Toxoplasma gondii und Plasmodium Spezies sind obligat intrazelluläre Parasiten, die Zucker zur Energiehomöostase als auch für die Synthese lebenswichtiger Makromoleküle verwenden. Die hier vorgestellten Daten zeigen, dass der Glukosetransporter von T. gondii, TgGT1, und die homologen Transporter von P. falciparum und P. berghei, PfHT1 und PbHT1, neben Glukose auch Mannose, Fructose und Galactose transportieren. Toxoplasma Tachyzoiten exprimieren neben TgGT1 noch einen weiteren putative Zuckertransporter (TgST2) an der Parasitenoberfläche. Beide Proteine sind nicht essentiell, wie durch ihre individuelle und gleichzeitige Gendeletion belegt wird. Die Deletion von TgGT1 bewirkt einen geringen Wachstumsdefekt. Die Mutante ?tggt1 zeigt keine Glukoseaufnahmeaktivität und folglich eine verminderte glukoseabhängige Motilität. In ?tggt1 Parasiten wird ein verstärkter Glutaminstoffwechsel nachgewiesen, der ausreichend ist dessen Motilität und Replikationsaktivität zu erhalten. Die ?tggt1 Mutante gewährt Einblick in die Anpassungsfähigkeit von T. gondii an unterschiedliche Wirtszellen. Im Gegensatz zu T. gondii benötigen erythrozytäre Plasmodien Glukose und der Transporter PfHT1 wird derzeit als drug-target eingestuft. Hier wird gezeigt, dass das PfHT1-Homolog, PbHT1, essentiell in Blutstadien des Nagerparasiten Plasmodium berghei ist, jedoch auch während des gesamten Lebenszyklus des Parasiten exprimiert wird. Ein PfHT1- und PbHT1-spezifischer Inhibitor (Compound 3361) kann die Entwicklung von P. berghei Leberstadien und Okineten stark hemmen. Um zukünftig PfHT1-Inhibitoren im Hochdurchsatzverfahren zu identifizieren und testen zu können, wurden auf Saccharomyces cerevisiae und P. berghei basierende Expressionssysteme für PfHT1 entwickelt. Abschließend stellt diese Arbeit die Unterschiedlichkeit des zentralen Kohlenstoffwechsels von Toxoplasma und Plasmodium Parasiten durch bisher unbekannter Aspekte heraus. / Toxoplasma gondii and Plasmodium species are obligate intracellular pathogens that utilize host sugars for energy homeostasis and macro molecular synthesis. Here, we report that the T. gondii glucose transporter, TgGT1, and of its homologs of P. falciparum and P. berghei (PfHT1 and PbHT1) transport glucose, mannose, galactose and fructose. Besides TgGT1, Toxoplasma harbours one additional surface localized putative sugar transporter (TgST2). Surprisingly both Proteins are nonessential and only the deletion of TgGT1 inflicts a mild defect in the parasite replication. The ?tggt1 mutant is unable to import glucose and consequently displays an attenuated glucose-dependent motility, which is completely rescued by glutamine. ?tggt1 performs increased glutamine metabolism that is sufficient to sustain motility and replication. The ?tggt1 strain provides a model for further investigating its adaptation to disparate host cells. In contrast to T. gondii, erythrocytic stages of Plasmodium species critically depend on glucose uptake, and the PfHT1 transporter is considered as a drug target against human malaria. Here, we report that PbHT1 (a PfHT1 homolog) is also essential for blood stage development in the rodent malaria parasite P. berghei. PbHT1 is expressed throughout the life cycle. Moreover, a PfHT1- and PbHT1-specific sugar analogue, compound 3361, can inhibit the hepatic development and ookinete formation in P. berghei. These results signify that PbHT1 and exogenous glucose are also required during the ex-erythrocytic stages of P. berghei. To permit a high-throughput screening of selective PfHT1 inhibitors and their subsequent in vivo assessment, we have established a PfHT1-expressing Saccharomyces cerevisiae mutant and generated a PfHT1-dependent ?pbht1 of P. berghei strain. This thesis underscores various previously unknown aspects of sugar metabolism in Toxoplasma and Plasmodium, and unravel their metabolic differences.

In type 1 diabetes (T1D), an autoimmune disease, autoantibodies are preferentially directed against proteins associated with Golgi and post-Golgi secretory vesicles, including insulin secretory granules and synaptic-like microvesicles. Thus, the study of beta-cell autoantigens with yet unknown function may provide novel insight into the secretory machinery of beta-cells and led to the discovery of novel pathways. Islet cell autoantigen of 69 kDa (ICA69) is a T1D autoantigen. It is a cytosolic protein of still unknown function. An impairment in neurotransmitter release upon mutation of its homologue in C. elegans suggests, however, an involvement of ICA69 in neurosecretion. Interestingly, ICA69 contains a BAR domain, present in several proteins involved in intracellular transport. The BAR domain functions as a dimerization motif, provides a general binding interface for different types of GTPases, and is a membrane binding/bending module. Its presence in ICA69 is a further hint supporting the putative involvement of ICA69 in intracellular membrane trafficking. The first part of this thesis was concerned with the characterization of ICA69, and the elucidation of its role in membrane traffic in pancreatic beta-cells. ICA69 was shown to be enriched in the perinuclear region, where also markers of the Golgi region are found. ICA69 was shown to interact with several membrane lipids, preferentially with PI(4)P, enriched on the Golgi complex. During the course of this thesis a combination of biochemical and imaging techniques were applied to investigate the interaction between ICA69 and Rab2, a small GTPase associated with the intermediate compartment and involved in the trafficking between the ER and the Golgi complex. ICA69 was shown to co-immunoprecipitate with Rab2 from INS-1 cells extracts. GST-pull down assays demonstrated that this interaction is GTP-dependent. Furthermore, confocal microscopy indicated that ICA69 and Rab2 extensively colocalize in particulate structures throughout the cytoplasm. Immunocytochemistry and subcellular fractionation experiments suggested that Rab2 recruits ICA69 to membranes. Functional studies indicated that ICA69 over-expression in INS-1 cells has effects that resemble, and in some cases amplify those observed upon Rab2 over-expression. Specifically, it impairs the trafficking between ER and Golgi, measured through the appearance and the conversion of the pro-form of ICA512 in the mature form of the protein. Moreover, it correlates with a redistribution of the beta-COP subunit of the coatomer, participating in the early secretory pathway, between membrane-bound compartments and the cytosol and it reduces stimulated insulin secretion. The data reported in this thesis conclusively point to ICA69 as a novel Rab2 effector, and may therefore contribute to the elucidation the yet poorly understood mechanism of action of Rab2 in the secretory pathway. The second part of the thesis was devoted to the study of an ICA69 paralogue gene, called ICA69-RP. Similarly to ICA69, ICA69-RP mRNA was shown to be primarily present in tissues such as brain and pancreatic islets, showing the expression pattern of a gene preferentially expressed in neuroendocrine cells. Unlike ICA69, however, and similar to other genes associated with the secretory machinery of beta-cells, ICA69-RP appeared to be glucose regulated, as shown by a 1.55 fold increase in mRNA levels upon stimulation of the cells with 25 mM glucose for two hours.Glucose stimulation of beta-cells prompts the activation of post-transcripional mechanisms which quickly up-regulate the expression of secretory granule genes and consequently renew granule stores. The increased expression of ICA69-RP upon glucose stimulation of cells may be part of this process. Unfortunately, all attempts to elucidate the intracellular localization of endogenous ICA69-RP failed, and it was not possible to obtain significant insights about its localization by over-expressing a fusion protein between ICA69-RP and GFP. Unlike other paralogues containing the BAR domain, such as amphiphysin 1 and 2 or Rvs167p and Rvs161p, ICA69 and ICA69-RP were shown not to form heterodimers. Furthermore, ICA69-RP did not show any interaction with Rab2 or Rab1, involved in the anterograde transport between ER and Golgi. Thus, its physiological role remains to be investigated.

Background: Tumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate. Methods: Glioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed. Results: The abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate. Conclusions: Our experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy.

In type 1 diabetes (T1D), an autoimmune disease, autoantibodies are preferentially directed against proteins associated with Golgi and post-Golgi secretory vesicles, including insulin secretory granules and synaptic-like microvesicles. Thus, the study of beta-cell autoantigens with yet unknown function may provide novel insight into the secretory machinery of beta-cells and led to the discovery of novel pathways. Islet cell autoantigen of 69 kDa (ICA69) is a T1D autoantigen. It is a cytosolic protein of still unknown function. An impairment in neurotransmitter release upon mutation of its homologue in C. elegans suggests, however, an involvement of ICA69 in neurosecretion. Interestingly, ICA69 contains a BAR domain, present in several proteins involved in intracellular transport. The BAR domain functions as a dimerization motif, provides a general binding interface for different types of GTPases, and is a membrane binding/bending module. Its presence in ICA69 is a further hint supporting the putative involvement of ICA69 in intracellular membrane trafficking. The first part of this thesis was concerned with the characterization of ICA69, and the elucidation of its role in membrane traffic in pancreatic beta-cells. ICA69 was shown to be enriched in the perinuclear region, where also markers of the Golgi region are found. ICA69 was shown to interact with several membrane lipids, preferentially with PI(4)P, enriched on the Golgi complex. During the course of this thesis a combination of biochemical and imaging techniques were applied to investigate the interaction between ICA69 and Rab2, a small GTPase associated with the intermediate compartment and involved in the trafficking between the ER and the Golgi complex. ICA69 was shown to co-immunoprecipitate with Rab2 from INS-1 cells extracts. GST-pull down assays demonstrated that this interaction is GTP-dependent. Furthermore, confocal microscopy indicated that ICA69 and Rab2 extensively colocalize in particulate structures throughout the cytoplasm. Immunocytochemistry and subcellular fractionation experiments suggested that Rab2 recruits ICA69 to membranes. Functional studies indicated that ICA69 over-expression in INS-1 cells has effects that resemble, and in some cases amplify those observed upon Rab2 over-expression. Specifically, it impairs the trafficking between ER and Golgi, measured through the appearance and the conversion of the pro-form of ICA512 in the mature form of the protein. Moreover, it correlates with a redistribution of the beta-COP subunit of the coatomer, participating in the early secretory pathway, between membrane-bound compartments and the cytosol and it reduces stimulated insulin secretion. The data reported in this thesis conclusively point to ICA69 as a novel Rab2 effector, and may therefore contribute to the elucidation the yet poorly understood mechanism of action of Rab2 in the secretory pathway. The second part of the thesis was devoted to the study of an ICA69 paralogue gene, called ICA69-RP. Similarly to ICA69, ICA69-RP mRNA was shown to be primarily present in tissues such as brain and pancreatic islets, showing the expression pattern of a gene preferentially expressed in neuroendocrine cells. Unlike ICA69, however, and similar to other genes associated with the secretory machinery of beta-cells, ICA69-RP appeared to be glucose regulated, as shown by a 1.55 fold increase in mRNA levels upon stimulation of the cells with 25 mM glucose for two hours.Glucose stimulation of beta-cells prompts the activation of post-transcripional mechanisms which quickly up-regulate the expression of secretory granule genes and consequently renew granule stores. The increased expression of ICA69-RP upon glucose stimulation of cells may be part of this process. Unfortunately, all attempts to elucidate the intracellular localization of endogenous ICA69-RP failed, and it was not possible to obtain significant insights about its localization by over-expressing a fusion protein between ICA69-RP and GFP. Unlike other paralogues containing the BAR domain, such as amphiphysin 1 and 2 or Rvs167p and Rvs161p, ICA69 and ICA69-RP were shown not to form heterodimers. Furthermore, ICA69-RP did not show any interaction with Rab2 or Rab1, involved in the anterograde transport between ER and Golgi. Thus, its physiological role remains to be investigated.

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

Hintergrund: Die Versorgung von Immunzellen mit Energie in Form von ATP ist Grundlage eines funktionstüchtigen Immunsystems. Diese wird durch die mitochondriale OXPHOS oder durch die zytosolische Glykolyse gewährleistet. Sauerstoff und Glukose stellen die Hauptsubstrate dieser Stoffwechselprozesse dar. Fragestellung: Unter pathologischen Bedingungen wie sie in Entzündungsgebieten herrschen, konnte ein relativer Sauerstoffmangel experimentell nachgewiesen werden. Ziel dieser Arbeit war es herauszufinden, in welcher Weise die Funktionen einer definierten Lymphozytenpopulation (CD4+) durch Sauerstoffmangel beeinflusst werden. Methoden: Nach Isolation von CD4+ Zellen aus peripherem Blut gesunder Spender, wurden definierte Zellmengen stimuliert und in einem mit einer Sauerstoffelektrode ausgestatteten Gefäß unter Luftabschluß inkubiert. Zu definierten Zeitpunkten wurden Proben zur ATP-Messung entnommen, sowie Protein- und RNA-Lysate hergestellt. Die Vitalität zu Anfang und zum Ende der Inkubation wurde mittels Propidium-Jodid-Färbung im FACS bestimmt. Aus gesammelten Überständen wurden mittels Multiplex-ELISA die Konzentrationen von IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha und MCAF gemessen. Als Kontrollen dienten unter Normoxie inkubierte Aliquots der Zellsuspensionen. HIF-1alpha wurde mit Immunoblotting nachgewiesen. Transkriptionsänderungen von SOD1 und HK1 wurden durch SYBR-Green Real-Time-PCR quantifiziert. Ergebnisse: Stimulierte CD4+-Zellen von Normalspendern schütten unter dem Einfluss von Hypoxie vermehrt proinflammatorische und chemotaktisch wirksame Zytokine, sowie zur Differenzierung notwendige antiinflammatorische Zytokine aus. Die Verfügbarkeit von Glukose hat hierauf einen verstärkenden Effekt. Eine hypoxische Umgebung sorgt in Abhängigkeit von der Versorgung mit Glukose für eine Anpassung der zellulären Atmungsrate. Glukose ist für die Aufrechterhaltung eines konstanten ATP-Levels verantwortlich. Die glykolytische Energiegewinnung unter Hypoxie kompensiert den Ausfall der OXPHOS. Hypoxie führt bei stimulierten CD4+-Zellen bei freier Glukoseverfügbarkeit zu einer vermehrten Transkription des Hexokinase1-Gens. Glukosemangel bewirkt dagegen in hypoxischer Umgebung eine Transkriptionssteigerung des SOD1-Gens. / Background: The energy supply of immune cells in form of ATP is the cornerstone of a functional immune system. This supply is realized by either mitochondrial OXPHOS or cytosolic glycolysis. Oxygen and glucose present the main substrates in these metabolic processes. Objective: Relative shortness of oxygen could be determined experimentally under pathological conditions present in inflamed tissues. The aim of this study was to determine the extent of hypoxic influence on the cellular function of CD4+ lymphocytes. Methods: Human CD4+ cells were isolated from peripheral blood of healthy blood donors by MACS sorting. Following a defined protocol cells were stimulated and incubated in a sealed container with a Clark type electrode. Samples were taken for measurements of ATP content. RNA- and Protein lysates were made to quantify the transcription of SOD1 and HK1 by SYBR green RT-PCR and look for the presence of HIF-1alpha by immunoblot analysis respectively. Supernatants were used to measure the expression of IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha and MCAF using a multiplex ELISA assay. Aliquots of cell supspensions incubated under normoxic conditions served as controls. Results / Conclusion: Under the influence of hypoxia stimulated CD4+ lymphocytes of healthy blood donors express proinflammatory and chemotactically active as well as anti-inflammatory cytokines important for cell differentiation. The availability of glucose leads to an increase of this effect. An hypoxic environment dependant on the availability of glucose leads to an adaptation of cellular respiration. Glucose deficiency provokes an increase in cellular oxygen utilization. The availability of glucose is responsible for a constant intracellular ATP level. This proves that in CD4+ lymphocytes glycolysis is capable of compensating for hypoxically impaired oxidative phosphorylation thus providing enough ATP to enable cellular function. Hypoxia under glucose provision leads to an increase in mRNA expression for HK1, a key enzyme of glycolysis. Lack of glucose under hypoxic conditions results in an increase in mRNA expression for SOD1. Glucose therefore serves in CD4+ cells as an agent of constant energy supply that leads to cell survival and an upkeep of a proinflammatory environment through cytokine expression.

Für die Untersuchung standen 20 tragende Booroola*Merinofleischschaf Kreuzungsgenotypen, meist pluripar, zwischen 2 und 6 Jahren zur Verfügung. Während der Trächtigkeit wurden in wöchentlichem Abstand die Größenveränderungen der Plazentomdurchmesser mittels transkutaner und transrektaler Ultrasonographie erfasst und Blutproben genommen. Post partum erfolgte die detaillierte Auswertung der Plazenta nach Anzahl und Durchmesser der Kotyledonen und des Kotyledonen- und Sekundinengewichts. Aus den gewonnenen Daten wurden Plazentomwachstumskurven im Verlauf der Trächtigkeit erstellt. Die Blutproben wurden hormonanalytisch auf Progesteron, 17ß-Östradiol, und IGF-1, sowie stoffwechselphysiologisch auf die, den Eiweiß- und Energiehaushalt charakterisierenden Substanzen Albumin, Gesamteiweiß, Harnstoff, BHB, Bilirubin und Glukose untersucht. Zudem erfolgte die Bestimmung von Substanzen, die in der Trächtigkeit stark beansprucht werden, wie ASAT, Calcium, Eisen und Cholesterol. Die Auswertung sämtlicher Daten erfolgte in Abhängigkeit von Wurfgröße (WG) und Wurfgewichtsklassen (WGK). Der größte Einfluss von WG und WGK auf die Plazenta zeigt sich bei der Auswertung der morphologischen Aspekte. Dabei sind in erster Linie eine Vergrößerung der Plazentomdurchmesser von uni- zu triparen Tieren sowie von WGK 1 zur WGK 3 festzustellen. In den jeweils höchsten Klassen fällt der Durchmesser wieder. Zudem nimmt in der Regel mit steigender WG und WGK die Anzahl der Plazentome mit kleinen Durchmessern (1-20 mm)ab, die Anzahl der Plazentome mit großen Durchmessern (20-50 mm) dagegen zu. Tripare Tiere und WGK 3 weisen die meisten Plazentome mit den größten Durchmessern (40-50 mm) auf. Bei der Gesamtkontaktfläche zeigt sich ein Anstieg der Fläche von uni- bis zu quadriparen Tieren und der WGK 1 bis WGK 4. Nur WGK 5 weist eine gegenüber WGK 4 verminderte Gesamtkontaktfläche auf. Unabhängig von WG und WGK verkleinern sich die Plazentomdurchmesser und die Gesamtkontaktflächen p.p. gegenüber den Plazentomdurchmessern und Gesamtkontaktflächen a.p. WG und WGK beeinflussen den Hormonhaushalt mäßig. Dabei zeigt sich der größte Einfluss beim Progesteron. Die kleinste WG bzw. WGK präsentiert die niedrigsten Progesteronkonzentrationen. Beim 17ß-Östradiol ist der Verlauf aller Konzentrationskurven im gesamten Trächtigkeitsverlauf und beim IGF-1 ab dem 100. Tag p.c. einheitlich. Durch WG oder WGK werden bei den Booroola*MF Kreuzungsgenotypen keine den Stoffwechsel charakterisierenden Substanzen in einer auffallenden oder für die Tierart untypischen Weise verändert. Die Anzahl Feten bzw. die WGK zeigen im vorliegenden Datenmaterial keinen gravierenden Einfluss auf den maternalen Stoffwechsel während der Gravidität. / The analysis was founded on 20 pregnant Booroola* Merino Mutton crossbreed sheep between 2 and 6 years, most of them pluriparous. During pregnancy the variations of size of the placentomdiameters were drawn up on a weekly basis using transcutaneous and transrectal ultrasonography. Samples of blood were taken likewise in a weekly rhythm. After birth a detailed examination of the placentae followed, considering especially their number and diameter as well as the weight of the cotyledons and the secundinae. The gained data were used to create placentom-growth-diagrams during pregnancy. The samples of blood were hormonally analysed with regard to progesterone, estradiol and IGF-1 as well as to the substances that characterize the protein- and energy metabolism as there are: albumin, protein, urea, BHB, bilirubin, glucose. Moreover substances which are highly required during pregnancy were determined as e.g. ASAT, calcium, iron, cholesterol. The evaluation of all the gained data was made in dependence on the littersize (LS) and total litterweight (TLW). The major influence of the LS and TLW on the placenta is noticed at the morphological aspects. The placentomdiameter increase from uni- to triparous ewes and from TLW 1 to TLW 3. On the other hand the placentomdiameters decrease in the highest groups. With an increase of LS and TLW the amound of placentoms with small diameters (1-20 mm) generally decreased in favour of the placentoms with big diameters (20-50 mm). Triparous ewes and TLW 3 show most of the placentoms with the largest diameters (40-50 mm). The "total contact area" demonstrates an increase from uni-to quadriparous ewes and TLW 1 to TLW 4. Only TLW 5 has a smaller "total contact area" as TLW 4. Independent of LS and TLW of the Booroola* Merino Mutton crossbreed sheep the placentomdiameter and the "total contact area" decrease p.p. in comparison to the placentomdiameter and "total contact area" a.p. The influence of LS and TLW on the endocrinological system is moderate. The highest influence is proved concerning progesterone, the smallest LS and TLW show the lowest blood-progesterone concentration. In contrast to this, the estradiol concentration of both groups seems to be uniform during the whole pregnancy and equally the IGF-1 concentration from the 100 day p.c. None of the metabolism characterising substances seems to be affected by the LS or TLW. The littersize as well as the litterweight do not influence in the gained data the maternal metabolism during pregnancy.

Für die Untersuchung standen 20 tragende Booroola*Merinofleischschaf Kreuzungsgenotypen, meist pluripar, zwischen 2 und 6 Jahren zur Verfügung. Während der Trächtigkeit wurden in wöchentlichem Abstand die Größenveränderungen der Plazentomdurchmesser mittels transkutaner und transrektaler Ultrasonographie erfasst und Blutproben genommen. Post partum erfolgte die detaillierte Auswertung der Plazenta nach Anzahl und Durchmesser der Kotyledonen und des Kotyledonen- und Sekundinengewichts. Aus den gewonnenen Daten wurden Plazentomwachstumskurven im Verlauf der Trächtigkeit erstellt. Die Blutproben wurden hormonanalytisch auf Progesteron, 17ß-Östradiol, und IGF-1, sowie stoffwechselphysiologisch auf die, den Eiweiß- und Energiehaushalt charakterisierenden Substanzen Albumin, Gesamteiweiß, Harnstoff, BHB, Bilirubin und Glukose untersucht. Zudem erfolgte die Bestimmung von Substanzen, die in der Trächtigkeit stark beansprucht werden, wie ASAT, Calcium, Eisen und Cholesterol. Die Auswertung sämtlicher Daten erfolgte in Abhängigkeit von Wurfgröße (WG) und Wurfgewichtsklassen (WGK). Der größte Einfluss von WG und WGK auf die Plazenta zeigt sich bei der Auswertung der morphologischen Aspekte. Dabei sind in erster Linie eine Vergrößerung der Plazentomdurchmesser von uni- zu triparen Tieren sowie von WGK 1 zur WGK 3 festzustellen. In den jeweils höchsten Klassen fällt der Durchmesser wieder. Zudem nimmt in der Regel mit steigender WG und WGK die Anzahl der Plazentome mit kleinen Durchmessern (1-20 mm)ab, die Anzahl der Plazentome mit großen Durchmessern (20-50 mm) dagegen zu. Tripare Tiere und WGK 3 weisen die meisten Plazentome mit den größten Durchmessern (40-50 mm) auf. Bei der Gesamtkontaktfläche zeigt sich ein Anstieg der Fläche von uni- bis zu quadriparen Tieren und der WGK 1 bis WGK 4. Nur WGK 5 weist eine gegenüber WGK 4 verminderte Gesamtkontaktfläche auf. Unabhängig von WG und WGK verkleinern sich die Plazentomdurchmesser und die Gesamtkontaktflächen p.p. gegenüber den Plazentomdurchmessern und Gesamtkontaktflächen a.p. WG und WGK beeinflussen den Hormonhaushalt mäßig. Dabei zeigt sich der größte Einfluss beim Progesteron. Die kleinste WG bzw. WGK präsentiert die niedrigsten Progesteronkonzentrationen. Beim 17ß-Östradiol ist der Verlauf aller Konzentrationskurven im gesamten Trächtigkeitsverlauf und beim IGF-1 ab dem 100. Tag p.c. einheitlich. Durch WG oder WGK werden bei den Booroola*MF Kreuzungsgenotypen keine den Stoffwechsel charakterisierenden Substanzen in einer auffallenden oder für die Tierart untypischen Weise verändert. Die Anzahl Feten bzw. die WGK zeigen im vorliegenden Datenmaterial keinen gravierenden Einfluss auf den maternalen Stoffwechsel während der Gravidität. / The analysis was founded on 20 pregnant Booroola* Merino Mutton crossbreed sheep between 2 and 6 years, most of them pluriparous. During pregnancy the variations of size of the placentomdiameters were drawn up on a weekly basis using transcutaneous and transrectal ultrasonography. Samples of blood were taken likewise in a weekly rhythm. After birth a detailed examination of the placentae followed, considering especially their number and diameter as well as the weight of the cotyledons and the secundinae. The gained data were used to create placentom-growth-diagrams during pregnancy. The samples of blood were hormonally analysed with regard to progesterone, estradiol and IGF-1 as well as to the substances that characterize the protein- and energy metabolism as there are: albumin, protein, urea, BHB, bilirubin, glucose. Moreover substances which are highly required during pregnancy were determined as e.g. ASAT, calcium, iron, cholesterol. The evaluation of all the gained data was made in dependence on the littersize (LS) and total litterweight (TLW). The major influence of the LS and TLW on the placenta is noticed at the morphological aspects. The placentomdiameter increase from uni- to triparous ewes and from TLW 1 to TLW 3. On the other hand the placentomdiameters decrease in the highest groups. With an increase of LS and TLW the amound of placentoms with small diameters (1-20 mm) generally decreased in favour of the placentoms with big diameters (20-50 mm). Triparous ewes and TLW 3 show most of the placentoms with the largest diameters (40-50 mm). The "total contact area" demonstrates an increase from uni-to quadriparous ewes and TLW 1 to TLW 4. Only TLW 5 has a smaller "total contact area" as TLW 4. Independent of LS and TLW of the Booroola* Merino Mutton crossbreed sheep the placentomdiameter and the "total contact area" decrease p.p. in comparison to the placentomdiameter and "total contact area" a.p. The influence of LS and TLW on the endocrinological system is moderate. The highest influence is proved concerning progesterone, the smallest LS and TLW show the lowest blood-progesterone concentration. In contrast to this, the estradiol concentration of both groups seems to be uniform during the whole pregnancy and equally the IGF-1 concentration from the 100 day p.c. None of the metabolism characterising substances seems to be affected by the LS or TLW. The littersize as well as the litterweight do not influence in the gained data the maternal metabolism during pregnancy.

Neurogenesis in the adult hippocampus contributes to learning and memory in the healthy brain but is dysregulated in metabolic and neurodegenerative diseases. The molecular relationships between neural stem cell activity, adult neurogenesis, and global metabolism are largely unknown. Here we applied unbiased systems genetics methods to quantify genetic covariation among adult neurogenesis and metabolic phenotypes in peripheral tissues of a genetically diverse family of rat strains, derived from a cross between the spontaneously hypertensive (SHR/OlaIpcv) strain and Brown Norway (BN-Lx/Cub). The HXB/BXH family is a very well established model to dissect genetic variants that modulate metabolic and cardiovascular diseases and we have accumulated deep phenome and transcriptome data in a FAIR-compliant resource for systematic and integrative analyses. Here we measured rates of precursor cell proliferation, survival of new neurons, and gene expression in the hippocampus of the entire HXB/BXH family, including both parents. These data were combined with published metabolic phenotypes to detect a neurometabolic quantitative trait locus (QTL) for serum glucose and neuronal survival on Chromosome 16: 62.1–66.3 Mb. We subsequently fine-mapped the key phenotype to a locus that includes the Telo2-interacting protein 2 gene (Tti2)—a chaperone that modulates the activity and stability of PIKK kinases. To verify the hypothesis that differences in neurogenesis and glucose levels are caused by a polymorphism in Tti2, we generated a targeted frameshift mutation on the SHR/OlaIpcv background. Heterozygous SHR-Tti2+/- mutants had lower rates of hippocampal neurogenesis and hallmarks of dysglycemia compared to wild-type littermates. Our findings highlight Tti2 as a causal genetic link between glucose metabolism and structural brain plasticity. In humans, more than 800 genomic variants are linked to TTI2 expression, seven of which have associations to protein and blood stem cell factor concentrations, blood pressure and frontotemporal dementia.

No description available.