Orientador: Jaime Amaya-Farfán / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T15:34:35Z (GMT). No. of bitstreams: 1 Moura_CarolinaSoaresde_M.pdf: 1533232 bytes, checksum: 1141b4840c5a0f9342072967abfece65 (MD5) Previous issue date: 2012 / Resumo: As heat shock proteins (HSPs), ou proteínas do estresse, correspondem a um importante sistema de defesa celular que é capaz de proteger e reparar danos causados ao organismo, conferindo à célula maior tolerância e resistência contra situações de alteração na homeostase, sendo também consideradas como um sistema antioxidante complementar. A glutamina é conhecida pelo seu potencial em promover o aumento na HSP70 contra diversas situações agressoras. As proteínas do soro do leite (PSL) contêm concentrações elevadas de aminoácidos de cadeia ramificada (BCAAs), sendo estes substratos para a síntese de glutamina, por meio da enzima glutamina sintetase. Objetivo: o objetivo deste trabalho foi observar a influência do consumo das proteínas do soro do leite (PSL), na forma concentrada (PSLC) e hidrolisada (PSLH), na concentração da HSP70 em ratos exercitados em esteira rolante. Metodologia: foram utilizados 48 ratos Wistar machos (290g ± 8g) divididos primeiramente pelo regime de atividade física em sedentários (S) e exercitados (E), e cada um desses, subdividido em outros três grupos, segundo a dieta. As dietas foram baseadas na AIN93-G, com substituição da fonte protéica da seguinte forma: PSLC, PSLH e caseína (CAS), como controle. O período em dieta experimental foi de 3 semanas, e os animais exercitados foram submetidos a 5 sessões de exercício a 22m/min durante 30 minutos como fonte de estresse térmico, na última semana de vida. Resultados: os resultados revelaram que o consumo da PSLH no grupo exercitado promoveu o aumento da HSP70 em pulmão, e nos músculos sóleo e gastrocnêmio. O consumo da PSLH aumentou os níveis de glutamato, isoleucina e leucina livres no plasma dos animais sedentários. Quando exercitado, o grupo PLSH teve redução no glutamato, leucina e valina (substratos envolvidos na síntese de glutamina) plasmáticos e aumento da enzima glutamina sintetase (GS) no sóleo, sugerindo o provável uso desses aminoácidos para proporcionar o aumento na HSP70. Em adição à elevação da GS, houve também aumento concomitante da concentração de corticosterona no grupo PSLH exercitado, sugerindo a influência do hormônio na enzima. Em relação ao possível dano oxidativo, avaliado pela geração de proteínas carboniladas, os grupos que consumiram PSLC e PSLH reduziram seus valores no plasma e, somente a PSLH, no gastrocnêmio. Houve preservação das proteínas totais e albumina nos grupos PSLC, PSLH exercitados. O ácido úrico aumentou no grupo PSLH exercitado, enquanto que a creatinina aumentou na PSLC, independente do exercício. A glicose foi reduzida nos animais sedentários que consumiram PSLH, porém as variações dos parâmetros sempre permaneceram dentro da normalidade. Nenhum efeito adverso ao consumo das diferentes fontes protéicas foi observado no rim ou no fígado, oriundo da mensuração das enzimas AST, ALT e o metabólito ureia respectivamente. Conclusão: os resultados indicam que o consumo da PSLH pode potencializar a resposta da HSP70, sugerindo aumento na proteção endógena e antioxidante, e que a PSLH possa ser mais estresse-responsiva em ratos submetidos ao exercício / Abstract: The heat shock proteins (HSPs), or stress proteins, correspond to an important cell defense system, whose function is to protect and repair injuries caused to the body, conferring the cell greater tolerance and resistance against altered homeostasis states, and for this reason they have been considered as a complementary antioxidant system. Glutamine in turn has been found to promote the increase of HSPs associated to various situations of stress. The milk whey proteins contain elevated concentrations of branched-chain amino acids (BCAAs), which can participate in the synthesis of glutamine via glutamine synthetase. Objective: the objective of this work was to assess the influence of the intake of the whey proteins either in the form of a concentrate (WPC) or a hydrolyzate (WPH) in enhancing the concentration of HSP70 in rats exercised in the treadmill. Methods: Forty-eight male Wistar rats (290 ± 8g) were divided, first, into two categories according to the level of physical activity: sedentary (S) and exercised (E), and each one subdivided into three groups according to the source of protein in the diet. The diets were based on the standard AIN93-G, formulated containing either WPC, WPH or casein (CAS), as the sole source of protein. The animals consumed the experimental diets for three weeks and those belonging to the exercised group were submitted to training 5 sessions on the last week of life. Results: the results showed that consumption of the WPH promoted the increase of HSP70 in lung, soleus and gastrocnemius in the exercised animals. Increases in plasma free glutamate, isoleucine and leucine of the sedentary rats were also observed. When exercised, the WPH group exhibited a reduction in the plasma levels of glutamate, leucine and valine (all involved in the synthesis of glutamine), plus an increase in the enzyme glutamine synthetase (GS) in the soleus muscle, thus suggesting a probable utilization of this amino acids, as a substrate, in the increase of HSP70. Considering that there was also an elevation of the corticosterone levels in the exercised cohorts that consumed the WPH, the concomitant increase of GS, suggested that the hormone exerted an influence on the enzyme. With regard to a possible oxidative damage, as assessed by the presence of carbonyls proteins, the group that consumed both of the whey proteins (WPC, WPH) exhibited lower plasma levels, but only the WPH reduced the levels in the gastrocnemius. Both total plasma proteins and albumin were preserved in the exercised animals. Uric acid was found to increase in the WPH exercised group, while creatinine increased in the WPC group, regardless of the exercise. Plasma glucose levels were also lowered in the sedentary animals that consumed the WPH diet, but at no time, did the increased or decreased levels of these parameters extrapolated normality. Additionally, from the AST, ALT and urea data, no adverse effects on either liver or kidney could be detected with the intake of the different proteins sources. Conclusion: from these results, it can be concluded that consumption of the WPH, in contrast to WPC or CAS, can enhance the HSP70 response suggesting a magnified endogenous and antioxidant protection, and that the hydrolyzed whey protein can be more stress-responsive / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

An exceptionally important stress response of Pseudomonas putida strains to toxic chemicals is the induction of efflux pumps that extrude solvents, as well as other toxicants, into the surrounding medium. However, the bacterial tolerance mechanisms are still not fully understood, thus in this thesis metabolomic approaches were used to detect and identify metabolites involved in P. putida DOT-T1E tolerance to abiotic stresses, in particular focussing on the role of efflux pumps. To elucidate any metabolome alterations several strains of P. putida, including the wild type DOT-T1E, and the efflux pump knockouts DOT-T1E-PS28 and DOT-T1E-18, were challenged with different levels of propranolol. Fourier-transform infrared (FT-IR) spectroscopy, which provided a rapid, high-throughput metabolic fingerprint of P. putida strains, was used to investigate any phenotypic changes resulting from exposure to propranolol. FT-IR data illustrated phenotypic changes associated with the presence of propranolol within the cell that could be assigned to the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling on the same samples was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during growth of bacteria following this toxic perturbation with propranolol. GC-MS revealed significant changes in ornithine levels which can be directly linked to bacterial tolerance mechanisms, and alterations in the levels of several other metabolites which were also modified in response to propranolol exposure. Moreover, the effect of the organic solvent toluene was also investigated using the same approach. Examination of FT-IR data indicated that protein and fatty acids were the most affected components of P. putida strains due to the presence of toluene within the cell. Moreover, application of GC-MS allowed for the identification and quantification of several metabolites which were differentially produced or consumed in the presence of toluene. To investigate the role of efflux pumps in P. putida DOT-T1E, several analytical techniques were employed including Raman spectroscopy, gas and liquid chromatography to identify and quantify the level of propranolol or toluene in P. putida cells. These analyses showed that propranolol and toluene accumulated in the mutant P. putida DOT-T1E-18 (lacking the TtgABC pump) at higher levels in comparison with the levels found in the wild-type DOT-T1E and the mutant DOT-T1E-PS28 (lacking the TtgGHI pump), indicating the key role of efflux pumps in solvent tolerance. Furthermore, the effect of Mg2+ and Ca2+ on the stabilisation of the toluene tolerance of P. putida DOT-T1E strains was examined in order to elucidate whether divalent cations interact with efflux pumps or other resistant mechanisms to improve solvent tolerance. FT-IR analysis suggested that the influence of divalent cations on the stabilisation of the toluene tolerance could be due to the contribution of metal ions towards other tolerance mechanisms such as lipopolysaccharide (LPS) instead of enhancing the activity of efflux pumps. In conclusion, this thesis presents evidence that phenotypic fingerprinting and metabolic profiling approaches in combination with chemometric methods can generate valuable information on phenotypic responses occurring within microbial cultures subjected to abiotic stress.

Nitrogen Catabolite Repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae reduces the expression of genes encoding components involved in the utilization of poor nitrogen sources when rich ones are available. Expression of NCR-sensitive genes is controlled by the negative regulator Ure2 and four DNA-binding GATA-like transcription factors: two activators (Gln3 and Gat1) and two repressors (Dal80 and Gzf3). In the presence of preferred nitrogen sources, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner, whereas upon growth under non-preferred nitrogen conditions, the GATA activators relocate to the nucleus and mediate the transcription of NCR-sensitive genes. Even though the Target of Rapamycin Complex 1 (TORC1) as well as several phosphatases are involved in regulating Gln3 and Gat1 subcellular localization, a detailed mechanistic understanding of the NCR process is still lacking. <p>In the first part of this work, we have shown that class C and D VPS (vacuolar protein sorting) components, involved in Golgi-to-vacuole vesicular trafficking, are required for intact Gat1 and Gln3 nuclear localization in response to TORC1-inhibiting rapamycin treatment or upon shifting cells from rich to poor nitrogen conditions. The requirements of Vps proteins for Gln3 function are media-specific: a requirement after rapamycin treatment was observed in minimal but not in rich medium. Moreover, we have seen that a significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. These observations support the view that GATA factor regulation in response to nitrogen signals seems to occur at intracellular compartments.<p>In a second step, we confirmed an important role for the anabolic glutamate dehydrogenase (Gdh1) within NCR, through the control of Gat1 function. However, since we observed a strong correlation between the anabolic activity of Gdh1 and its NCR regulatory capacity, we do not exclude that an alteration of Gdh1-substrates or any other metabolite could be responsible for the phenotype exhibited by gdh1 mutants. We also showed that there is no simple and direct link between the intracellular levels of glutamine/glutamate (reported in the literature as signals for NCR), TORC1 activity and NCR. In conclusion, the mechanisms regulating the perception of the quality of the nitrogen sources are still not fully understood. <p>Several screens for multi-copy suppression of mutated phenotypes were conducted during this work and led to the identification of several elements (URE2, BAP2, STP2, GZF3 and KDX1) that could interfere with NCR-sensitive gene expression. Among these, the gene encoding the Kdx1 kinase was identified in two independent screens. <p>In the last part of this work, we uncovered a role for leucine in NCR signaling. We showed that the addition of leucine in the culture medium could impair Gat1-dependent expression of certain NCR genes, while leucine starvation had no effect at this level. The repressive effect of leucine appeared to involve elements of the SPS signaling pathway which is required for the induction of genes encoding amino acid transporters in response to extracellular amino acids. The mechanism(s) by which leucine regulates Gat1 function is still not fully clear and requires further investigation:La levure Saccharomyces cerevisiae adapte l’expression de ses gènes selon la disponibilité en azote dans son environnement au moyen d’un contrôle majeur appelé répression catabolique azotée (NCR, pour « nitrogen catabolite repression ». L’expression des gènes NCR est contrôlée par un régulateur négatif de type prion (Ure2) et quatre facteurs de transcription de type GATA :deux activateurs, Gat1 et Gln3 et deux répresseurs, Dal80 et Gzf3. Bien que le complexe TORC1 et les phosphatases qu’il régule soient impliquées dans la régulation NCR, le mécanisme précis par lequel la NCR se produit est loin d’être compris.<p>Dans la première partie de ce travail, nous avons montré que les composants VPS (vacuolar protein sorting) de classe C et D, impliqués dans le trafic vésiculaire entre le Golgi et la vacuole, sont requis pour que Gat1 et Gln3 rejoignent le noyau en réponse à un traitement par la rapamycine, un inhibiteur de TORC1. En accord avec cette observation, nous avons montré que Gat1, comme Gln3, est associé aux membranes intracellulaires légères. <p>Dans un second temps, nous avons confirmé un rôle important pour la glutamate déshydrogénase anabolique (Gdh1) au sein de la NCR, par l’intermédiaire du contrôle de la fonction de Gat1. Cependant, étant donné qu’il semble exister une forte corrélation entre l’activité anabolique de Gdh1 et sa capacité à réguler la NCR, nous n’excluons pas qu’une altération des substrats de Gdh1 ou de tout autre métabolite pourrait être responsable du phénotype observé du mutant gdh1. Nous avons également montré qu’il n’existait pas de lien simple et direct entre niveaux intracellulaires de glutamine/glutamate, activité de TORC1 et signalisation NCR. En conclusion, les mécanismes conditionnant la perception de la qualité de l’aliment azoté sont encore méconnus à ce jour. <p>Plusieurs cribles de suppression multicopie ont été menés pendant ce travail et ont conduit à l’identification de plusieurs éléments pouvant éventuellement intervenir dans la voie de signalisation NCR. Parmi ceux-ci, le gène codant pour la kinase KDX1 a été identifié à deux reprises. Nous avons caractérisé en détail le rôle qu’elle joue dans la régulation des gènes NCR.<p>Dans la dernière partie de ce travail, nous avons montré que l’addition de leucine dans le milieu de culture pouvait affecter l’expression Gat1-dépendante de certains gènes NCR, alors que par ailleurs une carence en leucine est sans effet à ce niveau. Cet effet de répression par la leucine semble nécessiter des éléments de la voie de signalisation SPS, requise pour l’induction, en réponse aux acides aminés extracellulaires, de gènes codant pour des transporteurs d’acides aminés. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

L'encéphalopathie hépatique (HE) est un syndrome neuropsychiatrique complexe, une complication majeure de la maladie du foie. L'œdème cytotoxique est une complication grave de l'encéphalopathie hépatique, connu comme étant le résultat d'un gonflement des astrocytes. Les facteurs pathogéniques dérivés du sang tels que l'ammoniaque (NH4+) et le stress oxydatif (SO) sont connus pour être synergiquement impliqués. Les cellules endothéliales (CE) de la barrière hémato-encéphalique (BHE), régulant le passage vers le cerveau, sont les premières cellules à entrer en contact avec les molécules circulantes. L'effet de l'ammoniaque et du SO sur le transport et le métabolisme des CE n'a jamais été complètement exploré. Par conséquent, notre objectif était d'évaluer les effets de NH4+ et des espèces réactives de l'oxygène (ROS) sur les CE de la BHE en utilisant des systèmes de modèles in vivo et in vitro. Il a été démontré que le cotransporteur Na-K-2Cl (NKCC1) était impliqué dans la pathogenèse de l'œdème cérébral dans de nombreuses affections neurologiques. Le NKCC1 peut transporter NH4+ vers le cerveau et est régulé par les ROS. Par conséquent, l'expression de NKCC1 a été évaluée dans des CE primaires soumises à différentes concentrations de ROS et de NH4+ ainsi que dans des microvaisseaux cérébraux (MVC) isolés chez le rat BDL (bile-duct ligated), un modèle d'EH induit par une maladie hépatique chronique. Aucune régulation à la hausse de NKCC n'était présente chez les CE traitées ou les MVC. La glutamine synthétase (GS) est une enzyme qui joue un rôle compensatoire important dans la détoxification du NH4+ au cours de la maladie du foie. La GS est exprimée dans le muscle et le cerveau (astrocytes), mais n'a jamais été totalement explorée dans les CE de la BHE. L'expression et l'activité de la protéine GS ont été trouvées dans les CE de la BHE in vitro (CE primaires) et in vivo (MVC isolés de rats naïfs). Dans le modèle BDL, l'expression de GS dans les MVC n'était pas significativement différente des témoins (SHAM). Par ailleurs, nous avons traité des CE avec du milieu conditionné à partir de plasma de rats BDL et avons trouvé une diminution de l’expression de la protéine GS et de l'activité par rapport aux SHAM. De plus, les CE traitées avec NH4+ augmentaient en activité de GS tandis que les traitements avec SO avec et sans NH4+ diminuent l'activité de GS. Globalement, ces résultats démontrent pour la première fois que la GS est présente dans les CE, à la fois in vivo et in vitro. La GS est régulée à la baisse dans les CE traitées avec du plasma de BDL (mais pas dans les MVC de BDL). Il est intéressant de noter que le NH4+ stimule l'activité de GS dans les CE, alors que le SO inhibe l'activité de GS, ce qui justifie possiblement les résultats de nos études avec les milieux conditionnés. Nous supposons que le SO empêche la régulation à la hausse de GS de la BHE, en diminuant la capacité des CE à détoxifier l'ammoniaque et à limiter l'entrée d'ammoniaque dans le cerveau. Nous envisageons qu'une régulation à la hausse de GS dans les CE de la BHE pourrait devenir une nouvelle cible thérapeutique de l'EH. / Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome, which is a major complication of liver disease. Cytotoxic edema is a serious complication of HE, known to be the result of astrocyte swelling. Blood derived pathogenic factors such as ammonia (NH4+) and oxidative stress’ (OS) are known to be synergistically implicated. Endothelial cells (EC) of the blood brain barrier (BBB) are the first cells regulating passage into the brain and to contact blood-derived molecules. The effect of ammonia and oxidative stress on EC transport and metabolism has never been thoroughly explored. Therefore, our aim was to evaluate the effects of NH4+ and reactive oxygen species (ROS) on EC of the BBB using in vivo and in vitro models systems. The Na–K–2Cl cotransporter (NKCC1) has been demonstrated to be involved in the pathogenesis of brain edema in numerous neurological conditions. NKCC1 can transport NH4+ into the brain and is regulated by ROS. Therefore, the expression of NKCC1 was evaluated in primary EC submitted to different concentrations of ROS and NH4+ as well as in cerebral microvessels (CMV) isolated from the bile-duct ligated (BDL) rat, a model HE induced by chronic liver disease. No upregulation of NKCC1 was present in either the treated EC or CMV. Glutamine synthetase (GS) is an enzyme with an important compensatory role in NH4+ detoxification during liver disease. GS is expressed in muscle and brain (astrocytes) but has never been thoroughly explored in ECs of the BBB. GS protein expression and activity was found in EC of the BBB in vitro (primary EC) and in vivo (CMV isolated from naive rats). In the BDL model, GS expression in CMVs was not significantly different from SHAM-operated controls. In addition, we treated ECs with conditioned medium from plasma of BDL rats and found a decrease in GS protein and activity when compared to SHAM. Furthermore, EC treated with NH4+ increased GS activity while treatments with ROS with and without NH4+ decreased GS activity. Overall these results demonstrate for the first time that GS is present in EC both in vivo and in vitro. GS is downregulated in EC treated with BDL plasma (but not in BDL CMV). Interestingly, NH4+ stimulates GS activity in ECs, while ROS inhibits GS activity, possibly justifying the results found from the conditioned medium studies. We speculate that ROS prevents the upregulation of GS in the BBB, decreasing the capacity of the EC to detoxify ammonia and to limit ammonia entry into the brain. We foresee that upregulating GS in ECs of the BBB could become a new therapeutic target for HE.

PII-Proteine, zu denen GlnB und GlnK zählen, sind ubiquitär verbreitete kleine Regulatorproteine, die den internen Stickstoffzustand der Zelle sensieren und weiterleiten und hierdurch maßgeblich an der Regulation des Stickstoffmetabolismus beteiligt sind.Ziel dieser Arbeit war es, das GlnK1-Protein aus dem methanogenen Archeaon Methanosarcina mazei Stamm Gö1 umfassend zu charakterisieren und seine potentielle Rolle in der Regulation des Stickstoffmetabolismus aufzuklären. Das M. mazei GlnK1-Protein weist den typischen Tyrosin51-Rest auf, der in Bakterien stickstoffabhängig posttranslationell modifiziert wird. Sowohl in vitro als auch in vivo Experimente haben jedoch gezeigt, dass GlnK1 in M. mazei nicht stickstoffabhängig modifiziert wird. Weitere strukturelle Unterschiede zu bakteriellen PII-Proteinen haben Experimente zur Bildung von Heterotrimeren aufgezeigt. Trotz dieser deutlichen Unterschiede haben Komplementationsversuche ergeben, dass das archaeelle GlnK-Protein in der Lage ist, E. coli GlnK funktionell zu komplementieren. Dieses läßt darauf schließen, dass das GlnK1-Protein in M. mazei auch in der Regulation des Stickstoffmetabolismus involviert ist. Um dieses zu bestätigen, wurde eine chromosomale M. mazei glnK1-Mutante generiert. Hierfür war es erforderlich, zunächst ein funktionelles System zur Transformation von M. mazei Gö1 zu entwickeln. Es gelang, (i) durch Selektion einer potentiellen spontanen Zellwandmutante von M. mazei, die eine stark verbesserte Plattierungseffizienz aufwies, sowie (ii) durch mehrere Modifizierungen des von W. Metcalf (Urbana) entwickelten Liposomen-vermittelten Transformationsprotokolls für Methanosarcina-Stämme M. mazei Gö1 genetisch zugänglich zu machen. Wachstumsanalysen des konstruierten M. mazei glnK1-Mutantenstamms zeigten einen partiell reduzierten Wachstumsphänotyp unter stickstofflimitierenden Bedingungen. Quantitative Reverse Transkriptions-PCR Analysen ausgewählter Gene ergaben allerdings, dass das GlnK1 keinen Einfluss auf die Transkription stickstoffregulierter Gene ausübt.Sowohl biochemische Experimente mit gereinigtem Enzym als auch in vivo Versuche zeigten jedoch, dass das GlnK1-Protein mit der Glutamin-Synthetase (GlnA) interagiert und hierdurch deren Enzymaktivität inhibiert. Ein aktivierender Effekt auf die GlnA Enzymaktivität wurde hingegen bei Anwesenheit von 2-Oxoglutarat beobachtet, welches den internen Stickstoffstatus wiederspiegelt. Aus der Gesamtheit der Ergebnisse läßt sich folgendes hypothetisches Regulationsmodel ableiten: Unter Stickstofflimitierung wird 2-Oxoglutarat akkumuliert, welches die Glutamine-Synthetase Aktivität stark stimuliert; bei einem Übergang zu Stickstoffüberschuss wird die Glutamine-Synthetase sowohl durch einen reduzierten 2-Oxoglutarat-Spiegel als auch durch direkte Protein-Interaktion mit GlnK1 inaktiviert. Letzteres dient der Feinregulation und ermöglicht schnell auf eine veränderte Stickstoffversorgung reagieren zu können.

Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

This study has been concerned with the design and synthesis of A TP analogues with the potential to act as inhibitors of glutamine synthetase - a novel target for therapeutic intervention in the treatment of tuberculosis. Using a structural -analogy approach, various 3-indolylalkanoic acid, benzimidazole and pyrazolo[3,4-dJpyrimidine derivatives have been prepared and characterized. Alkylation of the heterocyclic bases using 4-(bromomethyl)-2,2-dimethyl-1 ,3-d ioxolane, 2-(bromomethoxy)ethyl acetate and 2-(chloroethoxy)ethanol in the presence of either NaH or BulOK afforded the corresponding N-alkylated derivatives of benzimidazole and 4-aminopyrazolo[3,4-dJpyrimidine (4-APP). Similar reactions with 3-indo lylalkanoic esters resulted in O-alkyl cleavage with the formation of new esters. Alkylation of benzimidazole with allyl bromide, 4-bromobutene and 2-methylbut-2-ene has also been shown to afford the corresponding l-alkenylbenzimidazoles in moderate to excellent yield (43-96%). Subsequent oxidation of these products using CTAP, gave the dihydroxy derivatives in poor to good yields (26-77%). Phosphorylation of various hydroxy derivatives of benzimidazole and 4-APP has been achieved using diethyl chlorophosphate to afford the corresponding monophosphate and 1,2-diphosphate esters. Glycosylation of each of the heterocyclic bases has been successfully achieved using 1,2,3,4,6-penta-O-acetyl-D-glucopyranose and SnCl4 in acetonitri le, while methanolysis of the resulting tetraacetates, using methanolic NaOMe, afforded the hydroxy derivatives in good yield (50-70%). Various 1- and 2-dimensional NMR spectroscopic methods (e.g., IH, 13C, lip, COSY, HSQC and HMBC) have been used to confirm the structures of the synthesized A IP analogues. The application of NMR prediction programmes has been explored, permitting assessment of their agreement with the experimental data and confirmation of assigned structures. High-resolution electron impact (EI) mass spectrometric data have been used to explore the mass fragmentation pathways exhibited by selected derivatives, and certain common fragmentations have been identified. Molecular modelling of selected products as potential glutamine synthetase ligands has been performed on the Accelrys Cerius2 platform, and interactive receptor-ligand docking studies have been conducted using the Ligand-Fit module. These studies have revealed possible hydrogen-boding interactions between the selected analogues and various amino acid residues in the glutamine synthetase active site.

Orchideoid mycorrhizal symbiosis (OM) can be found in nearly one tenth of higher plant species. This symbiosis is absolutely critical for orchids as they are unable to grow from seeds without external energy which is in nature provided by symbiotic fungi. The mechanism of transport between symbionts remains unknown. It is supposed that trehalose is one of the substances transported from fungi to plants as the source of energy. This thesis mainly aims to find out which other fungal saccharides could contribute to the process. The ability to utilize selected compounds was tested on protocorms of the common marsh orchid, Dactylorhiza majalis. The results showed that arabitol, erythritol, mannitol and sucralose are not utilized, while xylitol, sorbitol, glycerol and mannose are. Glutamin, an amino acid also suspected of participation in the OM transport, does not suffice as a source of energy. In orchids there were identified three groups of sequences coding for manitol dehydrogenase and two groups of sequences coding for sorbitol dehydrogenase. Key words: orchideoid mycorrhizal symbiosis, sugar alcohols, mannose, glutamine, carbon flow, energy flow, sorbitol dehydrogenase, in vitro

The transcription factor hypoxia-inducible factor 1 (HIF1) is the crucial regulator of genes that are involved in metabolism under hypoxic conditions, but information regarding the transcriptional activity of HIF1 in normoxic metabolism is limited. Different tumor cells were treated under normoxic and hypoxic conditions with various drugs that affect cellular metabolism. HIF1ff was silenced by siRNA in normoxic/hypoxic tumor cells, before RNA sequencing and bioinformatics analyses were performed while using the breast cancer cell line MDA-MB-231 as a model. Differentially expressed genes were further analyzed and validated by qPCR, while the activity of the metabolites was determined by enzyme assays. Under normoxic conditions, HIF1 activity was significantly increased by (i) glutamine metabolism, which was associated with the release of ammonium, and it was decreased by (ii) acetylation via acetyl CoA synthetase (ACSS2) or ATP citrate lyase (ACLY), respectively, and (iii) the presence of L-ascorbic acid, citrate, or acetyl-CoA. Interestingly, acetylsalicylic acid, ibuprofen, L-ascorbic acid, and citrate each significantly destabilized HIF1ff only under normoxia. The results from the deep sequence analyses indicated that, in HIF1-siRNA silenced MDA-MB-231 cells, 231 genes under normoxia and 1384 genes under hypoxia were transcriptionally significant deregulated in a HIF1-dependent manner. Focusing on glycolysis genes, it was confirmed that HIF1 significantly regulated six normoxic and 16 hypoxic glycolysis-associated gene transcripts. However, the results from the targeted metabolome analyses revealed that HIF1 activity affected neither the consumption of glucose nor the release of ammonium or lactate; however, it significantly inhibited the release of the amino acid alanine. This study comprehensively investigated, for the first time, how normoxic HIF1 is stabilized, and it analyzed the possible function of normoxic HIF1 in the transcriptome and metabolic processes of tumor cells in a breast cancer cell model. Furthermore, these data imply that HIF1 compensates for the metabolic outcomes of glutaminolysis and, subsequently, theWarburg effect might be a direct consequence of the altered amino acid metabolism in tumor cells.

Background and aims: Type 2 Diabetes Mellitus (T2DM) presents a significant global health challenge, characterized by impaired insulin secretion and/or action. A critical aspect of managing T2DM involves understanding the regulatory mechanisms of insulin secretion in pancreatic β-cells. Pancreatic β-cells play a pivotal role in maintaining glucose homeostasis. Although glucose is the primary stimulator of insulin secretion, certain amino acids also have regulatory roles. Traditional views have held that while glutamine contributes to insulin secretion, it does not directly influence this process in the absence of glutamate dehydrogenase (GDH) activation. We found that glutamine increases insulin secretion independently of GDH activation in INS-1 832/13 cells. Therefore, the aim of the thesis is to elucidate the role of glutamine in insulin secretion and examining its regulatory effects on glucose metabolism in pancreatic β-cells. To achieve this, we leverage advanced methodologies, including metabolomics and network analysis, to provide a comprehensive understanding of these complex mechanisms. Methods and results: Our initial findings presented a surprising challenge to the conventional belief that glutamine induces insulin secretion only in the presence of leucine. We discovered that glutamine (independent of leucine) could increase insulin secretion in a dose-dependent manner in INS-1 832/13 cells. To delve further into this phenomenon, we employed inhibitors of key enzymes in glutamine metabolism - GDH (responsible for glutamate oxidation) and glutaminase (converts glutamine to glutamate). Our results highlighted that while inhibiting GDH did not alter insulin secretion, inhibiting glutaminase significantly reduced the insulin-secretory response to glutamine in INS-1 832/13 cells. This finding indicated that the effect of glutamine on insulin secretion operates independently of glutamate oxidation. Our study also investigated the regulatory role of glutamine in insulin secretion and on the rate of glucokinase (GK) in response to glucose levels. We observed that increasing concentrations of glutamine affected both the dynamics of insulin secretion and the kinetic parameters of GK in INS-1 832/13 cells, suggesting a regulatory relationship between glutamine and glucose phosphorylation that had not been previously observed. To deepen our understanding of the intricate relationship, we developed a novel analytical approach that combined network analysis with metabolomics. This innovative method provided an unbiased assessment of the interrelationships between various metabolites, enabling a more comprehensive understanding of the metabolic pathways and their interactions. A striking outcome of our network analysis was the identification of proline as a key metabolite in the glutamine-glucose crosstalk. To validate this link, we conducted siRNA knockdown experiments targeting proline synthesis in INS-1 832/13 cells. Knockdown of these genes resulted in a significant reduction in insulin secretion in response to glutamine. Further, this effect could be rescued by the addition of proline, thereby underscoring the essential role of proline in glutamine-mediated insulin secretion. Furthermore, in vitro enzymatic assays using INS-1 832/13 cell extracts and purified rat GK revealed proline- mediated changes in kinetic parameters consistent with glutamine-mediated alterations in GK activity in live INS-1 832/13 cells. Additionally, a thermal stability assay demonstrated that the melting temperature of purified rat GK varied with proline concentration, suggesting a direct interaction of proline with GK. This effect of glutamine on insulin secretion was also observed in isolated rat islets, thereby affirming the physiological relevance of our results. Moreover, the thermal stability assay using purified human GK confirmed that this interaction is conserved in humans as well. Conclusion and outlook: This study reveals a novel mechanism by which glutamine metabolism, through proline synthesis, regulates GK activity and thereby influences insulin secretion in pancreatic β-cells. The outlook of this thesis opens promising avenues for future research and potential clinical applications, particularly in the context of T2DM management. Key areas for future exploration include translating these findings to in vivo models and clinical settings could open new therapeutic avenues for T2DM, emphasizing the importance of modulating glutamine and proline metabolism for more effective regulation of insulin secretion. Investigating the direct causal relationship between plasma proline levels and diabetic conditions could not only deepen our understanding of diabetes but also provide a potential biomarker for early risk assessment. Understanding the precise molecular interactions between proline and GK could allow the identification of potential novel binding sites for therapeutic intervention to enhance GK activity and improve glucose regulation. Extending this research to human cells and examining its implications in diabetes and other metabolic disorders is a vital next step, offering potential for significant advancements in diabetes treatment and understanding of metabolic diseases.:Table of Contents List of abbreviations List of figures List of tables 1. Introduction 1.1. Type 2 Diabetes 1.1.1. Definition, epidemiology, and risk factors 1.1.2. Pathophysiology of T2DM 1.1.3. Preserving or enhancing β-cell function 1.2. Physiology of pancreatic β-cells 1.2.1. Overview of glucose-stimulated insulin secretion 1.2.2. Regulation of glucose entry into the β-cells 1.2.3. Role of glucokinase as a glucose sensor 1.2.4. Regulation of mitochondrial metabolism in insulin secretion 1.2.5. Regulation of amino acid mediated insulin secretion 1.3. Metabolomics approach in studying β-cell function 1.4. Network analysis in metabolomics data analysis and interpretation 2. Aims of the study 3. Materials and Methods 3.1. Materials 3.1.1. INS-1 832/13 cells 3.1.2. Chemicals, solutions, and buffers for cell culture 3.1.3. Chemicals, solutions, and buffers for molecular and metabolic experiments 3.1.4. Software 3.2. Methods 3.2.1. Cell culture 3.2.1.1. Culturing INS-1 832/13 cells 3.2.1.2. Cryopreservation and thawing of INS1 832/13 cells 3.2.1.3. Isolation of rat islets 3.2.2. Expression and Purification of GST-fusion GK Proteins in E. coli. 3.2.3. Insulin secretion studies in INS1 832/13 cells 3.2.3.1. Effect of Glutamine on insulin secretion 3.2.3.2. Effect of chronic and acute exposure of glutamine on insulin secretion 3.2.3.3. Glutamine-responsive insulin secretion 3.2.3.4. Effect of glutamate oxidation in glutamine-mediated insulin secretion 3.2.3.5. Effect of glutamine on glucose-responsive insulin secretion 3.2.3.6. Effect of 2DG on glucose stimulated insulin secretion 3.2.3.7. Insulin and total protein quantification 3.2.4. Metabolomic experiments in INS-1 832/13 cells 3.2.4.1. Effect of specific perturbations on metabolomic profile 3.2.4.2. Effect of glutamine on metabolomic profile 3.2.5. Metabolomic analyses 3.2.5.1. LC-MS/MS method for characterization of metabolites 3.2.5.2. Metabolite concentration calculation 3.2.6. Network analysis 3.2.6.1. Metabolite network construction 3.2.6.2. Comparative metabolite analysis with weighted network metrics 3.2.7. GK kinetic studies 3.2.7.1. GK activity with GK activator in INS-1 832/13 cells 3.2.7.2. GK activity with glutamine in INS1 cells & rat islets 3.2.7.3. GK kinetics measurement 3.2.7.4. In vitro GK kinetic studies using cell extracts & purified GK enzyme 3.2.8. Gene expression analysis 3.2.8.1. RNA isolation 3.2.8.2. cDNA synthesis 3.2.8.3. qPCR 4. Results 4.1. Glutamine mediated insulin secretion in INS-1 832/13 cells 4.1.1. Glutamine alone stimulates insulin secretion 4.1.2. Glutamine amplifies insulin secretion independently of glutamate oxidation 4.2. Glutamine mediated insulin secretion and its impact on glucose responsiveness 4.2.1. Glutamine modulates the regulation of insulin secretion in INS-1 832/13 cells 4.2.2. Live cell GK activity measurement using 2DG uptake in INS-1 832/13 cells 4.2.3. Glutamine modulates GK activity in INS-1 832/13 cells 4.3. Identifying the glutamine-derived factor regulating GK activity 4.3.1. Network analysis to identify key metabolites associated with specific perturbations 4.3.2. Glutamine-induced insulin secretion is mediated by proline 4.3.3. Proline modulates GK activity in INS-1 832/13 cell extracts 4.3.4. Proline modulates activity of purified rat GK 4.3.5. Thermal stability assays in rat GK 4.3.6. siRNA knockdown of proline synthesis 4.4. Glutamine modulates insulin secretion and GK activity in rat islets 4.5. Proline interacts and modulate GK in human 5. Discussion 5.1. Reevaluating glutamine-mediated insulin secretion in pancreatic β-cells 5.2. Novel role of glutamine-mediated modulation of GK activity and insulin secretion in pancreatic β-cells 5.3. Network analysis as a tool to unravel complex interactions in metabolic research 5.4. Proline as a novel modulator of GK 5.5. Contrasting role of glutamine in pancreatic and liver metabolism 6. References 7. Summary 8. Zussammenfassung 9. Acknowledgements 10. Declaration / Hintergrund und Ziele: Typ-2-Diabetes mellitus (T2DM) stellt eine bedeutende globale Herausforderung für die Gesundheit dar und ist durch eine gestörte Insulinsekretion und/oder -wirkung gekennzeichnet. Ein entscheidender Aspekt bei der Behandlung von T2DM ist das Verstehen von Regulationsmechanismen der Insulinsekretion in den β-Zellen der Pankreas. Die β-Zellen der Bauchspeicheldrüse spielen eine zentrale Rolle bei der Aufrechterhaltung der Glukosehomöostase. Obwohl Glukose der primäre Stimulator der Insulinsekretion ist, spielen bestimmte Aminosäuren auch eine regulierende Rolle. Nach traditioneller Auffassung trägt Glutamin zwar zur Insulinsekretion bei, hat aber keinen direkten Einfluss auf diesen Prozess, es sei denn, er wird durch Glutamatdehydrogenase (GDH) aktiviert. Wir fanden heraus, dass Glutamin die Insulinsekretion unabhängig von der GDH-Aktivierung in INS-1 832/13-Zellen erhöht. Ziel dieser Arbeit war es daher, die Rolle von Glutamin bei der Insulinsekretion aufzuklären und seine regulierenden Effekte auf den Glukosestoffwechsel in β-Zellen der Pankreas zu untersuchen. Um dies zu erreichen, nutzen wir fortschrittliche Methoden, einschließlich Metabolomik- und Netzwerkanalysen, um ein umfassendes Verständnis dieser komplexen Mechanismen zu erlangen. Methoden und Ergebnisse: Unsere anfänglichen Ergebnisse stellten eine überraschende Inhomogenität zur herkömmlichen Annahme dar, dass Glutamin die Insulinsekretion nur in der Anwesenheit von Leucin induziert. Wir entdeckten, dass Glutamin (unabhängig von Leucin) die Insulinsekretion in INS-1 832/13-Zellen dosisabhängig steigern kann. Um dieses Phänomen näher zu untersuchen, setzten wir Hemmstoffe von Schlüsselenzymen des Glutaminstoffwechsels ein - GDH (verantwortlich für die Glutamatoxidation) und Glutaminase (konvertiert Glutamin zu Glutamat). Unsere Ergebnisse zeigten, dass die Hemmung der GDH die Insulinsekretion nicht modifizierte, während die Hemmung der Glutaminase die Insulinsekretionsantwort auf Glutamin in INS-1 832/13-Zellen deutlich verringerte. Diese Erkenntnis deutet darauf hin, dass die Wirkung von Glutamin auf die Insulinsekretion unabhängig von der Glutamatoxidation ist. In dieser Studie untersuchten wir weiterhin die regulatorische Rolle von Glutamin bei der Insulinsekretion und für die GK-Rate in Abhängigkeit vom Glukosespiegel. Wir stellten fest, dass steigende Glutaminkonzentrationen sowohl die Dynamik der Insulinsekretion als auch die kinetischen Parameter der Glucokinase (GK) in INS-1 832/13-Zellen beeinflussten, was auf eine bisher nicht erkannte regulatorische Beziehung zwischen Glutamin und Glukosephosphorylierung schließen lässt. Um unser Verständnis dieser komplexen Beziehung zu vertiefen, entwickelten wir einen neuartigen analytischen Ansatz, der die Netzwerkanalyse mit der Metabolomforschung kombinierte. Diese innovative Methode ermöglichte eine unvoreingenommene Bewertung der Wechselbeziehungen zwischen verschiedenen Metaboliten und damit ein umfassenderes Verständnis der Stoffwechselwege und ihrer Wechselwirkungen. Ein bemerkenswertes Ergebnis unserer Netzwerkanalyse war die Identifizierung von Prolin als Schlüsselmetabolit im Glutamin-Glukose-Crosstalk. Um diese Verbindung zu bestätigen, führten wir siRNA-Knockdown-Experimente durch, die auf die Prolinsynthese in INS-1 832/13-Zellen abzielten. Die Ausschaltung dieser Gene führte zu einer deutlichen Verringerung der Insulinsekretion als Reaktion auf Glutamin. Bemerkenswerterweise konnte dieser Effekt durch die Zugabe von Prolin wiederhergestellt werden, was die wesentliche Rolle von Prolin bei der Glutamin-vermittelten Insulinsekretion unterstreicht. Darüber hinaus ergaben in vitro Enzymassays mit INS-1 832/13-Zellextrakten und gereinigter Ratten-GK Prolin-vermittelte Veränderungen der kinetischen Parameter, die mit Glutamin-vermittelten Veränderungen der GK-Aktivität in lebenden INS-1 832/13-Zellen übereinstimmen. Darüber hinaus zeigte ein Thermal Stability Assay, dass die Schmelztemperatur von gereinigtem Ratten-GK mit der Prolin-Konzentration variierte, was auf eine direkte Interaktion von Prolin mit der GK hindeutet. Dieser Effekt von Glutamin auf die Insulinsekretion wurde auch in aus Ratten isolierten Langerhansschen Inseln beobachtet, was die physiologische Relevanz unserer Ergebnisse bestätigt. Darüber hinaus bestätigte der Thermal Stability Assay mit gereinigter menschlichen GK, dass diese Interaktion auch beim Menschen konserviert ist. Schlussfolgerung und Ausblick: Diese Studie enthüllt einen neuartigen Mechanismus, durch den der Glutamin-Stoffwechsel über die Prolin-Synthese die GK-Aktivität reguliert und dadurch die Insulinsekretion in den β-Zellen der Bauchspeicheldrüse beeinflusst, was bestehende Paradigmen in Frage stellt. Perspektivisch ermöglichen die Erkenntnisse dieser Arbeit vielversprechende Wege für die zukünftige Forschung und potenzielle klinische Anwendungen, insbesondere im Zusammenhang mit T2DM-Management. Zu den Schlüsselbereichen der zukünftigen Forschung gehören die Übertragung dieser Ergebnisse auf in vivo Modelle und klinische Studien, die neue therapeutische Wege für T2DM eröffnen könnten und die Bedeutung der Modulation des Glutamin- und Prolin-Stoffwechsels für eine effektivere Regulierung der Insulinsekretion unterstreichen. Die Untersuchung des direkten kausalen Zusammenhangs zwischen Plasmaprolinspiegeln und diabetischen Erkrankungen könnte nicht nur unser Verständnis von Diabetes vertiefen, sondern auch einen potenziellen Biomarker für eine frühzeitige Risikobewertung liefern. Die Entschlüsselung der genauen molekularen Wechselwirkungen zwischen Prolin und GK könnte die Identifizierung potenzieller neuer Bindungsstellen für therapeutische Eingriffe zur Steigerung der GK- Aktivität und zur Verbesserung der Glukoseregulierung ermöglichen. Die Erweiterung dieser Forschung auf menschliche Zellen und die Untersuchung ihrer Auswirkungen auf Diabetes und andere Stoffwechselstörungen ist ein wichtiger nächster Schritt, der das Potenzial für bedeutende Fortschritte bei der Behandlung von Diabetes und dem Verständnis von Stoffwechselkrankheiten bietet.:Table of Contents List of abbreviations List of figures List of tables 1. Introduction 1.1. Type 2 Diabetes 1.1.1. Definition, epidemiology, and risk factors 1.1.2. Pathophysiology of T2DM 1.1.3. Preserving or enhancing β-cell function 1.2. Physiology of pancreatic β-cells 1.2.1. Overview of glucose-stimulated insulin secretion 1.2.2. Regulation of glucose entry into the β-cells 1.2.3. Role of glucokinase as a glucose sensor 1.2.4. Regulation of mitochondrial metabolism in insulin secretion 1.2.5. Regulation of amino acid mediated insulin secretion 1.3. Metabolomics approach in studying β-cell function 1.4. Network analysis in metabolomics data analysis and interpretation 2. Aims of the study 3. Materials and Methods 3.1. Materials 3.1.1. INS-1 832/13 cells 3.1.2. Chemicals, solutions, and buffers for cell culture 3.1.3. Chemicals, solutions, and buffers for molecular and metabolic experiments 3.1.4. Software 3.2. Methods 3.2.1. Cell culture 3.2.1.1. Culturing INS-1 832/13 cells 3.2.1.2. Cryopreservation and thawing of INS1 832/13 cells 3.2.1.3. Isolation of rat islets 3.2.2. Expression and Purification of GST-fusion GK Proteins in E. coli. 3.2.3. Insulin secretion studies in INS1 832/13 cells 3.2.3.1. Effect of Glutamine on insulin secretion 3.2.3.2. Effect of chronic and acute exposure of glutamine on insulin secretion 3.2.3.3. Glutamine-responsive insulin secretion 3.2.3.4. Effect of glutamate oxidation in glutamine-mediated insulin secretion 3.2.3.5. Effect of glutamine on glucose-responsive insulin secretion 3.2.3.6. Effect of 2DG on glucose stimulated insulin secretion 3.2.3.7. Insulin and total protein quantification 3.2.4. Metabolomic experiments in INS-1 832/13 cells 3.2.4.1. Effect of specific perturbations on metabolomic profile 3.2.4.2. Effect of glutamine on metabolomic profile 3.2.5. Metabolomic analyses 3.2.5.1. LC-MS/MS method for characterization of metabolites 3.2.5.2. Metabolite concentration calculation 3.2.6. Network analysis 3.2.6.1. Metabolite network construction 3.2.6.2. Comparative metabolite analysis with weighted network metrics 3.2.7. GK kinetic studies 3.2.7.1. GK activity with GK activator in INS-1 832/13 cells 3.2.7.2. GK activity with glutamine in INS1 cells & rat islets 3.2.7.3. GK kinetics measurement 3.2.7.4. In vitro GK kinetic studies using cell extracts & purified GK enzyme 3.2.8. Gene expression analysis 3.2.8.1. RNA isolation 3.2.8.2. cDNA synthesis 3.2.8.3. qPCR 4. Results 4.1. Glutamine mediated insulin secretion in INS-1 832/13 cells 4.1.1. Glutamine alone stimulates insulin secretion 4.1.2. Glutamine amplifies insulin secretion independently of glutamate oxidation 4.2. Glutamine mediated insulin secretion and its impact on glucose responsiveness 4.2.1. Glutamine modulates the regulation of insulin secretion in INS-1 832/13 cells 4.2.2. Live cell GK activity measurement using 2DG uptake in INS-1 832/13 cells 4.2.3. Glutamine modulates GK activity in INS-1 832/13 cells 4.3. Identifying the glutamine-derived factor regulating GK activity 4.3.1. Network analysis to identify key metabolites associated with specific perturbations 4.3.2. Glutamine-induced insulin secretion is mediated by proline 4.3.3. Proline modulates GK activity in INS-1 832/13 cell extracts 4.3.4. Proline modulates activity of purified rat GK 4.3.5. Thermal stability assays in rat GK 4.3.6. siRNA knockdown of proline synthesis 4.4. Glutamine modulates insulin secretion and GK activity in rat islets 4.5. Proline interacts and modulate GK in human 5. Discussion 5.1. Reevaluating glutamine-mediated insulin secretion in pancreatic β-cells 5.2. Novel role of glutamine-mediated modulation of GK activity and insulin secretion in pancreatic β-cells 5.3. Network analysis as a tool to unravel complex interactions in metabolic research 5.4. Proline as a novel modulator of GK 5.5. Contrasting role of glutamine in pancreatic and liver metabolism 6. References 7. Summary 8. Zussammenfassung 9. Acknowledgements 10. Declaration