONCLUSION: Hypocitraturia and hypernatriuria are the main metabolic disorders among aging men with urolithiasis

The specific activities of citrate synthase and malate dehydrogenase extracted from mature green fruit of Lycopersicon esculentum, fell 60% during the first two weeks of a twelve week experiment in which the fruit were stored in an atmosphere designed to inhibit ethylene synthesis. Throughout the remainder of the experiment, the specific activities were relatively constant. In the initial two week period, the specific activity of NADP-linked malic enzyme rose by 400%, malic acid concentration fell by 50%, while the concentration of citric acid rose by 20%. Those features of ripening such as the de novo synthesis of lycopene and polygalacturonase, which were thought to depend on ethylene for initiation of response, could not be detected until the fruit were removed to a normal atmosphere. Additionally, citrate synthase and malate dehydrogenase from mature green tomato fruit stored in the presence or absence of ethylene, showed similar trends in specific activity, and the presence of the olefin made no significant difference to the rate of loss of enzyme specific activity. The purification and partial characterisation of citrate synthase from Lycopersicon esculentum is described. The enzyme is a dimer with sub-units of similar size and a total Mr of approximately. 100,000. The characterisation revealed no obvious regulatory features that would easily account for the fall in specific activity. Sub-cellular fractionation studies demonstrated unequivocally that the site of organic acid metabolism was the mitochondrion. Citrate synthase, NAD-dependent isocitrate dehydrogenase and NAD-dependent malic enzyme were shown to be located exclusively in the mitochondrion, while malate dehydrogenase was located both in the cytosol and the mitochondrion. All these enzymes including cytosolic malate dehydrogenase exhibited the co-ordinated fall in specific activity described above. A hypothesis is proposed which includes a novel coarse control of the citric acid cycle and related enzymes, as an early indicator of senescence.

Citrate synthase (CS) is a key mitochondrial enzyme in the tricarboxylic acid cycle (TCA). TCA provides NADH and FADH for the ETC to generate ATP through oxidative phosphorylation in muscle cells. The aim of this PhD project is to investigate the role of CS in skeletal muscle metabolism. The aim of the first study was to investigate the effects of a high fat diet (HFD, 45 % kcal fat) for 12 weeks on CS activity in the heart and gastrocnemius muscle of C57BL/6J (B6) mice and congenic (B6.A) characterised by 39% reduced CS activity. Spectrophotometric analysis of CS activity in the heart and gastrocnemius muscle revealed that HFD led to an increase in CS activity in gastrocnemius muscle but a decrease in the heart in both strains of mice. The aim of the second study was to investigate the effects of low CS activity on substrate metabolism in primary muscle cells established from B6 and B6.A mice. Primary muscle cells from both strains were incubated in radiolabelled glucose or palmitate to assess their oxidation in the mitochondria. The reduction of CS activity in B6.A muscle cells did not affect glucose and palmitate oxidation. The aim of the third study was to investigate the effects of D- and L-serine on CS activity in B6 muscle homogenates, primary muscle cells and purified CS from porcine heart. The muscle samples were incubated in D - or L-serine at 0.1 mM or 5 mM concentration and CS activity levels were assessed by spectrophotometer. D- or L-serine did not have any effect on CS activity in muscle samples. The aim of the fourth study was to investigate the effects of low CS activity on substrate metabolism in C2C12 muscle cells. Lentiviral transduction of C2C12 muscle cells with shRNA resulted in a reduction of CS activity and the metabolic pathways were assessed using XF24 Analyser, western blotting, Immunofluorescence and qRTPCR. Low CS activity was associated with a reduction in substrate oxidation by the mitochondria, an increase in glycolysis and ceramide accumulation in C2C12 muscle cells. The results highlight the significance of CS activity as a modulator of muscle metabolism.

Thesis advisor: Thomas C. Chiles
B-lymphocytes respond to environmental cues for their survival, growth, and differentiation through receptor-mediated signaling pathways. Naïve Blymphocytes must acquire and metabolize external glucose in order to support the bioenergetics associated with maintaining cell volume, ion gradients, and basal macromolecular synthesis. The up-regulation of glycolytic enzyme expression and activity via engaged B-cell receptor mediated-events was glucose-dependent. This suggests an essential role for glucose energy metabolism in the promotion of B cell growth, survival, and proliferation in response to extracellular stimuli. In addition, the activity of ATP-citrate lyase (ACL) was determined to be crucial for ex vivo splenic B cell differentiation to antibody-producing cells wherein B cells undergo endomembrane synthesis and expansion. This investigation employed knockout murine models as well as chemical inhibitors to determine the signaling components and enzymes responsible for glucose utilization and incorporation into membrane lipids. These results point to a critical role for phosphatidylinositol 3- kinase (PI3K) in orchestrating cellular glucose energy metabolism and glucosedependent de novo lipogenesis for B lymphocyte responses
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Der Citratzyklus (TCA) ist einer der bedeutendsten Stoffwechselwege für alle lebenden Organismen. Trotz der zentralen Rolle dieses Prozesses im Pflanzenmetabolismus ist er nur relativ wenig untersucht worden. In dieser Arbeit berichte ich über die Produktion und die funktionale Analyse von Tomatenpflanzen (Solanum lycopersicum), die unabhängig eine leicht eingeschränkte Aktivität der mitochondrialen Citrat-Synthase (CS) und zweier Isocitrat-dehydrogenasen (mitochondriale NAD-IDH und cytosolische NADP-ICDH) zeigen. Die transgene Pflanzen wiesen mehrheitlich keine erkennbare Veränderung eines Wachstumphänotyps auf. Obwohl die photosyntetische Leistung keine Änderungen gezeigt hatte, war die mitochondriale Respiration gestiegen, begleitet von einem reduzierten Kohlenstoff-fluss durch den Citratzyklus. Darüber hinaus waren die CS Pflanzen charakterisiert durch wesentliche Änderungen im Blattmetabolismus, einschließlich eines eingeschränkten Niveaus des photosynthetischen Pigments und Zwischenprodukten des Citratzyklus zusammen mit einer Akkumulation von Nitraten, verschiedenen Aminosäuren und Stärken. Zusammengefasst deuten diese Ergebnisse auf eine Einschränkung der Nitrat-Aufnahme hin. Das mit Hilfe von TOM1 Mikroarrays und quantitativer RT-PCR durchgeführte Transcript-profiling hat gezeigt, dass die fehlende Aktivität der mitochondrialen CS teilweise von einer gestiegenen, peroxisomalen CS Isoform ausgeglichen wird. Die metabolische Verschiebung ergab eine Verstärkung der photorespiratorischen Leistung, die vermutlich eine ausgleichende Rolle in der Produktion organischer Säuren und der Wiederherstellung der Redox-Balance spielt. Interessantenweise war die metabolische Antwort von Blättern auf Stickstoffmangel in NADP-ICDH Pflanzen dramatischer als in NAD-IDH Pflanzen, was darauf hindeutet, dass die cytosolische Isoform der Hauptlieferant von 2-Oxoglutarat im Tomatenmetabolismus sein könnte.
Although the TCA cycle is a respiratory metabolic pathway of central importance for all living organisms, relatively few molecular physiological studies of plants were performed to date. Here, I report the generation and functional analysis of tomato plants (Solanum lycopersicum) independently displaying mildly limited activity of mitochondrial citrate synthase (CS) and two isocitrate dehydrogenases, namely mitochondrial NAD-IDH and cytosolic NADP-ICDH. The transgenic plants revealed minor phenotypic alterations. Although the leaf photosynthetic performance was largely unaltered, the changes in mitochondrial respiration and carbon flux through the TCA cycle were observed. Moreover, the plants were characterized by significant modifications in the leaf metabolic content and in maximal catalytic activities of several enzymes involved in primary C and N metabolism. These results hint towards limitations in nitrate assimilation pathway. The transcript profiling performed by utilizing TOM1 microarrays and quantitative RT-PCR approach revealed that the deficiency in mitochondrial CS activity was partially compensated by up-regulation of peroxisomal CS isoform. The limitations in the activities of isocitrate dehydrogenases resulted in up-regulation of the photorespiratory pathway, which presumably played a compensatory role in supporting organic acid production and re-establishing redox balance in the transgenic leaves. Interestingly, the leaf metabolic response towards nitrogen starvation conditions was far more dramatic in NADP-ICDH transgenic plants than NAD-IDH plants, hinting that the cytosolic isoform may be the major 2-oxoglutarate supplier in tomato metabolism.

The main aim of this thesis was to examine the hypothesis that the A/J strain variant of H55N substitution affects citrate synthase (CS) enzyme activity and metabolic health in mice fed a high fat diet (HFD). C57BL/6J (B6) mice and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice, a strain which carries the A/J allele of Cs on the B6 strain background, were fed a HFD (45% kcal from fat) for 12 weeks. CS activity, but not that of ß-hydroxyacyl-coenzyme dehydrogenase was lower in the gastrocnemius muscle of B6.A mice compared to B6 mice (P< 0.001). During HFD feeding the glucose tolerance of mice decreased progressively and to a greater extent in B6.A females compared to B6 females, with males showing a similar trend. Interestingly, after 12 weeks of HFD feeding only B6.A males showed increases (P< 0.05) in their resting metabolic rate; moreover; core body temperature were also increased (P< 0.05) for congenic B6.A of both sexes by the end of the study. However, body weight and fat gain did not differ between B6.A and B6 mice. The second aim of the thesis was to test the hypothesis that low CS activity promotes palmitate-induced lipotoxicity in muscle cells. After 18 hours of incubation in 0.8 mM palmitate, C2C12 muscle cells with a ~50% reduction in CS activity showed low (P< 0.001) viability, increased (P< 0.001) levels of cleaved Caspase-3, high levels of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation (P< 0.05), low levels of protein kinase B phosphorylation, high mitogen-activated protein kinases activation (P< 0.001) compared to the control shRNA cells. This was coupled with higher levels of mitochondrial proteins (P< 0.05), which are involved in oxidative phosphorylation. C2C12 cells with reduced CS activity also showed high reactive oxygen species production (P< 0.05), low intracellular ATP levels (P< 0.05), and lower basal mitochondrial respiration (P< 0.001). In summary, the A/J strain variant of H55N is associated with low CS enzyme activity and impaired metabolic health when fed HFD. Palmitate has a lipotoxic effect on Cs shRNA transfected cells and can lead to cell death.

L’objectif de ce projet de thèse est d’étudier et caractériser les mécanismes impliqués dans la bascule respiro-fermentaire chez des cellules eucaryotes dotées d’un métabolisme mitochondrial. Les cellules eucaryotes ont des besoins différents en oxygène pour la production d’énergie et leur survie dans un environnement donnée. Elles sont qualifiées de type aérobie stricte lorsque la présence d’oxygène leur est nécessaire ou aéro-anaérobie facultatif dans le cas où l’oxygène n’est pas indispensable à la production d’énergie. La levure Yarrowia lipolytica a été choisie comme modèle d’étude de par sa particularité à être un micro-organisme aérobie strict avec une grande capacité d’accumuler de lipides et de production d’acides organiques. Les études expérimentales et analytiques, par l’emploi de méthodes mathématiques de modélisation du métabolisme, ont permis d’identifier des contraintes métaboliques impliquées dans la transition respiro-fermentaire chez cette levure au métabolisme énergétique oxydatif. La production de l’acide citrique par Y. lipolytica, déjà rapportée dans la littérature, a été choisi comme un marqueur de cette transition respiro-fermentaire. Nous avons découvert que l’inhibition de la protéine oxydase alternative (AOX), impliquée dans la respiration mitochondriale, par la molécule n-propyl gallate (nPG) permet d’améliorer le rendement de production d’acide citrique par fermentation du glucose dans une culture de Y. lipolytica. Ces résultats montrent que la nPG, déjà utilisée dans l’industrie agro-alimentaire et pharmaceutique en tant que conservateur joue sur la bascule respiro-fermentaire par inhibition de la consommation d’oxygène et stimule ainsi la production d’acide citrique. La modélisation du réseau métabolique de Y. lipolytica, décrit à l’échelle du genome, par dynamic Flux Balance Analysis (dFBA) a permis d’identifier l’accumulation des espèces oxydantes dites ROS (Reactive Oxygen Species) comme un levier majeur de la bascule respiro-fermentaire et donc de la production d’acide citrique chez la levure Y. lipolytica. De plus, nos résultats préliminaires montrent que l’oxydation des lipides accumulés par Y. lipolytica pourrait être à l’origine de la génération des ROS. Cette étude doit être approfondie expérimentalement et constitue un apport important pour l’industrie agro-alimentaire et pharmaceutique.Mots clés : Bascule respiro-fermentaire, Acide citrique, lipides, Yarrowia lipolytica, n-propyl gallate, Reactive Oxygen Species, modélisation, dynamic Flux Balance Analysis
The main goal of this thesis project is to study and characterize mechanisms involved in respiratory to fermentative shift in eukaryotic cells endowed with mitochondrial metabolism. Eukaryotic cells have different oxygen requirements for energy production and survival in a given environment. They are described as strict aerobic when the presence of oxygen is necessary or optional aero-anaerobic in when oxygen is not essential for energy production. The yeast Yarrowia lipolytica was chosen as our study model thanks to its particularity since it is a strict aerobic microorganism with a high capacity to accumulate lipids and to produce organic acids. Experimental and analytical studies, using mathematical methods for modeling cell metabolism, allowed us to identify metabolic constraints involved in respiratory to fermentative transition in this yeast showing oxidative energy metabolism. Production of citric acid by Y. lipolytica, already reported in the literature, has been chosen as a marker for this in respiratory to fermentative shift. We found that the inhibition of the alternative oxidase protein (AOX) involved in mitochondrial respiration, by adding n-Propyl gallate (nPG) molecule improves the yield of citric acid production by fermentation of glucose in a Y. lipolytica culture. These results show that nPG, already used in food and pharmaceutical industry as a preservative, plays on respiratory to fermentative balance by inhibition of oxygen consumption and thus stimulates the production of citric acid. Modeling of the metabolic network of Y. lipolytica, described at genome-scale, by dynamic Flux Balance Analysis (FBA) has identified the accumulation of intracellular ROS (Reactive Oxygen Species) species as major levers for respiratory to fermentative shift and therefore the production of citric acid by Y. lipolytica. Therefore, our preliminary results show that oxidation of lipids accumulated by Y. lipolytica could be involved in generation of ROS species. This study must be experimentally deepened and constitutes an important contribution for the agri-food and pharmaceutical industry.Key words: Respiratory to fermentative shift, Citric acid, lipids, Yarrowia lipolytica, n-Propyl gallate, Reactive Oxygen Species, modeling, dynamic Flux Balance Analysis

Oenococcus oeni est la principale bactérie lactique responsable de la fermentation malolactique en vin. Cette deuxième étape de fermentation permet la diminution de l’acidité totale, d’apporter rondeur, souplesse et stabilité microbiologique aux vins concernés. Les conditions physico-chimiques du vin affectent le développement des microorganismes par la combinaison de plusieurs facteurs de stress qui peuvent sérieusement compromettre le bon déroulement de la fermentation malolactique. Une attention particulière a été portée sur le stress acide, l’objectif étant de mettre en évidence des mécanismes impliqués dans la réponse au stress de O. oeni. Pour cela, deux évolutions expérimentales ont été mises en œuvre afin d’adapter les bactéries à un bas pH et ainsi améliorer leur acido-tolérance. Ce procédé a conduit à l’apparition de mutations qui ont été identifiées par séquençage du génome des populations évoluées obtenues.La première expérience réalisée sur la souche ATCC BAA-1163 a permis de mettre en évidence la fixation de mutations au sein du locus citrate affectant toutes les populations évoluées. De ce fait, le métabolisme du citrate chez O. oeni a fait l’objet d’une étude approfondie au cours de cette thèse. Les résultats ont permis de démontrer qu’une modulation de la vitesse de consommation de citrate pouvait renforcer l’acido-tolérance de O. oeni. En parallèle, les mécanismes impliqués dans la régulation du locus citrate chez cette bactérie ont été étudiés et ont révélé le rôle du régulateur transcriptionnel CitR et l’implication d’un ARN messager antisens. L’étude du métabolisme carboné de souches d’origines différentes a également montré une grande diversité entre ces dernières, qui pourrait être corrélée à leurs écosystèmes et qui conforte le lien entre la consommation de citrate et l’acido-tolérance des souches. Pour finir, une seconde évolution expérimentale réalisée sur la souche commerciale IOEB_S450 a mis en évidence l’apparition de mutations dans différents gènes incluant le locus malate, ce qui ouvre la voie vers de nouvelles perspectives de recherche
Oenococcus oeni is the wine bacterium responsible for malolactic fermentation in wine. This second fermentation step reduces total acidity, bringing roundness, softness and microbiological stability to the wine. Nevertheless, wine represents a harsh environment for the development of microorganisms, combining several stress factors that can seriously compromise malolactic fermentation. Particular attention was paid to acid stress, with the aim of identifying unknown mechanisms involved in the response of O. oeni to this stress. Therefore, two experimental evolutions were conducted on two different strains of this bacterium to adapt cells to low pH in order to improve their acid tolerance. It resulted in the appearance of mutations that were identified by whole genome sequencing of the evolved populations obtained.The first experiment conducted on ATCC BAA-1163 revealed the fixation of mutations in the citrate locus of all evolved populations. Therefore, citrate metabolism in O. oeni was extensively studied during this PhD. The results highlighted that a modulation of citrate consumption rate can improve acid tolerance of O. oeni. In addition, the mechanisms involved in the regulation of citrate locus expression in this bacterium were explored, revealing the function of the transcriptional regulator CitR and the involvement of an antisense RNA. A study of the global carbon metabolism of strains from different origins also showed a great diversity between them, which could be correlated to their ecosystems and comforted the link between citrate consumption and acid tolerance. Finally, the second adaptive evolution carried out on the commercial strain IOEB_S450 revealed the acquisition of mutations in different genes including the malate locus and opens the way to new research opportunities

Le récepteur hépatique aux lipoprotéines LSR est impliqué dans la clairance des lipoprotéines riches en triglycérides telles que les résidus de chylomicrons pendant la phase postprandiale. La réduction de l’expression du LSR chez la souris (LSR+/-) est associée à une dyslipidémie et une lipémie postprandiale élevée. Afin de mieux comprendre la régulation de la distribution des lipides alimentaires, nous avons cherché quels étaient les facteurs pouvant affecter le niveau protéique de LSR. La leptine, hormone sécrétée par le tissu adipeux et connue pour son action d’hormone de satiété au niveau du système nerveux central, a été démontrée dans cette thèse comme modulant l’expression de LSR par la régulation de la transcription du gène lsr. La leptine est impliquée dans la régulation de la lipogénèse à travers SREBP-1. Grâce à l’utilisation d’un extrait de Garcinia cambogia contenant un inhibiteur de l’ATP citrate lyase, nous avons démontré une interaction importante entre les enzymes lipogéniques, l’expression de LSR et d’autres récepteurs lipoprotéiques, afin de maintenir un équilibre entre la synthèse de lipides endogènes et l’apport alimentaire de lipides exogènes. Soumises à un régime hyperlipidique, les souris sauvages montrent une diminution de l’expression des enzymes lipogéniques hépatiques, aggravée chez les souris LSR+/-. Ces résultats indiquent qu’il existe un mécanisme de maintien de l’équilibre entre la lipogénèse (synthèse endogène de lipides), la lipolyse (utilisation lipidique comme substrat énergétique) et le stockage de lipides à travers une forte interaction entre les enzymes lipogéniques et LSR
The hepatic lipoprotein receptor LSR is involved in the clearance of triglyceride-rich lipoproteins including chylomicrons remnants during the post-prandial phase. Reduced LSR protein expression in mice (LSR+/-) is associated with dyslipidemia and increased postprandial lipemia; these mice exhibit increased weight gain with aging or when placed under a high-fat diet. In order to better understand the regulation of the distribution of dietary lipids, we looked for factors that could regulate LSR protein levels. Leptin is a hormone secreted by the adipose tissue that is a centrally-acting satiety factor, and was demonstrated to modulate LSR mRNA and protein expression through the modulation of transcription of the gene lsr. Leptin has been reported be involved in the control of lipogenesis through SREBP-1c. Using Garcinia cambogia extract containing an inhibitor of ATP citrate lyase, we demonstrated that there is an important link between lipogenic enzymes and LSR protein levels and with other lipoprotein receptors that provides the means to maintain a balance between endogenous lipid synthesis and dietary intake of exogenous lipids. When exogenous lipid intake is increased in the form of a high-fat diet, mice exhibited a decrease in hepatic lipogenic enzymes expression, but a deficiency of LSR led to increased lipid content in the peripheral tissues. These results suggest the presence of mechanisms for the maintenance for the balance between lipogenesis (de novo endogenous lipid synthesis), lipolysis (lipids used as energy substrate), and lipid storage through an important link between lipogenic enzymes and LSR

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

Tomato is an important food crop and a model for fleshy fruit development. The process of fruit ripening involves changes in chemical composition and in particular the accumulation of sugars, organic, amino acids and carotenes. The research described in this thesis aimed to identify key regulatory aspects associated with the accumulation of the major acids in tomato fruit by analysis of introgression lines resulting from a cross between a cultivated variety, Solanum lycopersicum, and a wild progenitor species, Solanum pennellii. Line 2-5 showed increases in citrate, malate, aspartate and glutamate in fruit grown under greenhouse conditions. The genetic differences between line 2-5, its overlapping lines, sub-introgression lines and the recurrent parent were used to link the metabolite phenotypes to smaller chromosomal regions. This analysis suggested multiple epistatic loci control fruit metabolite accumulation. Investigation of the biochemical differences between line 2-5 and the recurrent parent revealed that organic and amino acid accumulation did not dependent upon increased TCA cycle capacity. Regulation at the metabolic level was identified for citrate accumulation with changes in cytosolic aconitase in line 2-5. As these metabolites accumulate in the vacuole, tonoplast transport was investigated. Correlation of ATPase-dependent malate influx with altered malate content suggested malate tonoplast transport plays a role in malate accumulation and highlights the importance of vacuolar storage and transport in the regulation of organic and amino acid accumulation.

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A composição mineral da dieta influencia as características da urina de gatos, estando envolvida no desenvolvimento e prevenção de urolitíases. Diante disso, em um primeiro experimento, foram investigados os efeitos da manipulação mineral da dieta baseada em seu excesso de bases sobre excreção urinária de macroelementos e supersaturação relativa da urina para oxalato de cálcio (SSR OxCa). Em uma dieta basal (EB=381meq/kg) foram adicionadas quatro doses de mistura de sais aniônicos, resultando em dietas com EB de 248mEq/kg, 169mEq/kg, 115mEq/kg e -22mEq/kg. Foram utilizados 30 gatos adultos saudáveis em um delineamento inteiramente casualizado com cinco tratamentos e seis gatos por dieta. Os gatos permaneceram em gaiolas metabólicas durante sete dias de adaptação à dieta, seguidos de cinco dias de coleta total de urina (em garrafas com 100mg de timol). A urina produzida em cada período de 24 horas teve aferido seu volume, densidade e pH, concentrações de sete macroelementos, ácido úrico, citrato e oxalato. A SSR OxCa foi calculada pelo programa Equil-93.O equilíbrio ácido-básico foi estudado pela hemogasometria de sangue venoso e suas médias foram comparadas por contrastes polinomiais (P,0,05). Um segundo experimento investigou durante um período de quatro meses as consequências da ingestão de dieta com excesso de ânions sobre o equilíbrio ácido-básico, o balanço entre formação e reabsorção óssea, a excreção urinária de cálcio e a supersaturação relativa da urina para oxalato de cálcio. Foram utilizados 18 gatos adultos saudáveis em um delineamento inteiramente casualizado com três dietas (EB de 196, EB de 9 e EB de -187mEq/kg MS) e seis gatos por dieta. Foram realizados os mesmos procedimentos do experimento anterior e para a avaliação do metabolismo ósseo foi realizada a...
Food mineral composition influences the acid-base equilibrium and the characteristics of cat’s urine and is involved in the development and prevention of urolithiasis. We investigate, in the first experiment, the effects of diet mineral manipulation based on food base excess (BE) on cats mineral balance and urinary relative supersaturation for calcium oxalate (CaOx RSS). In a basal formulation for cat maintenance (BE=381mEq/kg) four dosages of a commercial mixture of acidifying salt was added, resulting in diets with BE of 248mEq/kg, 169mEq/kg, 115mEq/kg and -22mEq/kg). Thirty adult healthy cats, were used, in a completely randomized design with five treatments and six cats per diet. A 7-d adaptation phase followed 5-d of total collection of faeces and urine. Urine were pooled by cat and analyzed for density, volume, pH, Ca, P, Mg, Na, Cl, K, S, citrate and oxalate. The CaOx RSS was calculated with Equil-93. The acid-base balance was studied by blood gas analysis of venous blood and their means were compared by polynomial contrasts (P<0.05). The experiment 2 investigated over a period of four months, consequences of ingestion of diet with anions excess on acid-base balance, balance between bone formation and resorption, calcium urinary excretion and CaOx RSS. Eighteen adult healthy cats, were used, in a completely randomized design with three treatments (BE of 196, BE of 9 and BE of -187 mEq/kg) and six cats per diet. The same procedures were performed in the previous experiment and the assessment of bone metabolism was performed by bone densitometry (DEXA) and serum markers carboxy terminal telopeptide type 1 (CTX-1) and bone alkaline phosphatase (BAP). The addition of salt mixture resulted in dose-dependent reduction of the pH of urine (p <0.0001) and not altered the volume and density of cat's urine... (Complete abstract click electronic access below)

Orientador: Áulus Cavalieri Carciofi
Banca: Áureo Evangelista Santana
Banca: Márcia Mery Kogika
Banca: Ricardo Souza Vasconcellos
Banca: Márcio Antônio Brunetto
Resumo: A composição mineral da dieta influencia as características da urina de gatos, estando envolvida no desenvolvimento e prevenção de urolitíases. Diante disso, em um primeiro experimento, foram investigados os efeitos da manipulação mineral da dieta baseada em seu excesso de bases sobre excreção urinária de macroelementos e supersaturação relativa da urina para oxalato de cálcio (SSR OxCa). Em uma dieta basal (EB=381meq/kg) foram adicionadas quatro doses de mistura de sais aniônicos, resultando em dietas com EB de 248mEq/kg, 169mEq/kg, 115mEq/kg e -22mEq/kg. Foram utilizados 30 gatos adultos saudáveis em um delineamento inteiramente casualizado com cinco tratamentos e seis gatos por dieta. Os gatos permaneceram em gaiolas metabólicas durante sete dias de adaptação à dieta, seguidos de cinco dias de coleta total de urina (em garrafas com 100mg de timol). A urina produzida em cada período de 24 horas teve aferido seu volume, densidade e pH, concentrações de sete macroelementos, ácido úrico, citrato e oxalato. A SSR OxCa foi calculada pelo programa Equil-93.O equilíbrio ácido-básico foi estudado pela hemogasometria de sangue venoso e suas médias foram comparadas por contrastes polinomiais (P,0,05). Um segundo experimento investigou durante um período de quatro meses as consequências da ingestão de dieta com excesso de ânions sobre o equilíbrio ácido-básico, o balanço entre formação e reabsorção óssea, a excreção urinária de cálcio e a supersaturação relativa da urina para oxalato de cálcio. Foram utilizados 18 gatos adultos saudáveis em um delineamento inteiramente casualizado com três dietas (EB de 196, EB de 9 e EB de -187mEq/kg MS) e seis gatos por dieta. Foram realizados os mesmos procedimentos do experimento anterior e para a avaliação do metabolismo ósseo foi realizada a... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Food mineral composition influences the acid-base equilibrium and the characteristics of cat's urine and is involved in the development and prevention of urolithiasis. We investigate, in the first experiment, the effects of diet mineral manipulation based on food base excess (BE) on cats mineral balance and urinary relative supersaturation for calcium oxalate (CaOx RSS). In a basal formulation for cat maintenance (BE=381mEq/kg) four dosages of a commercial mixture of acidifying salt was added, resulting in diets with BE of 248mEq/kg, 169mEq/kg, 115mEq/kg and -22mEq/kg). Thirty adult healthy cats, were used, in a completely randomized design with five treatments and six cats per diet. A 7-d adaptation phase followed 5-d of total collection of faeces and urine. Urine were pooled by cat and analyzed for density, volume, pH, Ca, P, Mg, Na, Cl, K, S, citrate and oxalate. The CaOx RSS was calculated with Equil-93. The acid-base balance was studied by blood gas analysis of venous blood and their means were compared by polynomial contrasts (P<0.05). The experiment 2 investigated over a period of four months, consequences of ingestion of diet with anions excess on acid-base balance, balance between bone formation and resorption, calcium urinary excretion and CaOx RSS. Eighteen adult healthy cats, were used, in a completely randomized design with three treatments (BE of 196, BE of 9 and BE of -187 mEq/kg) and six cats per diet. The same procedures were performed in the previous experiment and the assessment of bone metabolism was performed by bone densitometry (DEXA) and serum markers carboxy terminal telopeptide type 1 (CTX-1) and bone alkaline phosphatase (BAP). The addition of salt mixture resulted in dose-dependent reduction of the pH of urine (p <0.0001) and not altered the volume and density of cat's urine... (Complete abstract click electronic access below)
Doutor

Le laboratoire d'analyses isotopique et electrochimique de metabolismes (l. A. I. E. M. ) a developpe une expertise dans l'etude de la filiation isotopique liant le produit d'une transformation biologique a son substrat d'origine. Soucieux d'eprouver et de developper nos methodes analytiques, nous desirons etudier les filiations isotopiques entre deux substrats et plusieurs produits. Nous avons choisi l'etude du co-metabolisme citrate-glucose chez une bacterie lactique : lactococcus lactis subsp. Lactis biovar. Diacetylactis comme modele d'etude. Ce choix nous a conduit a developper le dosage simultane de tous les composes d'interets : citrate, glucose, diacetyle, acetoine, acides lactique et acetique. Ce dosage par rmn 1h en utilisant une reference electronique : eretic est realise directement sur le milieu de culture. Dans ce modele, l'etude par spectrometrie de masse de rapports isotopiques (smri) du 1 3c, nous a permis de prouver la participation du citrate et du glucose, a la fois, a la biosynthese de l'acide lactique et des aromes (diacetyle et acetoine). De plus, nous avons quantifie les flux metaboliques s'engageant dans les 2 voies metaboliques au cours de la cinetique de fermentation. Finalement, l'utilisation de la methodologie des plans d'experiences a permis d'etudier efficacement l'impact des certains parametres de culture - notamment la valeur 1 3c des substrats, le rapport des concentrations des substrats, le ph et la temperature - sur les concentrations finales des produits synthetises et sur leur valeur 1 3c.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2016.
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O citrato é um metabólito intermediário do ciclo de Krebs que possui função alostérica. Quando em altas concentrações, esta molécula modula a glicólise por meio da inibição da atividade da enzima fosfofrutoquinase no citoplasma. As células de câncer apresentam reprogramação do seu metabolismo energético, produzindo energia, preferencialmente, por via glicolítica. Esta estratégia pode ser consequência de privação de oxigênio no microambiente do tumor; da rápida obtenção de energia, quando comparada à fosforilação oxidativa; de disfunção mitocondrial; ou mesmo da manutenção da homeostase mitocondrial, preservando a organela de estresse oxidativo. Nesse sentido, o citrato pode ser explorado como potencial agente antitumoral, atuando em processos-chave do funcionamento das células neoplásicas. O objetivo deste trabalho foi investigar alterações estruturais e metabólicas induzidas por citrato em células da linhagem tumoral MCF-7 (adenocarcinoma mamário humano) e em células normais (cultura primária de fibroblastos derivados de polpa dentária humana). Ensaios de citotoxicidade por meio do método colorimétrico MTT demonstraram redução da viabilidade dos dois tipos celulares de maneira dependente do tempo e da concentração do tratamento. O citrato foi mais citotóxico em células MCF-7 do que em fibroblastos na concentração de 20 mM e nos tempos de 24 e 48 h. O teor de citrato nas células MCF-7 é elevado no grupo controle e o tratamento não reduziu a produção de lactato. Foi observado inibição da atividade da enzima fosfofrutoquinase das células MCF-7 tratadas com citrato. Análises morfométricas apontaram alterações ultraestruturais das mitocôndrias em fibroblastos, o que sugere adaptação ao estresse celular produzido pelo tratamento. Não foi observado alteração de potencial de membrana mitocondrial em ambas as células após tratamento. No entanto, MCF-7 apresentou formação de espécies reativas de oxigênio. __________________________________________________________________________________________________ ABSTRACT
Citrate is an intermediate metabolite in the Krebs cycle that has allosteric function. When this molecule is present in high concentrations, it modulates the glycolysis through inhibition of the enzyme phosphofructokinase activity in the cytoplasm. Cancer cells exhibit metabolic reprogramming to the energy production preferably by glycolytic pathway. This strategy may result from oxygen deprivation in the tumor microenvironment; the rapid achievement of energy compared to oxidative phosphorylation; mitochondrial dysfunction; or even the maintenance of mitochondrial homeostasis, preserving organelle against oxidative stress. Therefore, the citrate can be explored as a potential antitumor agent, working in the key processes of cellular functions of the cancer cells. The objective of this study was to investigate structural and metabolic changes induced by citrate in tumor cell line MCF-7 (human breast adenocarcinoma) and normal cells (fibroblasts from human dental pulp). Cytotoxicity assays using the MTT colorimetric method performed in three independent experiments, demonstrated that citrate reduced the viability of both cell types in a manner dependent on the time and concentration of the treatment. Citrate was more cytotoxic in MCF-7 cells than in fibroblasts in a concentration of 20 mM at 24 and 48 h. The citrate content in MCF-7 cells is elevated in the control group and the treatment did not reduce the production of lactate. It was observed a reduction in the activity of the enzyme phosphofructokinase MCF-7 cells treated with citrate. Morphometric analysis showed ultrastructural changes of mitochondria in fibroblasts. These changes suggest adaptation to cellular stress produced by the treatment. There was no alteration in mitochondrial membrane potential in both cells after treatment. However, MCF-7 showed formation of reactive oxygen species.

Orientador: Edgard Ferro Collares
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: O objetivo do presente trabalho foi estudar um modelo de desidratação por privação de água e o esvaziamento gástrico de líquidos em ratos desidratados. Foram utilizados ratos Wistar (n=380) para o estudo de um modelo de privação de água (n=80) e para o estudo do esvaziamento gástrico das soluções (n=300). No primeiro grupo, os animais permaneceram em privação de água por periodos de 24 (n=10), 48 (n=lO) e 72 horas (n=10), comparados com controles (n=15), com acesso à água. A seguir, no estudo da recuperação da desidratação (n=35), utilizou-se o periodo de privação por 72 horas, sendo que, no tempo de 67 horas de privação, água foi oferecida por 60 minutos (n=15) e 120 minutos (n=15), enquanto controles (n=5) permaneceram sem acesso à água. Foram determinados perda de peso, hematócrito e densidade plasmática em cada animal. No estudo do esvaziamento gástrico, foram estudadas três refeições de prova, salina (n=30), água (n=30) e solução reidratante da OMS (n=30), nos tempos de privação de 24, 48 e 72 horas. O mesmo número de animais foi utilizado para composição dos gruposcontroles. A seguir, utilizando o tempo de privação de 72 horas, determinou-se o esvaziamento gástrico da solução reidratante (n=lO) e de água (n=10), com oferta de água por 60 minutos no tempo de 67 horas de privação. O mesmo procedimento foi usado para as soluções reidratante (n=10) e glico-salina (n=lO), com oferta de água por 120 minutos. Nos dois estudos foram utilizados o mesmo númere de animais-controles, com privação total e sem privação de água. No modelo de desidratação, houve diferença estatisticamente significativa nos três parâmetros estudados (perda de peso, hematócrito e densidade plasmática), entre os animais do grupo de estudo e de controle, quando se utilizaram os tempos de privação de 48 e 72 horas, sendo mais pronunciadas neste último (p<0,05). Na recuperação da desidratação, após privação de água por 67 horas e oferta da mesma por 60 minutos, ocorreu uma diminuição progressiva dos valores de perda de peso, hematócrito e densidade plasmática, avaliados com 72 horas do início do estudo. Houve tendência à normalização quando a oferta de água se deu por 120 minutos
Abstract: Os valores da retenção gástrica de salina e de água foram inferiores aos da solução reidratante, nos três tempos estudados. Não houve diferença nas medidas de retenção gástrica entre os grupos de estudo com privação de água e os controles, exceto para as refeições de prova de água e solução reidratante, no tempo de privação de 72 horas, que mostraram retarde de esvaziamento gástrico (p<0,05). No grupo de animais com oferta parcial de água por 60 minutos, no tempo de privação de 67 horas, observou-se uma recuperação do padrão de esvaziamento gástrico da água em relação ao controle, avaliado com 72 horas do início do estudo, mantendo-se o retarde da solução reidratante (p<0,02). No esvaziamento gástrico da solução reidratante e glico-salina, com oferta parcial de água por 120 minutos, no tempo de 67 horas de privação, observou-se uma tendência de retarde das refeições de prova, avaliado 72 horas após o início do estudo (p=0,04). Nos estados de desidratação provocados por privação de água, sem mudança ou com alterações pequenas do conteúdo eletrolítico corporal, a solução reidratante mostrou retarde de esvaziamento gástrico, após reidratação com água. Por outro lado, a água, na situação do animal reidratado, mostrou esvaziamento gástrico semelhante ao grupocontrole. Neste estudo, há indicações de que os mecanismos de controle em que participam receptores na mucosa duodenal, e que interferem na motilidade gástrica, estão alterados na desidratação por privação de água, em ratos
Mestrado
Pediatria
Doutor em Saude da Criança e do Adolescente

INTRODUÇÃO: A obesidade é uma das causas de hipogonadismo (HG) secundário no homem. A terapia de reposição padrão de testosterona (TRT) é associada à melhora dos parâmetros metabólicos, mas pode levar à infertilidade. Apenas recentemente indicou-se que não há novas evidências nível 1 para apoiar uma conexão definitiva entre TRT e eventos cardiovasculares (CV). OBJETIVO: Avaliar os efeitos do Citrato de Clomifeno (CC) em homens jovens com hipogonadismo associado à obesidade diagnosticado por testosterona total (TT) <= 300 ng/dL em duas ocasiões, sintomas positivos no questionário ADAM, hormônio Luteinizante (LH) baixo ou inadequadamente normal (VR: 1,7 - 8,6 UI/L). MÉTODOS: Estudo randomizado, duplo cego, controlado por placebo (PLB), longitudinal em centro único. Setenta e oito pacientes com idade entre 36,5±7,8 anos, índice de massa corporal (IMC) 46,2±8,5 kg/m2 foram randomizados (1:1) para receber CC 50 mg ou PLB durante 12 semanas. Os pacientes foram avaliados através de: 1) Parâmetros clínicos: Questionário ADAM, número de intercursos sexuais, queixa de insatisfação com a vida sexual; 2) Parâmetros hormonais: dosagem sérica de TT, testosterona livre, Estradiol (E2), LH, hormônio folículo estimulante (FSH), SHBG, relação TT:E2; 3) Parâmetros de composição corporal: IMC, circunferência abdominal (CA) e análise de bioimpedanciometria; 4) Parâmetros metabólicos: pressão arterial sistólica e diastólica, glicemia em jejum (GJ), hemoglobina glicada (HbA1c), índice HOMA-IR, colesterol total e frações, triglicérides; 5) Parâmetros de resposta CV: dilatação fluxo mediada artéria braquial (FMDAB), níveis circulantes de sICAM-1, sVCAM-1, Selectina-sE e quantificação de células endoteliais progenitoras (CEPs) por citometria de fluxo; 6) Efeitos adversos: hematócrito, antígeno prostático específico sérico (PSA), questionário internacional de sintomas prostáticos (I-PSS), dosagem sérica de alanina aminotransferase (ALT), aspartato aminotransferase (AST), e efeitos adversos autorreferidos. RESULTADOS: Na randomização os dois grupos foram semelhantes em relação à idade (CC: 35,5±7,8 anos, PLB: 35,6±7,8; p= 0,951), IMC (CC: 45,5±11,3 kg/m2; PLB: 47,2±9,6; p= 0,470), CA (CC: 137,5±17,9 cm; PLB: 140,2±19,6; p= 0,526) e testosterona total (CC: 225,8±70,0 ng/dL; PLB: 216,0±72,1; p= 0,543). Não houve diferenças nos parâmetros de resposta clínica, exceto com relação à queixa de perda de vigor nas ereções (p < 0,001). Observou-se elevação significativa (p= < 0,001) de TT, Testosterona livre, E2, LH, FSH e SHBG no grupo CC em comparação com PLB. Houve um aumento significativo (p < 0,001) na massa magra e na massa muscular; e também na massa livre de gordura (p= 0,004). O CC reduziu HDL em comparação com PLB (p < 0,001) e não mostrou efeito em outros parâmetros metabólicos. Não houve significância estatística nos parâmetros CV, indicando efeito nulo do tratamento. CC reduziu ALT (p < 0,001) e aumentou o PSA (p= 0,023) dentro dos limites da normalidade. CONCLUSÕES: CC foi efetivo para melhorar os parâmetros de resposta hormonal e afetou positivamente um parâmetro de resposta clínica (perda de vigor nas ereções). Apesar das alterações na composição corporal, não se observou melhora do perfil metabólico. No entanto, o CC não ocasionou resposta adversa nos parâmetros CV. O tratamento CC para HG parece ser uma alternativa efetiva em jovens obesos que desejam preservar sua fertilidade, mas ensaios clínicos de seguimento em longo prazo e com maior número de participantes são necessários para melhor análise do perfil metabólico e de sintomas, além de impactos CV
INTRODUCTION: Obesity can cause secondary hypogonadism in man. The standard testosterone replacement therapy (TRT) improves metabolic parameters but can lead to infertility. Only recently TRT was not clearly associated with adverse cardiovascular (CV) events, but its impacts on endothelial function are still controversial. AIM: To evaluate the effects of Clomiphene Citrate (CC) in out clinic young man with obesity related hypogonadism: total testosterone (TT) <= 300 ng/dL on two occasions, positive symptoms in ADAM questionnaire, Luteinizing Hormone (LH) low or inappropriate normal (RV: 1.7-8.6 IU/liter). METHODS: This is a randomized, double blind, placebo-controlled, parallel group, single-center study. Seventy eight patients aged 36.5±7.8 years, Body mass index (BMI) 46.2±8.5 kg/m2 were randomized (1:1) to receive CC 50 mg or Placebo (PLB) during 12 weeks. MAIN OUTCOME MEASURES: 1) Clinical symptomology: ADAM Questionnaire, number of sexual intercourses and satisfaction with sexual life; 2) Hormonal monitoring: serum TT, Free testosterone, Estradiol (E2), LH and Follicle-stimulating hormone (FSH), SHBG, TT/E2 ratio; 3) Body composition and anthropometric measurements: BMI, waist circumference (WC) and Bioelectric Impedance analysis parameters; 4) Metabolic response parameters: systolic and diastolic blood pressure, fasting blood glucose (FBG), glycated hemoglobin (HbA1c), serum cholesterol and fractions, triglycerides; 5) CV assessment by endothelial function parameters: Flowmediated dilatation of the brachial artery (FMDAB), circulating levels of sICAM-1, sVCAM-1, E-selectin and flow cytometry endothelial progenitor cells (EPCs); 6) Adverse outcomes: Hematocrit, serum Prostate-Specific Antigen (PSA), International Prostate Symptom Score (I-PSS), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Selfreported Adverse Effects. RESULTS: Two groups were similar with regard to age (CC: 35.5±7.8 years; PLB: 35.6±7.8; P=0.951), BMI (CC: 45.5±11.3 kg/m2; PLB: 47.2±9.6; P=0.470), WC (CC: 137.5±17.9 cm; PLB: 140.2±19.6; P=0.526) and total testosterone (CC: 225.8±70.0 ng/dL; PLB: 216.0±72.1; P=0.543) in baseline data. There was an improvement in one sexual complaint (weaker erections) (P < 0.001) and there were significant improvements (P < 0.001) in TT, Free Testosterone, E2, LH, FSH and SHBG in CC group (vs. PLB). There was a gain in lean mass (P < 0.001), free fat mass (P=0.004) and muscle mass (P < 0.001). CC reduced HDL compared to PLB (P < 0.001) and showed no effect in other metabolic parameters. No statistical significance was seen in CV parameters. CC reduced ALT (P < 0.001) and increased PSA (P=0.023). CONCLUSIONS: CC was effective in increase hormonal response parametersand improved one sexual complaint (weaker erections). Despite body composition changes, CC did not improved metabolic profile and lowered LDL cholesterol. CC showed no adverse response in CV parameters. CC treatment for HG appears to be an effective alternative in young obese men wishing to preserve their fertility but long-term follow-up trials to better analyze the metabolic profile and CV outcomes are needed

Orientador: Marcia Ortiz Mayo Marques
Banca: Beatriz Helena Lameiro de Noronha Sales Maia
Banca: Filipe Pereira Giardini Bonfim
Resumo: Lippia alba (Mill.) N.E.Br, pertencente à família Verbenaceae é uma planta medicinal amplamente distribuída na América do Sul. A composição química do óleo essencial da espécie, a qual derivam suas propriedades terapêuticas mostra-se variável, com divisão em quimiotipos. Entre eles, o quimiotipo citral destaca-se pelas ações calmante e ansiolítica, sendo suas folhas empregadas na medicina popular na forma de chá. Dessa forma, visando o potencial uso econômico de Lippia alba, este estudo teve como objetivos avaliar clones de Lippia alba do quimiotipo citral quanto a produtividade de biomassa e óleo essencial, perfil sensorial do chá e composição química do óleo essencial pelas técnicas de cromatografia gasosa unidimensional (CG) e bidimensional abrangente (CG×CG). Entre os clones, dois mostraram-se promissores para inserção em sistemas de produção, com foco em maior produção de biomassa e óleo essencial. Os perfis químicos dos óleos essenciais dos clones são variáveis entre si. O emprego do sistema CG×CG resultou em maior capacidade de detecção dos constituintes dos óleos essenciais. Ao todo foram identificadas 47 substâncias por CG×CG, contra 26 no CG. A maior sensibilidade e resolução fazem do sistema CG×CG uma importante ferramenta metabolômica no estudo dos constituintes voláteis de Lippia alba. Os clones X6M?C e X6M7 se destacaram quanto as características sensoriais de aroma e sabor, com potencial de inclusão na indústria alimentícia na forma de chá.
Abstract: Lippia alba (Mill.) N.E.Br, from the Verbanaceae family is a medicinal plant widely distributed in the South America. The species's essential oil chemical composition, which derives its therapeutic properties, shows itself as variable, with division in chemotypes. Among them, the citral chemotype highlights itself for the soothing and anxiolytic actions, being its leaves used in the popular medicine in the form of tea. Thus, aiming the potential economic use of the Lippia alba, this work has as objectives evaluate the clones of the citral chemotype of Lippia alba in terms of productivity of biomass and essential oil, sensorial profile and chemical composition of the essential oil by using the one-dimensional gas Chromatography (CG) and comprehensive twodimensional gas chromatography(CGxCG). Among the clone, two stood out as promising to insertion in production systems, with focus on a bigger production of biomass and essential oil. The chemical profiles of the clones's essential oil are variable among themselves. The use of the CGxCG system have resulted in a bigger capacity of identification of the essential oils constituents. Altogether, 47 substances have been identified by CGxCG, against 26 in CG. The better sensibility and resolution are part of the CGxCG, an important metabolomics tool of the volatile constituents of Lippia alba. The X6M?C e X6M7 clones stood out in terms of sensorial features of flavor, with potential of inclusion in the food industry in the form of tea.
Mestre

The influence of body size on metabolic rate, muscle enzyme activities, and the underlying patterns of mRNA for these enzymes were explored in an effort to explain the genetic basis of allometric variation in metabolic enzymes. Two pairs of sister species of centrarchid fishes were studied: black bass (largemouth bass, Micropterus salmoides and smallmouth bass, Micropterus dolomieui), and sunfish (pumpkinseed, Lepomis gibbosus and bluegill, Lepomis macrochirus). The goal was to assess the regulatory basis of both intraspecific and interspecific variation in relation to body size, as well as gain insights into the evolutionary constraints within lineages. Whole animal routine metabolic rate showed scaling coefficients not significantly different from 1, ranging from +0.87 to +0.96. However, there were significant effects of body size on the specific activities of oxidative and glycolytic enzymes. Mass-specific activity of the oxidative enzyme citrate synthase (CS) scaled negatively with body size in each species, with scaling coefficients ranging from -0.15 to -0.19 whereas the glycolytic enzyme pyruvate kinase (PK) showed positive scaling, with scaling coefficients ranging from +0.08 to +0.23. The ratio of mass-specific enzyme activity in PK to CS increased with body size, whereas the ratio of mRNA transcripts of PK to CS was unaffected, suggesting the enzyme relationships were not due simply to transcriptional regulation of both genes. The mass-dependent differences in PK activities were best explained by transcriptional regulation of the muscle PK gene; PK mRNA was a good predictor of PK specific enzyme activity within species and between species. Conversely, CS mRNA did not correlate with CS specific enzyme activities, suggesting post-transcriptional mechanisms may explain the observed inter-specific and intraspecific differences in oxidative enzymes.
Thesis (Master, Biology) -- Queen's University, 2007-10-31 11:55:28.757

Psychopharmaca are a large group of drugs widely used not only in psychiatry. Their systemic administration affects both the main diagnosis and the organism as a whole. The subject of our experiments is the effect of psychopharmaca on the changes in mitochondrial functions, which is beneficial for understanding of molecular mechanisms of therapeutic and adverse effects of drugs. The aim of this thesis was to study the in vitro effects of selected drugs on the cell energy metabolism. Selected antipsychotics (chlorpromazine, levomepromazine, haloperidol, risperidone, ziprasidone, zotepine, aripiprazole, clozapine, olanzapine, and quetiapine), antidepressants (bupropion, fluoxetine, amitriptyline, imipramine) and mood stabilizers (lithium, valproate, valpromide, lamotrigine, carbamazepine) were tested. In vitro effects of selected psychopharmaca were measured on isolated pig brain mitochondria. The activities of citrate synthase (CS) and electron transport chain (ETC) complexes (I, II+III, IV) were measured spectrophotometrically. Drug-induced changes of mitochondrial respiration rates linked to complex I (supported by malate and pyruvate) and complex II (supported by succinate) were evaluated by high resolution respirometry. Complex I was significantly inhibited by lithium, carbamazepine, fluoxetine,...

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.