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

Most research on human pancreatic islets is conducted on samples obtained from normoglycaemic or diseased brain-dead donors and thus cannot accurately describe the molecular changes of pancreatic islet beta cells as they progress towards a state of deficient insulin secretion in type 2 diabetes (T2D). Here, we conduct a comprehensive multi-omics analysis of pancreatic islets obtained from metabolically profiled pancreatectomized living human donors stratified along the glycemic continuum, from normoglycemia to T2D. We find that islet pools isolated from surgical samples by laser-capture microdissection display remarkably more heterogeneous transcriptomic and proteomic profiles in patients with diabetes than in non-diabetic controls. The differential regulation of islet gene expression is already observed in prediabetic individuals with impaired glucose tolerance. Our findings demonstrate a progressive, but disharmonic, remodelling of mature beta cells, challenging current hypotheses of linear trajectories toward precursor or transdifferentiation stages in T2D. Furthermore, through integration of islet transcriptomics with preoperative blood plasma lipidomics, we define the relative importance of gene coexpression modules and lipids that are positively or negatively associated with HbA1c levels, pointing to potential prognostic markers.

Bakgrund: Diabetes typ 2 är en endokrin sjukdom och ett globalt hälsoproblem, där antalet insjuknande personer ökar kraftigt. Behandlingen vid diabetes typ 2 utgörs till största del av egenvård vilket ställer stora krav på patienten och på sjukvården. En bristande följsamhet till råd om egenvård kan leda till sämre hälsa för patienten och ökade kostnader för samhället. Syfte: Syftet med denna litteraturöversikt var att beskriva vilka faktorer som påverkar följsamheten till råd om egenvård hos patienter med diabetes typ 2. Metod: En litteraturöversikt baserad på 15 vetenskapliga artiklar där både kvalitativa och kvantitativa artiklar har granskats. Databaserna CINAHL och PubMed har använts. Resultat: Faktorer som påverkade följsamheten till råd om egenvård identifierades och resulterade i fem huvudkategorier: Information; Kunskap; Socialt stöd; Teknologiskt stöd och Livssituation. Dessa faktorer framkom som viktiga för en god följsamhet till råd om egenvård. Slutsats: Det är av stor betydelse att försöka identifiera varje individs olika förutsättningar, för att på så sätt ha möjlighet att anpassa både information, utbildning och egenvårdsplanering utifrån individen. / Background: Type 2 diabetes is an endocrine disorder and a global health problem, with the number falling ill persons greatly increases. Treatment of type 2 diabetes consists mainly of self-care, which places great demands on the patient and the healthcare system. A lack of compliance to advice on self-care can lead to poorer health for the patient and increased costs for society. Aim: The purpose of this literature review was to describe the factors that influence compliance to advice on self-care in patients with type 2 diabetes. Method: A literature review based on 15 scientific articles, both qualitative and quantitative articles were reviewed. The databases CINAHL and PubMed were used. Results: Factors affecting compliance to advice on self-care were identified and resulted in five broad categories: Information; Knowledge; Social support; technological support and Life situation revealed as important factors for low compliance to advice on self-care. Conclusion: It is very important to try to identify each individual's different conditions, in order to thus be able to cu

Introduction: The continual increase of type 2 diabetes (T2D) prevalence is a global public health concern. The aetiology of T2D has not been fully elucidated and this is hampering the development of effective preventative and curative interventions to curb the T2D burden. Although much has been done to elucidate the environmental risk factors associated with T2D, little is known about the precise genetic risk factors that predispose people to it. There is limited knowledge about the common variants associated with T2D risk in the black South African population. However, evidence of shared common variants associated with T2D among people of different ethnicities has been documented. Nonetheless, the majority of the common variants that have been reported to be associated with T2D in other ethnicities are still yet to be evaluated in the black South African population. Objectives: The aim of this study was to evaluate the association of previously reported common genetic variants with T2D susceptibility, as indicated by impaired glucose tolerance (IGT), in a black South African population of Tswana descent. Methods: This study was a case-control study of 180 cases and 180 controls nested in the Prospective Urban Rural Epidemiology (PURE) study baseline data, which was collected in 2005. The DNA samples of the participants were genotyped for 77 single nucleotide polymorphisms (SNPs), using Illumina® VeraCode technology on the BeadXpress® platform. The gPlink software was used to evaluate the standard genetic models of disease penetrance for the association of the common variants with impaired glucose tolerance (IGT) while adjusting for age, sex and body mass index. Results: Four out of the 66 SNPs that were evaluated through the genetic association tests in this study were noted to be significantly associated with IGT (p< 0.05). Of the four SNPs, only rs1436955 was associated with an increase in T2D risk, while the other three variants, rs831571, rs8050136 and rs7542900, were noted to be associated with a decreased risk of T2D. However, none of the four SNPs was significantly associated with IGT after correcting for multiple testing (p <0.05). Conclusions: Black South Africans of Tswana descent might not share common variants associated with T2D risk, as indicated by IGT in other ethnicities. Wellpowered studies are required to evaluate the association of common variants with T2D risk in this population group. The results from this study emphasise the need for population-specific variants to assess the genetic susceptibility of complex diseases such as T2D in the black South African population. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2014

Introduction: The continual increase of type 2 diabetes (T2D) prevalence is a global public health concern. The aetiology of T2D has not been fully elucidated and this is hampering the development of effective preventative and curative interventions to curb the T2D burden. Although much has been done to elucidate the environmental risk factors associated with T2D, little is known about the precise genetic risk factors that predispose people to it. There is limited knowledge about the common variants associated with T2D risk in the black South African population. However, evidence of shared common variants associated with T2D among people of different ethnicities has been documented. Nonetheless, the majority of the common variants that have been reported to be associated with T2D in other ethnicities are still yet to be evaluated in the black South African population. Objectives: The aim of this study was to evaluate the association of previously reported common genetic variants with T2D susceptibility, as indicated by impaired glucose tolerance (IGT), in a black South African population of Tswana descent. Methods: This study was a case-control study of 180 cases and 180 controls nested in the Prospective Urban Rural Epidemiology (PURE) study baseline data, which was collected in 2005. The DNA samples of the participants were genotyped for 77 single nucleotide polymorphisms (SNPs), using Illumina® VeraCode technology on the BeadXpress® platform. The gPlink software was used to evaluate the standard genetic models of disease penetrance for the association of the common variants with impaired glucose tolerance (IGT) while adjusting for age, sex and body mass index. Results: Four out of the 66 SNPs that were evaluated through the genetic association tests in this study were noted to be significantly associated with IGT (p< 0.05). Of the four SNPs, only rs1436955 was associated with an increase in T2D risk, while the other three variants, rs831571, rs8050136 and rs7542900, were noted to be associated with a decreased risk of T2D. However, none of the four SNPs was significantly associated with IGT after correcting for multiple testing (p <0.05). Conclusions: Black South Africans of Tswana descent might not share common variants associated with T2D risk, as indicated by IGT in other ethnicities. Wellpowered studies are required to evaluate the association of common variants with T2D risk in this population group. The results from this study emphasise the need for population-specific variants to assess the genetic susceptibility of complex diseases such as T2D in the black South African population. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2014

High circulating glucose and non-esterified free fatty acid (NEFA) levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In this thesis by exploring mitochondrial energy metabolism in INS-1E insulinoma cells and isolated pancreatic islets, a role of mitochondria in pancreatic β-cell glucolipotoxicity is uncovered. It is reported that prolonged palmitate exposure at high glucose attenuates glucose-stimulated mitochondrial respiration which is coupled to ADP phosphorylation. These mitochondrial defects coincide with an increased level of mitochondrial reactive oxygen species (ROS), impaired glucose-stimulated insulin secretion (GSIS) and decreased cell viability. Palmitoleate, on the other hand, does not affect mitochondrial ROS levels or cell viability and protects against the adverse effects of palmitate on these phenotypes. Interestingly, palmitoleate does not significantly protect against mitochondrial respiratory or insulin secretion defects and in pancreatic islets tends to limit these functions on its own. Furthermore, strong evidence suggests that glucolipotoxic-induced ROS are of a mitochondrial origin and these ROS are somehow linked with NEFA-induced loss in cell viability. To explore the mechanism of glucolipotxic-induced mitochondrial ROS and associated cell loss, uncoupling protein-2 (UCP2) protein levels and activity were probed in NEFA exposed INS-1E cells. It is concluded that UCP2 neither mediates palmitate-induced mitochondrial ROS production and the related cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity. Collectively, these data shed important new light on the area of glucolipotoxicity in pancreatic β-cells and provide novel insights into the pathogenesis of Type 2 diabetes.

Aim: To test the interactive effects of the constructs of Modified Social Learning Theory (MSLT) in relation to predicting health behaviour in Type 2 Diabetes. Methods: The study is mixed methods and employs an exploratory sequential design. Qualitative Phase: (N=12) Semi-structured interviews with adults with insulin-treated type 2 diabetes, explored how beliefs and values influence self-management behaviour. Interim Phase: Thematic analysis allowed development of an adapted Health Value Measure. Quantitative phase: (N=107) Valid questionnaires measured Health Value, Health Locus of Control (HLC) and Self-efficacy (SE). Health Value was measured pre and post diagnosis in order to compare any changes with time. Anxiety and depression was controlled for using the Hospital Anxiety and Depression(HAD) scale. Five subscales measured diabetes outcome behaviour: general diet, specific diet, exercise, blood sugar and foot care. Hierarchical Multiple Regression(HMR) analyses consisted of four blocks, including three two-way interaction terms and one three-way interaction term to test the interactive effects of the three-predictor variables on outcome behaviours. ANOVA’s were conducted in an effort to add support to HMR results. Results: The interviews suggest that people may hold terminal (beliefs about desired end states)/instrumental health values (beliefs about desired modes of action) pre-diagnosis but these are mainly instrumental post-diagnosis in order to meet their new needs and maintain quality of life. The qualitative data also drew attention to the way in which LOC and SE beliefs impact on behaviour. Additionally, differing dimensions of various emergent themes highlight the demands Type 2 diabetes places on a person and how this influences beliefs and values. Interim phase results resulted in the new items being removed from the adapted health value measure prior to the quantitative data analysis, as item 5 was deemed problematic. Sensitivity analysis was carried out to increase the robustness of the quantitative findings due to removing 29 cases with missing data from Dataset 1. Dataset 1 includes 78 complete cases and Dataset 2 contains 107 cases, 29 of which had missing values and were replaced using regression imputation. HMR analyses produced significant results that support MSLT when the three-way interaction variable was added to block 4. ANOVA results produced minimum support for MSLT. Conclusion: Support for MSLT has been found and can be used to inform interventions to change self-management behaviours of patients with poor diabetes control. Change in health value orientation post-diagnosis purports further investigation, as it is supported by qualitative results but not quantitative.

Background: Type 2 diabetes mellitus (T2DM) is a complex health condition that impacts multiple organ systems and contributes to both acute and chronic health problems. In the United States (U.S.), T2DM is a growing health concern with increasing prevalence among both adult and pediatric populations (American Diabetes Association [ADA], 2015; Dea, 2011). Developing a comprehensive plan of care that incorporates a multifaceted treatment and prevention plan is necessary to address this growing health concern and reduce overall morbidity and mortality. Problem: The Edmund Primary Care (EPC) practice data for routine annual diabetic foot exams, annual eye exams, annual urine microalbumin, smoking cessation education and recommendations for pneumococcal polysaccharide do not meet the ADA (American Diabetes Association, 2015) recommendations for patients with T2DM.Design: Quality improvement (QI) project applying the Plan-Do-Study-Act (PDSA) cycle to develop a process change to improve diabetic preventative care measures for hemoglobin A1C, urine microalbumin, diabetic foot exams, and optometry referrals. Setting: A small, nurse practitioner owned, family practice clinic targeting patients 18 years and older with a diagnosis of T2DM.Intervention: A fishbone diagram to conduct a root cause analysis led to identification of key factors contributing to the problem. A comprehensive process change integrating a Diabetic Assessment Flow Sheet (DAFS) and diabetic foot exam sheet was developed to address the problem. Expected Outcome: Increase in rates of completion to at least 90% over eight weeks. Results: Analyzed with run charts demonstrating an increase in rates of completion to 100% for A1C, urine microalbumin, diabetic foot exams, and optometry referrals. A positive percent of change for each measure is as follows: A1C 7%; urine microalbumin 43%; diabetic foot exams 150%; and referrals to optometrist 43%. Significance: This QI project emphasizes the importance of implementing a system to evaluate the quality of care being delivered. It also highlights the usefulness of the PDSA cycle as a method to implementing quality improvement measures in health care. Lastly, this QI project demonstrated the effectiveness of flow sheets in improving the quality of care delivered to patients with T2DM.

Obesity increases the risk for the development of cardiovascular disease, type 2 diabetes and other co-morbid conditions. Type 2 diabetes also is often associated with excessive visceral abdominal fat. Weight loss in obese individuals decreases the risk for developing the co-morbid conditions. Individuals with type 2 diabetes often have a greater difficulty in controlling these complications compared to individuals without type 2 diabetes. The purpose of this study was to evaluate adherence to a medically-supervised low-energy diet (LED) weight loss program and changes in body composition and metabolic parameters after weight loss in women with and without type 2 diabetes. Subjects consisted of Caucasian women, between the ages of 40 to 65 years, with BMIs between 30 and 45 kg/m2. There was no significant difference in BMI between the groups at study initiation (38.1 kg/m2, diabetics (DM) and 36.0 kg/m2, non-diabetics (NDM), p=0.2314). All subjects participated in the HMR Program for 16 weeks. Twenty-nine subjects completed the weight loss phase (18 diabetics, 11 non-diabetics) and were evaluated for change in weight, body composition, and blood parameters. Data were analyzed by repeated-measures ANCOVA and students t-tests using SAS version 8.02. DM and NDM lost 11.7% and 16% of body weight, respectively (p=0.6474). Results indicate DM has more total lean tissue (p=0.004), more total body fat (p=0.04), more total abdominal tissue (p=0.001), more visceral adipose tissue (p=0.001) and lost less percent body fat (p=0.04) than NDM after 16 weeks of weight loss. After weight loss there was no significant difference in leptin, ghrelin or adiponectin levels. DM had greater insulin (p=0.05), HOMA-IR (pandlt;0.0001), glucose (pandlt;0.0001), HbA1c (pandlt;0.0001), resistin (p=0.04) and PAI-1 (p=0.02). There were no differences after weight loss in lipid levels, blood pressure, diet compliance or exercise. The data show that medically-supervised LEDs are safe and effective for treating obesity in individuals with type 2 diabetes. Cardiovascular risk factors improved in both NDM and DM subjects with weight loss. The findings also suggest that insulin and metabolically dysfunctional lean tissue may play a critical role in the complex axes affecting changes in body composition and inflammation in individuals with type 2 diabetes.

Type 2 diabetes (T2DM) has reached epidemic levels worldwide during the past two decades. It affects nearly 26 million adults in the U.S. Advances in both the treatments for T2DM and guidelines for its optimal management are extensive. Despite these advances, barely half of type 2 diabetics achieve recommended glycemic targets. Specific Aims: The specific aims were to: Describe the available research on clinical inertia and interventions that have been implemented to reduce it. Analyze various behavioral theories that explain and predict self-care practices in diabetes in order to develop a conceptual model on which to base an investigation of predictors of readiness to initiate insulin therapy in type 2 diabetics. Determine predictors of readiness to initiate insulin therapy in patients with T2DM when oral medications fail to control hyperglycemia using the conceptual model based on Self-Determination Theory (SDT) as a framework. Results: A review of research articles published from 1990 to 2010 concluded that clinical inertia of primary care providers treating T2DM resulted in a majority of patients experiencing unnecessary chronic uncontrolled hyperglycemia. Behavioral theories were analyzed for their ability to predict self-care behaviors in type 2 diabetics. A conceptual model was developed based on the major constructs of SDT in order to guide the design of study to investigate predictors of readiness to begin insulin therapy in T2DM. Finally, a descriptive, correlational study was performed to determine readiness to initiate insulin therapy in patients with T2DM when oral medications fail to control hyperglycemia. Results of the study revealed that participants who had a friend or family using insulin were 5.5 times more likely to rate their readiness to initiate insulin as high than those who had neither (p=.020). In addition, those with greater negative beliefs and attitudes toward insulin therapy were more likely to rate their readiness to initiate insulin as low (p=.012). A majority (58%) of participants rated their readiness to begin insulin therapy as immediate if it would give them better control over their hyperglycemia. The study also confirmed findings from previous studies that clinical inertia was present in this setting.