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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-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

info:eu-repo/classification/ddc/610

ddc:610

Identifer	oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:92211
Date	28 June 2024
Creators	Mohanraj, Karthikeyan
Contributors	Mirtschink, Peter, Guan, Kaomei, Technische Universität Dresden
Source Sets	Hochschulschriftenserver (HSSS) der SLUB Dresden
Language	English
Detected Language	English
Type	info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text
Rights	info:eu-repo/semantics/openAccess

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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-cells

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