Orientador: Márcio Alberto Torsoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:21:53Z (GMT). No. of bitstreams: 1 Pereira_ViniciusDias_M.pdf: 1179657 bytes, checksum: 3d71a3a82020acdf4b2e2a10b8eabfdf (MD5) Previous issue date: 2013 / Resumo: A AMPK hipotalâmica age como um sensor energético e é capaz de modular a ingestão alimentar, homeostase de glicose e a biossíntese de ácidos graxos. É conhecido que a injeção intra-hipotalâmica de ácidos graxos suprime a produção de glicose pelo fígado, principalmente pela ativação de canais de potássio sensíveis a ATP hipotalâmico (K(ATP)). Uma vez que em todos os modelos estudados a biossíntese de malonil-CoA estava envolvida, nós hipotetizamos que a Acetil-CoA Carboxilase poderia modular respostas contra-regulatórias independente da disponibilidade de nutrientes. Nesse estudo foram empregados os seguintes métodos: Immunoblot, PCR em tempo real, ELISA e avaliações bioquímicas. Através desses métodos, nós mostramos que a redução da expressão de acetil-CoA carboxilase pela injeção de oligonucleotídeo antisense intraventricular resultou no aumento da ingestão alimentar e diminuiu a expressão dos mRNA de CART, CRH e TRH. Além disso, como nos ratos em jejum, os ratos tratados com oligonucleotídeo antisense apresentaram concentrações de corpos cetônicos e glucagon séricos aumentados, além de níveis de insulina e glicogênio hepático diminuídos. A redução de acetil-CoA carboxilase hipotalâmica também aumentou a expressão de PEPCK, fosforilação de AMPK e a produção de glicose no fígado. Interessantemente, esses efeitos foram observados sem modificação da fosforilação da AMPK hipotalâmica. Com isso, concluímos que a inibição da ACC hipotalâmica pode ativar resposta contra-regulatória hepática independente da ativação da AMPK hipotalâmica / Abstract: BACKGROUND: Hypothalamic AMPK acts as a cell energy sensor and can modulate food intake, glucose homeostasis, and fatty acid biosynthesis. Intrahypothalamic fatty acid injection is known to suppress liver glucose production, mainly by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels. Since all models employed seem to involve malonyl-CoA biosynthesis, we hypothesized that acetyl-CoA carboxylase can modulate the counter-regulatory response independent of nutrient availability. METHODOLOGY/PRINCIPAL FINDINGS: In this study employing immunoblot, realtime PCR, ELISA, and biochemical measurements, we showed that reduction of the hypothalamic expression of acetyl-CoA carboxylase by antisense oligonucleotide after intraventricular injection increased food intake and diminished the expression of CART, CRH, and TRH mRNA. Additionally, as in fasted rats antisense oligonucleotide-treated rats, increased serum glucagon and ketone bodies were observed along with diminished levels of serum insulin and hepatic glycogen. The reduction of hypothalamic acetyl-CoA carboxylase also increased PEPCK expression, AMPK phosphorylation, and glucose production. Interestingly, these effects were observed without modification of hypothalamic AMPK phosphorylation. CONCLUSION/SIGNIFICANCE: Hypothalamic ACC inhibition can activate hepatic counter-regulatory response independent of hypothalamic AMPK activation / Mestrado / Clinica Medica / Mestre em Clinica Medica

<p>  </p> <p>Biosynthesis of fatty acids and specialized metabolites, such as polyketides, is dependent on the C-C bond forming enzymatic activity of carboxylases and <u>k</u>eto<u>s</u>ynthases (KS). Carboxylases and KS perform complex carbon-carbon bond forming reactions via a ping-pong mechanism; the catalytic interactions of which are still unclear. The KS reaction involves the Claisen condensation of an acylated enzyme with a malonyl-thioester, driven forward by the energy of the malonyl-thioester decarboxylation. Similarly, the carboxylase proceeds via a carboxyl-biotin-enzyme intermediate, and a subsequent C-C bond forming reaction. Engineering the substrate specificity of these enzyme involved in producing polyketides is sought after for the purpose of producing novel, derivative polyketides. These derivative polyketides may have serve as effective new antibiotics, of which discovery has waned. Unfortunately, incomplete understanding of protein-protein interactions, conformational changes, and substrate orientation in catalysis leads to not well informed engineering attempts. A challenge in deducing the catalytic details of enzymes acting on malonyl-thioesters in general is the hyper-reactivity of their β-ketoacid and thioester substrates, which are prone to hydrolysis and decarboxylation. Many structures of malonyl-CoA bound enzymes feature hydrolysis of the thioester, preventing determination of enzyme:substrate interactions in structure-function studies. To work around this problem of innate reactivity, groups have synthesized a variety of acyl-thioester analogs for probing the details of enzyme catalysis with mixed success. The success of these enzyme:analog mechanistic studies appears to hinge upon the similarity of the analog to the natural substrate. Here, we demonstrate the synthesis of near-natural, acyl-thioester analogs, featuring single atom substitutions. Using a novel UV-vis assay, we have determined <em>K</em>i values of our analogs with paradigmatic KSs <em>E. coli</em> FabH. These <em>K</em>i values are marginally higher than the substrate <em>K</em>m values, suggesting the KSs bind the analogs as they would natural substrates. Using this information, we have conducted preliminary X-ray crystallography experiments to determine the carboxylase:analog and KS:analog catalytic interactions, which will allow for new insight into debated C-C bond forming catalytic details. The information presented in this thesis and additional studies on protein-protein interactions can be leveraged into informed engineering studies of PKS enzymes.</p>

The effect of exogenous unsaturated fatty acids on cellular fatty acid biosynthesis in mammary cells was examined. Under normal situations, even though the diet of a dairy cow contains considerable amounts of unsaturated fatty acids, viz. oleic acid (18:1) and linoleic acid (18:2), the major 18-carbon fatty acid that enters the circulation post-ruminally for delivery to the mammary gland is saturated fatty acid, viz. stearic acid (18:0). This is due to extensive ruminal biohydrogenation of unsaturated fatty acids. Studies have indicated that saturated fatty acids such as 18:0 are enhancers and that certain unsaturated fatty acids are inhibitors of de novo fatty acid synthesis in tissues such as the liver and adipose tissue. The present study investigated the effect of cis and trans isomers of 18:1 and 18:2 on de novo fatty acid synthesis and desaturation in mouse and bovine mammary epithelial cell cultures, and compared it with the effect caused by 18:0. In the first experiment 12.5, 25, 50 or 100 micromoles stearic acid (SA), oleic acid (OA), elaidic acid (EA), trans-vaccenic acid (TVA), linoleic acid (LA) or conjugated linoleic acid (CLA) were supplemented in the media of mouse mammary epithelial (MME) cells that were grown to confluence in Dulbecco's modified Eagle's medium (DMEM). As indicated by cellular palmitic acid (16:0) content and fatty acid synthetase (FAS) activity, when compared with SA all unsaturated fatty acid treatments inhibited de novo fatty acid synthesis in MME cells. In addition, OA at all concentrations and LA and CLA at 50 and 100 micromoles inhibited cellular stearoyl-CoA desaturase (SCD) activity and mRNA abundance. However, EA and TVA, when compared with SA, enhanced SCD activity and mRNA abundance at 12.5 and 25 micromoles. In the second experiment 25, 50 or 100 micromoles SA, OA, TVA, LA or CLA were supplemented in the media of bovine mammary epithelial cells that were grown to confluence in DMEM. As indicated by cellular 16:0 content, acetyl-CoA carboxylase (ACC) activity and FAS activity, treatment with the unsaturated fatty acids inhibited de novo fatty acid synthesis at all concentrations, when compared with SA. Unsaturated fatty acid treatments also reduced the abundance of ACC and FAS mRNA in the cells. When compared with SA at all treatment-concentrations, OA and LA inhibited whereas TVA and CLA enhanced cellular SCD activity and mRNA abundance in the bovine cells. In both cell types, CLA and TVA appeared to be the most potent inhibitors of saturated fatty acid biosynthesis. / Ph. D.

Conjugated linoleic acids (CLA) have been shown to have remarkable yet inconsistent metabolic effects in mice, rats, hamsters, chickens, cattle, and humans. In particular, effects on lipogenesis vary with tissue, physiological state and specie. In this study we tested the hypothesis that CLA would differentially affect ducks of the same genetic background but of differing age. Growing (7 wk) and maintenance (11 wk) Moulard ducks were grouped by age and fed a standard diet supplemented with either 5% soybean oil (control) or 5% CLA isomer mixture. Animals were harvested after 3 weeks or 6 weeks for assessment of body composition including adipose, liver, viscera, and empty carcass weight. Serum nonesterified fatty acid (NEFA) and glucose concentrations were evaluated, and gene targets were cloned from the duck to use in quantifying mRNA abundance for genes involved in lipogenesis (fatty acid synthase, FAS; acetyl-CoA carboxylase, ACC) and lipid oxidation (carnitine palmitoyl transferase-1, CPT-1) in liver tissue from maintenance animals. After 3 weeks, the growing CLA group exhibited a 24% decrease in dissectible adipose tissue (P < 0.05) while maintenance animals showed no significant diet effect. After 6 weeks, the growing CLA group exhibited a 20% increase in liver mass compared to the control (P < 0.05), but no diet effect on adipose tissue. Maintenance animals receiving dietary CLA had a 42% decrease in adipose tissue mass after 6 weeks, increased serum NEFA, ACC and CPT-1 mRNA after 3 and 6 weeks (P < 0.05), and increased FAS mRNA after 3 weeks of treatment (P < 0.05). These data indicate that CLA have potent effects on lipid metabolism in ducks, but that these effects differ dependent on physiological age.

Background: Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) via Ser108 of the AMPK β1 subunit, a mechanism that is distinct from metformin, which increases AMP:ATP. Many cancers have high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is unknown if targeting the AMPK-ACC-lipogenic pathway using salicylate and metformin may be effective for inhibiting cancer cell survival. Results: Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations of aspirin. These clinically achievable concentrations of salicylate activated AMPK per the increasing phosphorylation of ACC and suppressing the activity of mTOR effectors kinase p70-S6 kinase and S6; effects that were enhanced with the addition of metformin and blunted in mouse embryonic fibroblasts (MEFS) deficient in AMPK β1. MEF cells deficient in AMPK β1 were more resistant to salicylates inhibitory effect on proliferation. Supplementation of media with fatty acids and mevalonate reverses the suppressive effects on cell survival indicating the inhibition of de novo lipogenesis is likely important. Conclusions: Salicylate increases ACC phosphorylation, reduces phosphorylation of mTOR targets and inhibits de novo lipogenesis in prostate and lung cancer cells, with concentrations of salicylate achievable through the ingestion of Aspirin (0.25-1.0mM) these effects are blunted in AMPK β1 deficient cells. Effects on AMPK activity via ACC phosphorylation as well as reductions in mTOR signalling targets and de novo lipogenesis are enhanced when used in combination with metformin. Suppressive effects on prostate and lung cancer cell survival are ameliorated when media is supplemented with mevalonate and fatty acids. Pre-clinical studies evaluating the use of salicylates alone and with metformin to inhibit de novo lipogenesis and the growth of prostate and lung cancers are warranted. / Thesis / Master of Science (MSc)

Thesis (PhD (Plant biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In soils, the concentration of available phosphate (P) for plants is normally very low (ca. 1 µM in the soil solution), because most of the P combines with iron, aluminium and calcium to form relatively insoluble compounds. Inorganic P (Pi)-deficiency is thought to be one of the limiting factors of nitrogen fixation due to the high energy requirement for nitrogenase function of plants taking part in nitrogen fixation. Pideficiency has important implications for the metabolic Pi and adenylate pools of plants, which influence respiration and nitrogen fixation. An alternative route of pyruvate supply during Pi stress has been proposed involving the combined activities of phosphoenolpyruvate carboxylase (PEPc), malate dehydrogenase (MDH) and NAD-malic enzyme (ME) supplying pyruvate to the mitochondrion during Pi stress. Previously, three isoforms of PEPc were isolated from lupin nodules and roots, with two forms being nodule specific. The aim of this project was to determine the effect of Pi stress on these PEPc isoforms in Lupinus luteus at transcript and protein expression level with a view to produce genetically modified crops for nutrient-poor soils. Cytosolic P levels were measured over a time course to give an indication of temporal development of P stress in nodules. The changes in enzyme activities of PEPc, MDH and PK (pyruvate kinase) under P stress were measured and the downstream effect on amino and organic acid pools were analysed. Two novel PEPc isoforms, LUP1 (AM235211) and LUP2 (AM237200) were isolated from nodules, followed by transcriptional and protein expression analyses. Nodules under P stress had lower amounts of metabolically available Pi and as P stressed developed, the amount of Pi decreased. This decline in Pi levels was associated with lower growth, but higher biological nitrogen fixation (BNF). A greater proportion of root-nodule respiration was devoted to nutrient acquisition than to new growth. A typical P-stress response is higher anaplerotic carbon fixation via PEPc. However, in this study, no significant differences were found for PEPc, MDH or PK in P-stressed plants compared to P-sufficient plants which would lead to an increase in organic acids. An increase in key amino acids was reported along with unchanged levels of organic acids. These levels of organic and amino acid are in congruence with the increases in BNF under P-starvation. No significant differences were found in expression of PEPC1 or PEPC2 at 12 and 20 days for both P-sufficient and P-stressed plants which further supported the lack of engagement of the PEPc-MDH-ME bypass. PEPc activity appeared not to be regulated by gene expression or phosphorylation indicating that other posttranslational modifications such as a decrease in protein degradation may be of importance. / AFRIKAANSE OPSOMMING: Die konsentrasie van fosfaat (P) beskikbaar vir opname deur plante vanuit die grond is gewoonlik baie laag (in die omgewing van 1 µM) aangesien die P onoplosbare komplekse vorm met katione soos yster, aluminium en kalsium. ‘n Tekort aan anorganiese P (Pi) word gereken as een van die beperkende faktore van stikstofbinding as gevolg van die hoë energie behoefte wat nitrogenase plaas op plante wat van gefikseerde stikstof gebruik maak. Hierdie P-tekort het ook belangrike betrekking op die metaboliese fosfaat- en adenilaatpoele wat weer op hul beurt respirasie en stikstofbinding beÏnvloed. ‘n Alternatiewe roete van pirovaatvoorsiening aan mitochondria tydens fosfaatstres is voorgestel wat bestaan uit die aktiwiteite van fosfoenolpirovaat karboksilase (PEPc), malaat dehidrogenase en NAD-malaat ensiem. Vantevore is drie isovorme van PEPc uit Lupinus luteus wortelknoppies en wortels geïsoleer, met twee van die isovorme wat wortelknoppie-spesifiek was. The doel van hierdie projek was om die invloed van P-tekort op die transkripsie en proteien uitdrukkingsvlak van hierdie PEPc isovorme te bepaal met die doel van gemodifiseerde gewasse vir arm gronde ingedagte. Sitoplasmiese P konsentrasies is gemeet oor tyd om ‘n aanduiding te gee van die ontwikkeling van P-tekort oor tyd. Veranderinge in ensiemaktiwiteite van PEPc, MDH en pirovaatkinase (PK) is gemeet gedurende P-tekort as ook die moontlike effek van hierdie ensiemaktiwiteite op aminosuur en organiese suur poele. Twee nuwe PEPc isovorme, LUP1 (AM235211) en LUP2 (AM237200) is uit wortelknoppies geïsoleer en gekarakteriseer. Transkripsie en proteïenuitdrukking is geanaliseer. Wortelknoppies wat P-tekort behandeling ontvang het, het laer vlakke van metabolise beskikbare Pi gehad en soos die P-tekort ontwikkel het oor tyd, het die Pi vlakke gedaal. Hierdie afname in vlakke van Pi was geassosieer met laer groei, maar met ‘n toename in biologiese stikstofbinding. ‘n Groter proporsie van respirasie is toegestaan aan minerale opname as aan nuwe groei. ‘n Tipiese reaksie op P-tekort is hoër anaplerotiese koolstofbinding via PEPc. Alhoewel, in hierdie studie is geen gevind betekenisvolle verandering gevind in die aktiwiteite van PEPc, MDH en PK nie in plante wat P-tekort ervaar het nie. Verhoogde aktiwiteit van hierdie ensieme sou verhoogde organise suur konsentrasies tot gevolg hê. ‘n Toename in aminosuur konsentrasies is gevind tesame met onveranderde vlakke van organiese sure. Hierdie toename in aminosure word onderskryf deur die verhoogde biologiese stikstofbinding tydens P-tekort. Geen betekenisvolle verskille is gevind in die geenuitdrukking van pepc1 en pepc2 by beide 12 en 20 dae van P-tekort nie, wat verder die afwesigheid van die PEPc- MDH-ME alternatiewe roete beaam het. Dit blyk dat PEPc aktiwiteit nie deur geenuitdrukking of proteïenfosforilering beheer word nie, maar eerder dat ander posttranslasie modifikasies soos ‘n verlaagde afbraak van proteïen ‘n rol speel.

Electron crystallography of two-dimensional crystals is a structure-determination method well suited to the study of membrane protein structure-function. Two-dimensional crystals consist of ordered arrays of protein within reconstituted lipid bilayers, an arrangement that mimics the natural membrane environment. In this work we describe our recent progress in the use of this method with three different proteins, each providing a window into a separate paradigm in the electron crystallographic pipeline. Specific crystallization conditions for human leukotriene C₄ synthase (LTC₄S) have previously been determined, but our continued refinement of purification and crystallization has identified a number of additional parameters that greatly affect crystal size and quality, and we have developed a protocol to rapidly and reproducibly grow large, non-mosaic crystals of LTC₄S. The human gamma-glutamyl carboxylase (GGCX) has also been crystallized, but is sensitive to cryo-EM sample preparation conditions and we present here the successful reproduction of crystallization and refinement of cryo-EM sample preparation conditions. Lastly, we describe our crystallization screens with the Vibrio cholerae sodium-pumping NADH:ubiquinone reductase complex (Na⁺-NQR), and identify the factors critical to membrane reconstitution of the complex, a necessary first step towards crystallization. We also describe a semi-quantitative crystal screening protocol we have developed that provides quick and accurate method to assess two- dimensional crystallization trials, and discuss some general observations in optimization of membrane protein purification and two-dimensional crystallization for electron crystallography.

Post-translational redox-regulation is a well-known mechanism to regulate enzymes of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP synthesis in response to light. The aim of the present thesis was to investigate whether a similar mechanism is also regulating carbon storage in leaves. <br><br> Previous studies have shown that the key-regulatory enzyme of starch synthesis, ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to regulate starch synthesis in leaves was not investigated. The work presented in this thesis shows that AGPase is subject to post-translational redox-regulation in leaves of pea, potato and Arabidopsis in response to day night changes. Light was shown to trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted from a dimer to a monomer when isolated pea chloroplasts were illuminated and from a monomer to a dimer when preilluminated leaves were darkened. Conversion of AGPB from dimer to monomer was accompanied by an increase in activity due to changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts showed that AGPase redox-activation is mediated by thioredoxins f and m from spinach in-vitro. In a further set of experiments it was shown that sugars provide a second input leading to AGPase redox activation and increased starch synthesis and that they can act as a signal which is independent from light. External feeding of sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to conversion of AGPB from dimer to monomer and to an increase in the rate of starch synthesis, while there were no significant changes in the level of 3PGA, an allosteric activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the precursor of trehalose synthesis, provided genetic evidence that T6P rather than trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased activation of AGPase and higher starch level during the day, while trehalose-phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These changes occurred independently of changes in sugar and sugar-phosphate levels and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing leaves led to monomerisation of AGPB, while this response was attenuated in TPP expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-activation of AGPase. To provide biochemical evidence that T6P promotes redox-activation of AGPase independently of cytosolic elements, T6P was fed to intact isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls, significantly and specifically increased AGPB monomerisation and AGPase activity within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio consistent with T6P modulating redox-transfer to AGPase independently of changes in plastidial redox-state. <br><br> Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid and lipid synthesis in plants. At the start of the present thesis there was mainly in vitro evidence in the literature showing redox-regulation of ACCase by DTT, and thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to regulate lipid synthesis in leaves was investigated. ACCase activity measurement in leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a 3-fold higher activation state of the enzyme in the light than in the dark. Redox-activation was accompanied by change in kinetic properties of ACCase, leading to an increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as sucrose were fed to leaves, and both treatments led to a stimulation in the rate of lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-CoA content. <br><br> In a final approach, comparison of metabolic and transcript profiling after DTT feeding and after sucrose feeding to leaves provided evidence that redox-modification is an important regulatory mechanism in central metabolic pathways such as TCA cycle and amino acid synthesis, which acts independently of transcript levels. / Es ist bereits seit längerem bekannt, dass viele Enzyme des Calvinzyklus, des oxidativen Pentosephosphatwegs, des NAD(P)H-Exports und der ATP-Synthese durch post-translationale Redox-Modifikation in Antwort auf Licht reguliert werden. In der vorliegenden Arbeit sollte untersucht werden, ob ein ähnlicher Mechanismus auch die Kohlenstoffspeicherung in Blättern reguliert. <br><br> Vorangegangene Studien mit Kartoffelknollen zeigten, dass das Schlüsselenzym der Stärkesynthese ADP-Glukose-Pyrophosphorylase (AGPase) durch die Bildung einer Disulfidbrücke zwischen den zwei kleinen Untereinheiten (AGPB) des tetrameren Proteins inaktiviert wird, die Bedeutung dieses Mechanismus für die Stärkesynthese in Blättern blieb jedoch bislang ungeklärt. Die vorliegenden Arbeiten zeigen, das AGPase in Erbsen-, Kartoffel- und Arabidopsis-Blättern über post-translationale Redox-Modifikation in Antwort auf Tag-Nacht Änderungen reguliert wird. Dies erfolgt über ein Licht-abhängiges Signal, da, erstens, AGPB in isolierten Chloroplasten durch Belichtung sehr schnell von Dimer zu Monomer umgewandelt wird und, zweitens, ein Abdunkeln der Blätter zu einer schnellen Umwandlung von AGPB von Monomer zu Dimer führt. Die Monomerisierung von AGPB ging mit Änderungen in den kinetischen Eigenschaften des Enzyms einher, die zu einer Aktivierung führten. Studien mit Extrakten aus Erbsenchloroplasten zeigten, dass die AGPase-Redoxaktivierung in-vitro durch die Thioredoxine f und m aus Spinat vermittelt wird. In einem weiteren experimentellen Ansatz konnte gezeigt werden, dass auch Zucker zu Redox-Aktivierung der AGPase und erhöhter Stärkesynthese in Blättern führen, und dass diese unabhängig von Licht wirken. Externe Zugabe von Zuckern wie Saccharose oder Trehalose an Arabidopsis-Blätter im Dunkeln führten zu Monomerisierung von AGPB und einer Erhöhung der Stärkesyntheserate / während die Spiegel des allosterischen Aktivators 3PGA unverändert blieben und keine Änderungen im NADPH/NADP+-Verhältnis auftraten. Experimente mit transgenen Arabidopsis-Pflanzen mit veränderten Spiegeln des Vorläufers der Trehalosesynthese, Trehalose-6-phosphat (T6P), zeigten, dass T6P und nicht Trehalose zu Redox-Aktivierung von AGPase führt. Expression einer E. coli T6P synthase (TPS) im Zytosol führte zu erhöhter Redox-Aktivierung von AGPase und erhöhter Stäreksynthese in Blättern, während die Expression einer T6P-Phosphatase (TPP) gegenteilige Änderungen bewirkte. Diese Auswirkungen erfolgten unabhängig von Änderungen in den Spiegeln von Zuckern und Zuckerphosphaten oder im NADPH/NADP+-Verhältnis. Externe Zugabe von Saccharose führte zu Monomerisierung von AGPB in Wildtyp und TPS exprimierenden Blättern, während diese Antwort in TPP exprimierenden Blättern stark abgeschwächt war. Dies zeigt, dass T6P eine wesentliche Komponente darstellt, die die Redox-Aktivierung der AGPase in Antwort auf Saccharose vermittelt. T6P wurde auch für 15 min direkt an intakte, isolierte Erbsenchloroplasten gefüttert. T6P Konzentrationen im Bereich von 100 µM bis 10 mM führten zu einem signifikanten und spezifischen Anstieg der AGPB-Monomersierung und der AGPase Aktivität. Dies zeigt, dass T6P auch ohne zytosolische Elemente die Redox-Aktivierung der AGPase stimuliert und somit ein Signal zwischen Zytosol und Plastid darstellt. Diese Antwort erfolgte ohne Änderungen im NADPH/NADP+-Verhältnis, was zeigt, dass T6P eher den Redox-Transfer zu AGPase als den Redoxzustand des Chloroplasten moduliert. <br><br> Acetyl-CoA-Carboxylase (ACCase) ist als Schlüsselenzym der Fettsäure- und Lipidsynthese in Pflanzen bekannt. Zu Beginn der vorliegenden Arbeit lagen hauptsächlich in-vitro Befunde vor, die zeigten, dass ACCase durch DTT und thioredoxine f und m über Redox-Modulation reguliert wird. In der Arbeit sollte daher die in-vivo Relevanz dieses Mechanismus für die Regulation der Lipidsynthese in Blättern untersucht werden. ACCase zeigte einen höheren Redox-Aktivierungszustand in Arabidopsis-Blätter, die während des Tages im Vergleich zur Nacht geerntet wurden. Die Redox-Aktivierung der ACCase wurde von Änderungen in den kinetischen Eigenschaften begleitet und führte zu einer erhöhten Affinität des Enzymes gegenüber Acetyl-CoA als Substrat. <br><br> In weiteren Versuchen wurde sowohl DTT als auch Saccharose an Blätter gefüttert, und beide Behandlungen führten zu Redox-Aktivierung von ACCase, was mit erhöhten Lipidsynthesraten und einem Rückgang des Acetyl-CoA-Spiegels einherging.

Orientador: Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T14:08:09Z (GMT). No. of bitstreams: 1 Kato_LarissaSayuri_M.pdf: 3085711 bytes, checksum: 2c9b592f72e52ceb332be3af215a87fe (MD5) Previous issue date: 2011 / Resumo: O estudo das vias de sinalização envolvidas no metabolismo energético é de grande relevância fisiológica, uma vez que um desequilíbrio da homeostase energética pode resultar em obesidade e/ou síndrome metabólica e aumento da mortalidade por doença cardiovascular. Estudos recentes de nosso grupo em três modelos experimentais que exibem distintos tipos de dislipidemias revelaram alterações significativas da composição corporal, gasto energético e padrão de ingestão alimentar. Neste trabalho estudamos a homeostase energética desses modelos dislipidêmicos avaliando: (1) a expressão e fosforilação da proteína quinase dependente de AMP (AMPK), um importante regulador do metabolismo energético, bem como de seu alvo, a enzima acetil-CoA carboxilase (ACC), em fígado e músculo esquelético de camundongos hipoalfalipoproteinêmicos e hipercolesterolêmicos e (2) o efeito da alimentação pareada em animais hipertrigliceridêmicos que apresentam alterações de comportamento alimentar, metabolismo corporal e maior atividade do canal mitocondrial de potássio sensível ao ATP (mitoKATP). Considerando os animais hipoalfalipoproteinêmicos (transgênicos para CETP), os quais apresentam aumento de gasto energético global, verificamos que estes apresentam redução da massa relativa dos depósitos adiposos quando comparados os controles wild type (WT). O estudo da ativação da AMPK e da ACC mostra que o estado energético dos tecidos muscular e hepático parece não diferir nos animais CETP e WT. Tanto no fígado como no músculo dos animais CETP não houve alteração da massa e do estado de ativação da AMPK e da ACC. Estes resultados sugerem que não ocorrem variações significativas na síntese, armazenamento e "exportação" de lípides no fígado destes animais. Em relação ao músculo sóleo, pode-se concluir que não há alteração de síntese e catabolismo lipídico nos animais CETP. De modo geral, podemos dizer que a sinalização da AMPK não está ativada nestes tecidos e, portanto, que o maior metabolismo corporal observado nestes animais deve estar sendo causado por outros tecidos do organismo, por exemplo, o próprio tecido adiposo. Em animais machos e fêmeas hipercolesterolêmicas (LDLR0) observamos redução da massa corporal, porém sem alteração significativa da massa relativa dos depósitos adiposos quando comparados aos animais controles wild type. Os resultados sobre ativação da AMPK e da ACC mostram que o estado energético em tecidos periféricos é diferente nos animais LDLR0 e controles (WT). No fígado das fêmeas hipercolesterolêmicas observamos aumento da ativação da AMPK sem alteração significativa da fosforilação da ACC. Isso significa que não houve inibição da lipogênese ou ativação da beta-oxidação no fígado dos animais hipercolesterolêmicos, embora possa ter havido aumento de catabolismo de outros nutrientes. No músculo sóleo das fêmeas e dos machos não houve alteração de fosforilação de ambas AMPK e ACC. Pode-se então concluir que não há deprivação energética no músculo destes animais. Considerando o estudo em animais hipertrigliceridêmicos (HTG), quando submetidos ao regime de paired feeding (PF) observamos uma redução de 17% no consumo alimentar nas fêmeas e nos machos HTG quando comparados aos HTG alimentados ad libitum (ad lib). Isso levou a uma redução significativa do ganho de peso dos HTG-PF comparados aos WT-ad lib, em ambos os sexos. Os animais HTG-PF mantiveram a massa dos depósitos adiposos da carcaça semelhantes aos WT-ad lib e HTG-ad lib. No entanto, o depósito adiposo visceral das fêmeas HTG-ad lib é menor que dos WT-ad lib, enquanto nos machos, os HGT-PF apresentaram maior adiposo visceral que os HTG-ad lib. Quando comparados aos WT-ad lib, verificamos que as fêmeas HTG-PF mantiveram aumento significativo da atividade (abertura) do canal de potássio mitocondrial sensível ao ATP (mitoKATP) e da produção corporal de CO2. No entanto, nos machos HTG-PF houve fechamento dos mitoKATP, redução da produção de CO2 e manutenção da massa corporal. Assim, pode-se inferir que o metabolismo corporal (produção de CO2) reflete o aumento do metabolismo celular causado por aumento da atividade do mitoKATP que desacopla levemente as mitocôndrias e que estas adaptações são revertidas pela restrição alimentar nos machos, mas não nas fêmeas HTG / Abstract: An imbalance of energy homeostasis can result in obesity and/or metabolic syndrome and increased mortality from cardiovascular disease. Recent studies by our group in three experimental models that exhibit different types of dyslipidemia have shown significant changes in body composition, energy expenditure and food intake. In this work we studied the energy homeostasis in these models through: (1) quantifying the expression and phosphorylation of AMP-dependent protein kinase (AMPK), a key regulator of energy metabolism, as well as its target, the enzyme acetyl-CoA carboxylase (ACC) in liver and skeletal muscle in hypoalphalipoproteinemic and hypercholesterolemic mice and (2) the effect of paired feeding regimen on hypertriglyceridemic mice that present increased food intake, body CO2 production and increased activity of the mitochondrial potassium channel sensitive to ATP (mitoKATP). Considering the hypoalphalipoproteinemic mice (transgenic for CETP), which show an increased overall energy expenditure, we found that these mice have reduced relative fat depot mass when compared to wild type controls (WT). Western blot analyses showed that, in both tissues, liver and muscle, there were no changes in mass and state of activation of AMPK and ACC in CETP compared to WT mice. These results suggest that no significant variations in the synthesis, storage and secretion of lipids in the liver of these mice. Regarding the soleus muscle, these results suggest that there is no change in lipid synthesis and catabolism in CETP mice. Overall we may say that AMPK signaling is not activated in liver and muscle tissues and, therefore, that the increased body metabolism observed in these CETP mice must be caused by other body tissues, for example, the adipose tissue itself. In hypercholesterolemic male and female mice (LDLR0) we observed a reduction in body mass, but no significant change in the relative mass of fat depots when compared to WT. The results on activation of AMPK and ACC show that the liver of LDLR0 females had increased activation of AMPK without significant change in the phosphorylation of ACC. This means that there was no inhibition of lipogenesis and activation of ?-oxidation in the liver of hypercholesterolemic mice, although there may have been increased catabolism of other nutrients. In the soleus muscle of females and males there were no changes in the phosphorylation state of both AMPK and ACC. Then, we can conclude that there is no energy deprivation in the muscle of these LDLR0 mice. Considering the study with hypertriglyceridemic (HTG) mice, when subjected to the paired feeding (PF) we observed a 17% reduction in food intake of females and males when compared to HTG mice fed ad libitum (ad lib). This led to a significant reduction in HTG-PF weight gain compared to WT-ad lib in both sexes. HTG-PF mice retained the mass of carcass fat deposits similar to WT and HTG ad lib. Compared to WT-ad lib, HTG-PF mice maintained significant increased activity (opening) of the mitoKATP and body CO2 production. These data showed that the regimen of paired feeding in which HTG mice were submitted did not change the high rate body metabolism and mitochondrial resting respiration observed in HTG-ad lib mice. These results suggest that the metabolic adaptation of HTG (higher activity of mitoKATP) is not sensitive to changes in food restriction and compromises the rate of body growth / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Roots live in and have evolved in a high carbon dioxide (CO₂) environment, yet relatively little research has been conducted on the impacts of soil dissolved inorganic carbon (DIC) on root metabolism. In this thesis, I explore the impacts of root-zone DIC on whole plant biomass accumulation, water use efficiency, and above-ground gas exchange. In addition, I explore the impacts of root-zone DIC on root processes: root PEP-Carboxylase activity, root respiration rate and root exudation of Krebs cycle organic acids. Root-zone DIC did not impact biomass accumulation, leaf gas exchange parameters or water use efficiency under the growth conditions examined. Root-zone DIC did increase root PEP-Carboxylase activity, but decreased root respiration (both CO₂ production and O₂ consumption) and decreased organic acid exudation rates. Increase in measurement CO₂ partial pressure was found to cause an instantaneous decrease in root CO₂ production, and I provide evidence that changes in root metabolism (CO₂ uptake by roots) are part of the cause of this phenomenon. A hypothesized relationship between root respiration rate and Krebs cycle organic acid exudation was not supported by my data. I conclude that root-zone DIC has important impacts on critical functions of root metabolism, and should be considered as an important abiotic factor much in the same way atmospheric CO₂ is for leaves and whole plant biology.