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Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained ImmunityArts, Rob J.W., Novakovic, Boris, ter Horst, Rob, Carvalho, Agostinho, Bekkering, Siroon, Lachmandas, Ekta, Rodrigues, Fernando, Silvestre, Ricardo, Cheng, Shih Chin, Wang, Shuang Yin, Habibi, Ehsan, Gonçalves, Luís G., Mesquita, Inês, Cunha, Cristina, van Laarhoven, Arjan, van de Veerdonk, Frank L., Williams, David L., van der Meer, Jos, Logie, Colin, O'Neill, Luke A., Dinarello, Charles A., Riksen, Niels P., van Crevel, Reinout, Clish, Clary, Notebaart, Richard A., Joosten, Leo A.B., Stunnenberg, Hendrik G., Xavier, Ramnik J. 13 December 2016 (has links)
Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.
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Exploiting stable isotope imaging with high resolution secondary ion mass spectrometry for applications in biologyJiang, Haibo January 2014 (has links)
This thesis presents applications of high resolution secondary ion mass spectrometry (NanoSIMS) analysis for stable isotope imaging in biological samples. These projects were designed to explore the potential applications of NanoSIMS analysis, and to develop protocols and novel methodologies to visualize and quantify biological processes. Working with collaborators in the UK and USA, I have applied NanoSIMS analysis to study 3 research areas, including molecule interactions, single cell metabolisms and lipid imaging in tissues. Antimicrobial peptides (AMPs) play important role in the immune system, and understanding how AMPs interact with cell membranes can provide useful information to design new therapies to control infection. The pore structures and dynamics of the interaction of AMPs with membranes has been visualized for the first time and confirmed with combined AFM and NanoSIMS analysis. A correlative backscattered electron (BSE) imaging and NanoSIMS analysis methodology has been developed to study glutamine metabolism in single cancer cells. This method enables us to measure the chemical information in specific organelles in these cells and can be widely applied to study metabolisms and to trace the uptake of labelled molecules in biological matrices. Quantitative analysis on the effects of hypoxic conditions and the PYGL gene were studied. Applying correlative BSE and NanoSIMS analysis, I also studied lipid uptake mechanisms in various mouse tissues, including brown adipose tissue, heart, intestines, liver and skeletal muscle, mainly focused on a recently discovered protein, GPIHBP1, and its function in the lipid uptake process. TRL margination was proved to depend on the GPIBP1-LPL complex, and 3 stages of lipid transport from capillary lumen to lipid droplets was also visualized by combined BSE and NanoSIMS analysis.
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PTEN regulates glutamine flux to pyrimidine synthesis and sensitivity to dihydroorotate dehydrogenase inhibitionMathur, Deepti January 2017 (has links)
The importance of metabolism in tumor initiation and progression is becoming increasingly clear. Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Phosphatase and Tensin homolog deleted from chromosome ten (PTEN) is one of the most commonly mutated tumor suppressors in cancer and operates in multiple roles, rendering it a hub for understanding cancer biology and for developing targeted therapy. PTEN’s canonical function is its ability to antagonize the phosphoinositide 3-kinase (PI3K) pathway by dephosphorylating the lipid second messenger phosphatidylinositol (3,4,5) tri-phosphate (PIP3). This thesis focuses on the effects of PTEN loss on cellular metabolism, and the therapeutic vulnerability that stems from metabolic alterations.
First, we discovered that loss of Pten in mouse embryonic fibroblasts (MEFs) increases cellular proliferation and the number of replication forks per cell, launching our investigation into metabolic pathways that may be altered to support increased growth. Indeed, we found that Pten-/- cells exhibited a dependence on glutamine for their faster rate of growth, and that glutamine was channeled into the de novo synthesis of pyrimidines.
The next chapter examined dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for pyrimidine ring synthesis in the de novo pyrimidine synthesis pathway. We found that PTEN-deficient primary cells and cancer cell lines were more sensitive to inhibition of DHODH than PTEN WT cells were, and that the growth inhibition could be rescued by metabolites downstream of DHODH. Furthermore, we found that xenografted human triple negative breast cancer tumors in mice could be diminished by treatment with leflunomide, a DHODH inhibitor.
In the following chapter, we aimed to identify the mechanisms leading to cell death in PTEN mutant cells upon DHODH inhibition. We found that inherent defects in checkpoint regulation in PTEN-deficient cells were exacerbated by the stress of obstructed de novo pyrimidine synthesis, leading to a buildup of DNA damage at replication forks and ultimately chromosomal breaks. This was instigated by AKT-mediated phosphorylation of TOPBP1 that caused inadequate ATR activation, as well as AKT-mediated phosphorylation and inactivation of CHK1.
In sum, the findings of this thesis indicate that enhanced glutamine flux to de novo pyrimidine synthesis in PTEN mutant cells generates vulnerability to DHODH inhibition. The integration of altered glutamine regulation with PTEN’s effect on replication, DNA damage, and the checkpoint response manifests as synthetic lethality upon DHODH inhibition in cells with PTEN inactivation. Inhibition of DHODH could thus be a promising therapy for patients with PTEN mutant cancers.
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Preclinical evaluation of pharmacological strategies designed to enhance the activity of established and novel anti-cancer drugs : synopsis - evaluation of pharmacological strategies designed to modulate the Warburg effect, enhance the activity of tyrosine kinase inhibitors and novel analogues of TemozolomideSaleem, Mohammed Umer January 2014 (has links)
Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.
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Metabolic Changes in Pulmonary Arterial Smooth Muscle Cells Exposed to Increased Mechanical Forces from an Ovine Model of Congenital Heart Disease with Increased Pulmonary Blood FlowSeifert, Elena 01 January 2019 (has links)
An important cause of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To gain a better understanding of the disease process, the changes in biochemical pathways and metabolism of pulmonary arterial smooth muscle cells (PASMCs) were studied using a unique surgical ovine model of increased pulmonary blood flow. PASMCs isolated from 4-week-old lambs with increased PBF (shunt) showed lower oxygen consumption rates and lower extracellular acidification rates linked to glutamine metabolism when compared to controls. Shunt and control PASMCs both exhibited a switch into the reverse tricarboxylic acid (TCA) cycle, while only shunt cells showed a decrease of glucose being transformed into Acetyl CoA to enter the forward TCA cycle. Shunt PASMCs also demonstrated increased levels of yes-associated protein 1 (YAP1) expression in the nucleus. These results indicate changes in glutamine metabolism, glucose metabolism, and protein signaling cascades associated with increased mechanical forces in the setting of increased PBF, as seen in PAH in children with CHD.
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Preclinical evaluation of pharmacological strategies designed to enhance the activity of established and novel anti-cancer drugs. Synopsis: Evaluation of pharmacological strategies designed to modulate the Warburg effect, enhance the activity of tyrosine kinase inhibitors and novel analogues of Temozolomide.Saleem, Mohammed Umer January 2014 (has links)
Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.
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Proline is a novel modulator of glucokinase mediating the crosstalk between glutamine and glucose metabolism in the regulation of insulin secretion by pancreatic β-cellsMohanraj, Karthikeyan 28 June 2024 (has links)
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
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