Précédemment, nous avons montré que la surexpression de l'échangeur Na/Ca NCX1), une protéine responsable de la sortie de calcium (Ca2+) des cellules, augmentait la mort cellulaire programmée ou « apoptose » et réduisait la prolifération des cellules β. Afin d’étudier plus en profondeur le rôle de l’échangeur dans les cellules β in vivo, nous avons développé et caractérisé des souris présentant une inactivation de NCX1.<p>Des méthodes biologiques et morphologiques (imagerie du Ca2+, capture de Ca2+, métabolisme du glucose, sécrétion d'insuline et morphométrie par comptage de points) ont été utilisées pour évaluer la fonction de la cellule β in vitro. Les taux de glucose et d'insuline dans le sang ont été mesurés afin de déterminer le métabolisme du glucose et la sensibilité à l’insuline in vivo. Des îlots ont été transplantés sous la capsule rénale pour évaluer leur capacité à corriger le diabète chez les souris rendues diabétiques par l’alloxane.<p>L'inactivation hétérozygote de Ncx1 chez les souris provoque une augmentation de la sécrétion d’insuline induite par le glucose avec un renforcement important à la fois de la première et de la deuxième phase. Ces résultats s’accompagnent d’une augmentation de la masse et de la prolifération des cellules β. La mutation augmente également le contenu en insuline, l’immunomarquage de la proinsuline, la capture de Ca2+ induite par le glucose et la résistance à l'hypoxie des cellules β. En outre, les îlots de souris Ncx1+/- montrent une capacité à compenser le diabète 2 à 4 fois plus élevé que les îlots de souris Ncx1+/+ lorsque transplantés chez des souris diabétiques.<p>En conclusion, l’inactivation de l'échangeur Na/Ca conduit à une augmentation de la fonction de la cellule β, de sa prolifération, de sa masse et de sa résistance au stress physiologique, à savoir à divers changements de fonction des cellules β opposés aux principales anomalies rencontrées dans le diabète de type 2 (Type 2 Diabetes Mellitus,T2DM). Ceci nous procure un modèle unique pour la prévention et le traitement du dysfonctionnement des cellules β dans le T2DM et pour la transplantation d'îlots.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

La régulation de l’homéostasie du glucose dans l’organisme est la fonction principale des cellules beta sécrétrices d’insuline dans le pancréas endocrine. Le facteur de transcription à domaine « winged helix », RFX6, a récemment, été identifié comme un nouveau régulateur de la différenciation endocrine pancréatique en aval de Ngn3 chez le poisson zèbre, la souris et l’homme. De plus, diverses mutations de Rfx6 chez l’homme ont été identifiées et reliées au syndrome de Mitchell Riley notamment caractérisé par un diabète néonatal, une atrésie de l’intestin grêle et une malabsorption intestinale. Lors de mes travaux de thèse, une approche combinée de transcriptomique chez la souris et la recherche des sites de fixation de RFX6 dans une lignée cellulaire beta et dans les ilots pancréatiques a permis de démontrer son importance dans le maintien de l’identité et de la fonction de la cellule beta. Pour la première fois, l’identification des cibles directes de RFX6 in vivo a été réalisée et a permis l’identification de l’ensemble du répertoire des gènes régulés directement par RFX6 dans les cellules beta qui n’ont pas été révélés dans le système cellulaire. Cette étude aura également permis d’identifier Mlxipl comme principale cible directement régulée par Rfx6 à la fois chez la souris et l’homme. Les expériences réalisées ont ainsi permis de déterminer les gènes cibles directs de RFX6 et contribué à élucider le rôle de ce facteur de transcription dans la différenciation et la fonction des cellules beta sécrétrices d’insuline. / Glucose homeostasis regulation in the body is the main function of insulin secreting beta cells in the endocrine pancreas. The winged-helix transcription factor RFX6 has recently been identified as a new pancreatic endocrine differentiation regulator, downstream of Ngn3,in zebra fish, mouse and human. Moreover, several Rfx6 mutations in humans were discovered and linked to the Mitchell Riley syndrome, which is characterized by neonatal diabetes, intestinal atresia and malabsorption. My thesis consisted of using an approach combining transcriptomic analysis in mouse and the identification of RFX6 target genes in a beta cell line as well as in pancreatic islets. This work has demonstrated the crucial role of RFX6 in maintaining beta cell identity and function. For the first time, RFX6 target genes were identified in vivo as well as the whole repertoire of directly regulated RFX6 target genesin beta cells, which were previously unidentified in the beta cell line. These studies have also shown that Mlxipl is a main RFX6 regulated target gene in mice and human. Overall, this work has allowed the clear identification of RFX6 target genes, thus contributing inunderstanding the role of this crucial transcription factor in the differentiation and function of insulin secreting beta cells.

Le diabète est une maladie qui touche 347 millions de personnes dans le monde (90% ayant un diabète de type 2) (OMS, septembre 2012). Elle se définit par une perturbation de la régulation de l’homéostasie glucidique avec un déficit dans la fonction des cellules ß du pancréas. Dans le diabète de type 1, ce déficit est provoqué par une destruction autoimmune. Dans le diabète de type 2, il est dû à une insulino-résistance périphérique conduisant à un épuisement des cellules ß qui ne peuvent plus maintenir leur fonction. Une stratégie pour restaurer une masse fonctionnelle de cellules ß est, soit d’induire la prolifération de ces cellules in vitro avant de les greffer, soit d’induire leur prolifération in vivo. Cependant, ceci implique une meilleure compréhension des mécanismes moléculaires impliqués dans le cycle cellulaire des cellules ß pancréatiques humaines. L’objectif de ma thèse a été de disséquer ces mécanismes. Pour cela, nous disposons au laboratoire d’un outil unique, deux lignées de cellules ß pancréatiques humaines. Dans l’une d’elle, les transgènes immortalisant peuvent être délétés. Les cellules arrêtent alors de proliférer donnant des cellules ß pseudo-primaires. En comparant l’expression des régulateurs du cycle cellulaire de la lignée de cellules ß humaine immortalisées aux cellules ß humaine pseudo-primaires, nous avons pu montrer que le cycle de ces cellules était bloqué en phase G1. L’absence de plusieurs protéines responsables de la progression du cycle cellulaire en aval de cette phase a été confirmée dans les îlots humains. Nous avons également observé une diminution du temps de doublement des cellules ß humaines suite à leur traitement avec de la GH et de l’EPO. Suite à ce traitement nous observons également une activation du facteur de transcription STAT5 connue pour son implication dans la prolifération cellulaire des cellules ß de rongeurs. Enfin nous avons étudié l’effet que provoquait un apport nutritif sur la fonction et la prolifération des cellules ß humaines. Nous avons ainsi pu voir que les cellules répondaient mieux à la fonction ß avec un temps de doublement du nombre de cellules plus court dans un milieu enrichi en nutriment. De plus, dans ces conditions, l’autophagie, préexistante avant l’apport de nutriments et vraisemblablement due à un manque en énergie cellulaire, disparait. Ces résultats nous permettent de mieux comprendre les mécanismes contrôlant la prolifération des cellules ß pancréatiques humaines et d’envisager de nouvelles thérapies du diabète. / Diabetes is a disease that affects 347 million people worldwide (90% with type 2 diabetes) (WHO, September 2012). It is defined by a disturbance in the regulation of glucose homeostasis with a deficit in function of pancreatic beta cells. In type 1 diabetes, this deficit is caused by autoimmune destruction. In type 2 diabetes, it is due to peripheral insulin resistance leading to a depletion of beta cells that can no longer maintain their function. A strategy to restore a functional beta cell mass is, or of inducing proliferation of these cells in vitro prior to transplant, or to induce proliferation in vivo. However, this implies a better understanding of the molecular mechanisms involved in the cell cycle of human pancreatic beta cells. The aim of my thesis was to dissect these mechanisms. For this, we have a unique laboratory tool, two lines of human pancreatic beta cells. In one of them, immortalizing transgenes may be deleted. Then, the cells stop to proliferate giving pseudo- primary beta-cells. By comparing the expression of cell cycle regulators of the lineage of human beta cells immortalized pseudo- primary human beta- cells, we could show that the cycle of these cells was blocked in the G1 phase. The lack of several proteins responsible for cell cycle progression downstream of this phase has been confirmed in human islets. We also observed a decrease in the doubling time of human beta cells following treatment with GH and EPO. Following this treatment we also observe an activation of the transcription factor STAT5 known for its involvement in cell proliferation of rodent beta cells. Finally, we studied the effect that caused a nutrient supply on the function and proliferation of human beta cells. We have seen that the cells responded better to beta function with a doubling time shorter amount of cells in a nutrient enriched environment. In addition, under these conditions, autophagy, existing before the supply of nutrients and likely due to a lack of cellular energy, disappears. These results allow us to better understand the mechanisms controlling proliferation of human pancreatic ß and consider new diabetes therapies cells.

Type 1 diabetes develops through the progressive destruction of the insulin-producing beta-cells. Regeneration or replacement of beta-cells is therefore needed to restore normal glucose homeostasis. Presently, normoglycemia can be achieved by the transplantation of whole pancreas or isolated islets of Langerhans. Islet transplantation can be performed through a simple laparoscopic procedure, but the long-term graft survival is low due to poor revascularization and early cell death. This thesis examined the possibility of using different auxiliary cells (Schwann cells, endothelial progenitor cells, and neural crest stem cells) to improve the engraftment and function of endogenous and transplanted islets. Co-transplantation of Schwann cells with islets improved islet graft function early after transplantation, and caused an increased islet mass at one month posttransplantation. However, the vascular densities of these grafts were decreased, which also related to an impaired graft function. Islet grafts containing endothelial progenitor cells had a superior vascular density, with functional chimeric blood vessels and substantially higher blood perfusion and oxygen tension than control transplants. By culturing and transplanting islets together with neural crest stem cells it was found that islets exposed to these cells had a higher beta-cell proliferation compared with control islets. At one month posttransplantation, the grafts with neural crest stem cells also had a superior vascular- and neural density. The potential of intracardially injected neural crest stem cells to home to the pancreas and ameliorate hyperglycemia in diabetic mice was investigated. During a three-week period after such cell treatment blood glucose concentrations decreased, but were not fully normalized. Neural crest stem cells were present in more than 10% of the pancreatic islets at two days postinjection, at which time the beta-cell proliferation was markedly increased when compared with islets of saline-treated diabetic animals. Three weeks later, a doubled beta-cell mass was observed in animals receiving neural crest stem cells. In summary, islets can easily be transplanted together with different auxiliary cells. Some of these cells provide the possibility of improving vascular- and neural engraftment, as well as beta-cell growth and survival. Systemic administration of neural crest stem cells holds the potential of regenerating the endogenous beta-cells.

Indiana University-Purdue University Indianapolis (IUPUI) Indiana University School of Medicine / The islet β cell is central to the maintenance of glucose homeostasis as the β cell is solely responsible for the synthesis of Insulin. Therefore, better understanding of the molecular mechanisms governing β cell function is crucial to designing therapies for diabetes. Pdx1, the master transcription factor of the β cell, is required for the synthesis of proteins that maintain optimal β cell function such as Insulin and glucose transporter type 2. Previous studies showed that Pdx1 interacts with the lysine methyltransferase Set7/9, relaxing chromatin and increasing transcription. Because Set7/9 also methylates non-histone proteins, I hypothesized that Set7/9-mediated methylation of Pdx1 increases its transcriptional activity. I showed that recombinant and cellular Pdx1 protein is methylated at two lysine residues, Lys123 and Lys131. Lys131 is involved in Set7/9 mediated augmented transactivation of Pdx1 target genes. Furthermore, β cell-specific Set7/9 knockout mice displayed glucose intolerance and impaired insulin secretion, accompanied by a reduction in the expression of Pdx1 target genes. Our results indicate a previously unappreciated role for Set7/9 in the maintenance of Pdx1 activity and β cell function. β cell function is regulated on both the transcriptional and translational levels. β cell function is central to the development of type 1 diabetes, a disease wherein the β cell is destroyed by immune cells. Although the immune system is considered the primary instigator of the disease, recent studies suggest that defective β cells may initiate the autoimmune response. I tested the hypothesis that improving β cell function would reduce immune infiltration of the islet in the NOD mouse, a mouse model of spontaneous type 1 diabetes. Prediabetic NOD mice treated with pioglitazone, a drug that improves β cell function, displayed an improvement in β cell function, a reduction in β cell death, accompanied by reductions in β cell autoimmunity, indicating that β cell dysfunction assists in the development of type 1 diabetes. Therefore, understanding the molecular mechanisms involved in β cell function is essential for the development of therapies for diabetes.

No description available.

Les îlots de Langerhans, principalement composés de cellules bêta sécrétant l’insuline, jouent un rôle majeur dans l’homéostasie glucidique grâce à leur sécrétion hormonale finement régulée. Dans un contexte d’insulino-résistance associée à l’obésité, la masse fonctionnelle des cellules bêta pancréatiques augmente, en partie grâce à une prolifération accrue. Le diabète de type 2 survient lorsque les mécanismes de compensation échouent et que la sécrétion d’insuline devient insuffisante. Par conséquent, augmenter la prolifération des cellules bêta a été proposée comme approche thérapeutique afin de retarder l’apparition du diabète de type 2. Parmi les différents facteurs pouvant moduler la prolifération des cellules bêta, les nutriments, en particulier le glucose et les acides gras, jouent un rôle important et plusieurs études chez le rongeur montrent que les nutriments augmentent la prolifération et la masse des cellules bêta avant l’apparition de l’insulino-résistance. De plus, des travaux de notre laboratoire ont montré que l’infusion d’un mélange d’acide gras, le ClinOleic (65% oléate, 20% linoléate et 15% palmitate) et de glucose provoquait une augmentation marquée de la prolifération des cellules bêta chez le rat. L’objectif de cette thèse est donc d’évaluer les mécanismes par lesquels les acides gras stimulent la prolifération des cellules bêta. Dans un premier article, seul l’oléate, parmi plusieurs acides gras testé, a démonté un effet significatif sur l’augmentation de la prolifération des cellules bêta en présence de glucose ex vivo. La prolifération induite par l’oléate nécessite la formation de sphingolipides à très longue chaîne monoinsaturée, tandis que la perturbation de leur synthèse provoque une diminution de la réponse proliférative. Dans une seconde étude, l’analyse par séquençage d’ARN sur cellules uniques a mis en évidence le rôle important des espèces réactives de l’oxygène, des peroxyrédoxines et du proto-oncogène MYC dans le processus prolifératif des cellules bêta induit par l’oléate. Dans l’ensemble, les travaux présentés dans cette thèse apportent un éclairage nouveau sur le potentiel prolifératif encore énigmatique des cellules bêta pancréatiques et soulignent le rôle des sphingolipides et des espèces réactives de l’oxygène dans ce processus. / The islets of Langerhans, mainly composed of insulin-secreting beta cells, plays a major role in glucose homeostasis due to their finely regulated hormone secretion. In a context of insulin resistance associated with obesity, the functional mass of pancreatic beta cells increases, in part due to increased proliferation. Type 2 diabetes occurs when these compensatory mechanisms fail and insulin secretion becomes insufficient. Therefore, increasing beta cell proliferation has been proposed as a therapeutic approach to delay the onset of type 2 diabetes. Among the various factors that can modulate the proliferation of beta cells, nutrients, in particular glucose and fatty acids, play an important role, and several studies in rodents show that nutrients increase beta-cell proliferation before insulin resistance can be detected. Previous work from our laboratory has shown that infusion of the fatty-acid mixture ClinOleic (65% oleate, 20% linoleate and 15% palmitate) in the presence of glucose markedly increases beta-cell proliferation in rats. The objective of this thesis is to evaluate the underlying mechanisms by which fatty acids stimulate beta-cell proliferation. In a first study, among several fatty acids tested, only oleate increased beta cell proliferation in presence of glucose ex vivo. Oleate-induced beta-cell proliferation requires the formation of monounsaturated very long chain sphingolipids, while blockade of their biosynthesis dampens the proliferative response. In a second study, single-cell RNA sequencing analysis highlighted the role of reactive oxygen species, peroxiredoxins, and the proto-oncogene MYC in oleate-induced beta cell proliferation. Overall, the work presented in this thesis sheds new light on the enigmatic proliferative potential of pancreatic beta cells and identifies a role for sphingolipids and reactive oxygen species in this process.

<p dir="ltr">Obesity is a worldwide epidemic that is partially linked to changing lifestyles within the modern world, including increased access to calorically dense foods and decreased energy output due to more sedentary jobs. Obesity can lead to many different health complications, such as cardiovascular diseases or Type 2 Diabetes (T2D). Obesity-induced T2D is caused by dysfunction of the insulin-producing beta cells of the pancreas. However, mechanisms that promote obesity and the mechanisms by which obesity leads to beta cell dysfunction are not fully known.</p><p dir="ltr">Spinophilin is a filamentous (F)-actin binding, protein scaffolding, and protein phosphatase 1 (PP1)-targeting protein that can regulate protein. Spinophilin has multiple actions. Spinophilin can bundle filamentous actin to modulate the cellular cytoskeleton. Spinophilin also mediates substrate phosphorylation by targeting and modulating PP1 activity. In addition, spinophilin interacts with multiple proteins, including certain G-protein coupled receptors and can scaffold them with F-actin and/or PP1. Previous studies established that spinophilin KO mice have decreased fat mass, increased lean mass, and improved glucose tolerance. Yet, how spinophilin modulates the above metabolic parameters is unclear. We found that spinophilin is expressed in hypothalamic tissue and appears to also be expressed in the feeding center of the hypothalamus, as well as in other glucose-sensing cells known as tanycytes that neighbor the arcuate nucleus and the third ventricle. We found that loss of spinophilin limited weight gain observed in both a leptin receptor db/db mouse line (Leprdb/db<i>)</i> and mice fed a high-fat diet. Moreover, we found that the decreased fat mass seen in global spinophilin KO mice, at least in the Leprdb/db mice, was not due to major differences in feeding behaviors, consistent with what was observed by other groups using high-fat diet-fed mice. </p><p dir="ltr">As spinophilin was not associated with alterations in feeding, we posited that its ability to modulate glucose homeostasis may be linked to non-neuronal actions of the protein. Previous studies have found that spinophilin may regulate adipose tissue function and <i>in vitro</i> pancreatic beta cell function; however, its role in the pancreas and beta cells <i>in vivo</i> is not well characterized. We found that spinophilin is expressed in mouse pancreas. Using proteomics-based approaches we identified multiple putative spinophilin interacting proteins isolated from intact pancreas, including: PP1, the spinophilin homolog neurabin, and myosin-9. KEGG pathway analysis of proteomic proteins identified multiple pathways regulating ER stress, such as the unfolded protein response, and cytoskeletal arrangement. We observed decreased associations of spinophilin with PP1 and neurabin and increased association with myosin-9 in obese, Leprdb/db mice as early as 6 weeks, as well as significant decreases in body weight when spinophilin was knocked out in Leprdb/db mice. Moreover, we confirmed a robust and specific increased interaction of spinophilin with myosin-9, and other cytoskeletal proteins. Additionally, we found specific spinophilin interactions with ribosomal proteins, and exocrine and digestion proteins in high-fat diet-fed mice. Using our recently generated pancreatic beta cell-specific spinophilin KO mice, we found that loss of spinophilin in mice on a high-fat diet significantly reduces weight gain and improves whole- body glucose tolerance, and loss of spinophilin specifically within the beta cells also improves whole-body glucose tolerance, with no effect on body weight, further suggesting cell type-specific and independent roles for spinophilin on body weight and glucose homeostasis.</p>

<p>  </p> <p>The role of the endoplasmic reticulum (ER) Ca2+ release channels ryanodine receptor 2 (RyR2) and inositol 1,4,5-triphosphate receptor (IP3R) in pancreatic b-cell function are emerging, but are not well defined. It has been demonstrated that ER stress brought about by RyR2 dysfunction leads to impaired insulin secretion and contributes to the etiology of type 2 diabetes (T2D). Our work contributes to the understanding of the role of RyR2 in physiological pancreatic b-cell function and how loss of RyR2 contributes to the pathophysiology of T2D.</p> <p>To investigate the role of RyR2 in pancreatic b-cell function, we utilized CRISPR-Cas9 to delete RyR2 from the rat insulinoma INS-1 cell line (RyR2KO). We found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) and were sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. We discovered that total cellular insulin content and secretion were reduced in RyR2KO cells, but more modestly reduced in IRBITKO cells. We found that <em>INS2</em> mRNA levels were reduced in both RyR2KO and IRBITKO cells, but <em>INS1</em> mRNA levels were specifically decreased in RyR2KO cells. Additionally, nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in both RyR2KO and IRBITKO cells. DNA methylation of exon 2 of the <em>INS1</em> and <em>INS2</em> genes was more extensively methylated in RyR2KO and IRBITKO cells compared to controls. Proteomics analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 regulates IRBIT levels and activity, and together maintain insulin content and secretion, and regulate the INS-1 cell proteome, perhaps via DNA methylation.</p> <p>The role of interplay between RyR2 and IP3R in Ca2+ signaling and homeostasis in pancreatic b-cell function remains understudied. Stimulation with the sulfonylurea tolbutamide resulted in markedly delayed Ca2+ transients in both RyR2KO and IRBITKO cells. Xestospongin C significantly reduced the AUC of Ca2+ in RyR2KO and IRBITKO cells. Muscarinic receptor stimulation revealed a markedly increased AUC of Ca2+ in IRBITKO cells compared to both RyR2KO and control INS-1 cells. Assessment of PLC activity revealed that basal and stimulated PLC activity were reduced in the absence of RyR2 or IRBIT. Store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion revealed a decreased SOCE amplitude only in RyR2KO cells. Given evidence that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion from the plasma membrane can regulate voltage-gated Ca2+ channel inhibition, we explored electrophysiological properties of all three cell lines. The frequency of glucose-stimulated action potentials was doubled in RyR2KO cells. Additionally, whole-cell voltage-gated Ca2+ current density was doubled in RyR2KO cells, and this current was more sensitive to hydrolysis of PIP2. These results evidence crosstalk between RyR2 and IP3R, and that RyR2 plays a critical role in maintaining proper Ca2+ homeostasis, PLC activity, and electrophysiological properties in pancreatic b-cells.</p>

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats. / AFRIKAANSE OPSOMMING: Insigte oor die rol wat oksidatiewe stres en pankreas β-sel disfunksie in die patogenese van tipe 2-diabetes (T2D) speel, bied 'n geleentheid vir die ontwikkeling van nuwe terapeutiese middels wat β-selle direk daarteen beskerm. Die beskermende rol van antioksidante in die dieët soos plantaardige polifenole teen oksidatiewe stres geinduseerde siektes soos T2D, is toenemend onder die soeklig. Polifenolryk ekstrakte van Cyclopia spp wat mangiferin bevat mag nuwe terapeutiese middels lewer. ‘n Waterekstrak van ongefermenteerde Cyclopia maculata wat meer as 6% mangiferin bevat, is ondersoek vir sy beskermende effek op pankreas ß-selle in vitro, ex vivo en in vivo teen kondisies kenmerkend aan T2D. Die effek van mangiferin is ook in vitro en ex vivo geëvalueer, met N-asetielsistien (NAC) as 'n antioksidant kontrole. In hierdie studie is in vitro toksisiteitsmodelle in RIN-5F insulinoomselle gevestig. Die modelle is gebaseer op toestande waaraan β-selle blootgestel word tydens T2D; d.w.s. lipotoksisiteit, inflammasie en oksidatiewe stres. Hiervoor is die selle aan die volgende stressors blootgestel: palmitiensuur (PA), ‘n pro-inflammatoriese sitokien mengsel en streptozotosien (STZ). Vervolgens is die vermoë van die C. maculata ekstrak, mangiferin en NAC om die RIN-5Fselle teen hierdie stressors te beskerm, beoordeel deur die meting van β-sellewensvatbaarheid, funksie en oksidatiewe stres. Sellewensvatbaarheid is bepaal met 3-(4,5-dimetielthiazol-2-yl)-2,5-difenieltetrazolium bromied, adenosientrifosfaat en anneksien-V and propidium jodied toetse. Selfunksie is geëvalueer d.m.v. glukose gestimuleerde insuliensekresie, selproliferasie en sellulêre kalsium bepaling. Oksidatiewe stres in die RIN-5Fselle is geëvalueer d.m.v. diaminofluorescein-FM en dihidroethidium fluoressensie bepalings, asook meting van superoksied dismutase ensiemaktiwiteit. Die in vitro bevindings is daarna in geїsoleerde rot pankreaseilande bevestig deur die metodes en modelle wat in die RIN-5F eksperimente gebruik is. Die antidiabetiese effekte van die ekstrak, NAC en metformien in STZ-geїnduseerde diabetiese Wistar rotte is bepaal d.m.v. twee behandlingsregimes, d.w.s. die behandeling van rotte met gevestigde diabetes of deur die behandeling voor die induksie van diabetes te begin. Glukose metabolisme, oksidatiewe stres en veranderinge in die pankreasmorfologie is ondersoek d.m.v. orale glukose toleransie toetse en die bepaling van serum insulien, trigliseriedes, nitriete, katalase en glutationien. Hepatiese tiobarbituursuur reaktiewe stowwe en nitrotirosien is ook geëvalueer. Immunohistochemiese kleuring van pankreas snitte is gebruik vir morfologiese assessering van insulien, glukagon en MIB-5. Die ekstrak het mees opvallend β-sel lewensvatbaarheid en funksie verbeter, terwyl oksidatiewe stres verminder is in die STZ- en PA-geїnduseerde toksisiteitmodelle. Bogenoemde effekte van die ekstrak in vitro en in die geїsoleerde eilande was vergelykbaar met die van NAC. Mangiferin was minder effektief, met slegs ‘n marginale verbetering in die funksie van RIN-5Fselle en eilande, asook t.o.v. oksidatiewe stres. Behandeling van die Wistar rotte met die ekstrak voor induksie van diabetes met STZ was net so effektief, of selfs beter as metformien in terme van verbeterde glukosetoleransie, trigliseriedvlakke en die morfologie van pankreas eilande wat verband gehou het met β-sel funksie. Hierdie studie het getoon dat die waterekstrak van ongefermenteerde C. maculata pankreas β-selle teen veral STZ- en PA-geїnduseerde toksisiteit in vitro en ex vivo beskerm het. In vivo het behandeling met die ekstrak voor en na induksie van diabetes, glukosemetabolisme en die morfologie van pankreas eilande in STZ-geїnduseerde diabetiese Wistar rotte verbeter.