Tesis sobre el tema "Beta-cell mass"

Those key factors, supporting re-expansion of beta cell numbers after injury in various model systems, are largely unknown. Insulin-like growth factor II (IGF-II), an important member from the IGF family, plays a critical role in supporting cell division and differentiation during ontogeny, but its role in the adult is not known. In this study we investigated the effect of IGF-II in beta cell regeneration. An in vivo model of „switchable‟ c-Myc-induced beta cell ablation, pIns-c-MycERTAM (Pelengaris, Khan et al. 2002), which exhibits beta cell regeneration once Myc is deactivated, is employed in this study of the IGF-II function. Here we show for the first time that IGF-II is re-expressed in the adult pancreas following beta cell injury. Moreover, whereas a 90% beta cell ablation was induced in both pIns-cMycERTAM/IGF-II WT (MIG) and pIns-cMycERTAM/IGF-II KO (MIGKO) mice, a recovery up to 3 months was performed. By investigating the beta cell mass and numbers our results demonstrate that re-expression of IGF-II is important in supporting the beta cell regeneration in adult mice. Moreover this study supports the utility of using such ablation-recovery models for identifying other potential factors critical for underpinning successful beta cell regeneration in vivo. Both Myc and PML contribute to the regulation of apoptosis. Recent studies suggest that Myc and PML may interact at several levels in control of cell fate. Here we examined whether loss of the PML protein, which has been shown to regulate apoptosis via the p53 pathway, can prevent or affect c-Myc-induced beta cell apoptosis in pIns-c-MycERTAM transgenic mice. Together with the Applied Neuroinformatics Group at the University of Bielefeld in Germany, we have been developing and validating a machine learning based system to analyze beta and alpha cell numbers (Herold, Zhou et al. 2009). Comparative results between traditional techniques and this new method are presented here.

Current therapies do not prevent the devastating complications associated with type 1 and 2 diabetes. Novel therapies seek to restore a functional beta-cell mass through stimulating endogenous beta-cell mass expansion. Whilst there is considerable evidence that the beta-cell mass is under homeostatic regulation in the normal pancreas, it is unclear if such regulation exists in the context of induced beta-cell mass expansion. The aim of this study was to demonstrate that beta-cell mass expansion resulting from the induction of islet cell neogenesis is subject to long-term homeostatic control in the normoglycemic mouse adult pancreas.
A pentadecapeptide fragment of Islet Neogenesis Associated Protein (INGAP 104-118) was administered daily to adult C57BL/6J mice for 12 weeks. Four animals from the INGAP104-118 treatment group and control group were sacrificed each week. The pancreas was removed from each mouse and stained for insulin. beta-cell mass was calculated as the organ weight multiplied by the percent of insulin+ area of total tissue area. Contrary to our expectations, there was no change in the total beta-cell mass in INGAP104-118-treated animals compared to control. Reanalysis of the stained tissue sections was preformed, and insulin+ structures were classified as being: (1) a duct islet, (2) a cluster of insulin+ cells, or (3) a mature islet. The density (#/mm2) of duct islets, clusters, and total structures in INGAP 104-118-treated animals was significantly increased; conversely, the density of mature islets was significantly decreased. The increase in cluster density suggests that INGAP104-118 induced neogenesis in the pancreas of treated animals. Poisson regression revealed 9th order polynomial time trends in the structure densities. Though these time trends differed between the classes of structures, they were identical in INGAP104-118 and control animals for each class of structure, suggesting an external stimulus was acting equally on both groups.
While this study did not determine if there is homeostatic regulation of induced beta-cell mass expansion, it did reveal important aspects for the design of a future study to address this issue. The definitions for structure classification must be well-established and rates of beta-cell replication should be determined.

Current therapies for diabetes mellitus are insufficient to prevent the devastating complications associated with this disease. A novel approach for the treatment of diabetes is the restoration of an insulin-producing beta-cell mass through the stimulation of endogenous progenitor cells. Thus, the aim of this thesis was to determine if pharmacological initiation of islet neogenesis and subsequent beta-cell mass expansion will lead to the reversal of hyperglycaemia in a response that is under homeostatic regulation and has efficacy in humans.
A pentadecapeptide fragment of Islet Neogenesis Associated Protein (INGAP 104-108), was administered to normoglycaemic hamsters and was found to result in an expanded beta-cell mass as measured by immunohistochemical morphometric analysis. This expansion was shown to occur through the transformation of duct- and acinar-associated progenitors. In order to determine if this therapeutic approach would be effective in mammals other than hamsters, INGAP 104-108 was administered to normoglycaemic mice, dogs and monkeys, hyperglycaemic mice, and to human pancreatic tissue cultures.
INGAP104-108 administration led to a dose-dependent increase of beta-cell mass in mice, with similar trends observed in dogs. Similarly, administration of INGAP104-108 to normoglycaemic monkeys for 90 days resulted in profound areas of islet neogenesis. Administration of INGAP104-108 to diabetic mice resulted in restoration of euglycaemia and a dramatic increase in beta-cell mass. Furthermore, INGAP104-108 administration to cultured human acinar tissue, led to the formation of insulin-producing islet-like structures. These results suggest that INGAP 104-108 therapy has the ability to reverse a diabetic state and could be effective in humans. However, it was necessary to determine whether the continual stimulation of islet neogenesis through INGAP 104-108 administration is a safe therapeutic approach.
The beta-cell mass dynamics of euglycaemic mice administered INGAP 104-108 at various doses for 31 or 90 days were determined. beta-cell mass was greatly increased at 31 days of therapy, though by 90 days of therapy there was no difference in total beta cell mass between all treatment groups. However, there were marked instances of islet neogenesis in mice treated with INGAP104-108 for 90 days. This elevation in islet neogenesis was tempered by decreased beta-cell replication and increased beta-cell apoptosis, resulting in no overall difference in total beta-cell mass. These results suggest that inherent homeostatic regulation persisted to maintain a net beta-cell mass that matched the physiological need, even in the setting of continual induction of islet neogenesis.
INGAP104-108 therapy has been shown to expand the insulin-producing beta-cell mass in a safe homeostatic manner and reverse diabetic hyperglycaemia. These findings suggest that a novel pharmacological agent for the successful stimulation of beta-cell mass expansion is within reach, enabling new therapeutic modalities for the treatment of diabetes.

Le diabètes mellites de type 2 (T2DM) est caractérisé par une hyperglycémie provenant d’une dérégulation de la sécrétion d’insuline combinée avec une altération de l’action de l’insuline. CaMK1D est un nouveau gène identifié, dont le rôle reste à explorer. Dans l’étude exposée ici, j’ai montré que CaMK1D a un effet majeur sur la régulation du glucose. J’ai observé une réduction exceptionnelle des taux de glucose sanguins à jeun, ce qui entraine une amélioration globale de la tolérance au glucose. Les souris mutantes montrent une augmentation conséquente dans les niveaux sanguins d’insuline. Les souris invalidées pour CaMK1D présentent des ilots pancréatiques de taille plus importante due à une hypertrophie des cellules béta. De plus, les souris mutantes sont protégées contre la stéatose hépatique. Dans l’ensemble, mon travail met en évidence le nouveau rôle clé de CaMK1D chez les cellules béta et apporte plus de compréhension quant à son rôle lors du développement du T2DM
Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia resulting from defects in insulin secretion in combination with impaired insulin action. CaMK1D represents one potential candidate gene, the in vivo function remained elusive. In this work, I have found that CaMK1D plays a central role in blood glucose regulation. Pancreas-specific CaMK1D knockout mice display dramatically reduced fasting blood glucose levels leading to an overall improved glucose tolerance. CaMK1D knockout mice show markedly higher ad libitum and fasting insulin levels. Interestingly, pancreas-specific CaMK1D knockout mice display islet hyperplasia caused by beta-cell hypertrophy. Furthermore, conditional knockout mice are protected against high-fat feeding-induced hepatic steatosis. Overall, my work establishes an essential role of CaMK1D in pancreatic beta-cells and provides further understanding about its role in the development of T2DM

Type 1 and 2 Diabetes Mellitus are a growing health problem throughout the world. There is an increasing  need for methodologies, which are both reliable and non-invasive to measure the amount of insulin-producing tissue (Beta-cell mass, or BCM), as well as rapidly quantify changes in the BCM due to the onset of disease, beta-cell replacement therapy, or other treatments. Positron Emission Tomography (PET) is a non-invasive, quantitative functional imaging technique which can be used to study dynamical or static processes inside the body. In this thesis, we present a study protocol for in vivo imaging of the most common form of beta- cell replacement therapy; islet transplantation. Islets were labeled with the PET tracer, 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG), and administered intra-portally, while the recipient was monitored by PET/CT. The hepatic distribution of the islets was highly heterogeneous, and around 25% (human) or 50% (porcine) of the administered islets could not be found in the liver after completed transplantation, confirming previous reports of considerable cell injury during the procedure leading to low hepatic engraftment. Native BCM in the pancreas can potentially be quantified using a PET tracer with sufficiently high specificity, but the major obstacle is the relative low amounts of insulin producing tissue (only 1-2% of the pancreatic volume). Two tetrabenazine analogues, [18F]FE-(+)-DTBZ and [18F]FE-(+)-DTBZ-d4, are ligands to VMAT2, which is expressed in islet tissue. Both analogues were investigated and characterized as potential BCM imaging agents both in vitro and in vivo.  Both tracers exhibited high preferential binding to islet tissue compared to exocrine pancreatic tissue. However, the specificity was not high enough to overcome the obscuring exocrine signal in vivo (7-10% of the signal originating from specific islet tracer uptake). This thesis demonstrates that it is possible to quantitatively assess islet transplantation by PET imaging. In vivo determination of native pancreatic BCM is, in theory, possible with both [18F]FE-(+)-DTBZ and [18F]FE-(+)-DTBZ-d4, but tracer analogues with higher islet specificity is needed for quantification of smaller BCM changes with physiological impact.

Islet transplantation is a possible curative treatment for type 1 diabetes (T1D). Currently the liver dominates as implantation site, despite the many challenges encountered at this site. Acute hypoxia in islets transplanted to muscle and omentum, two possible alternative sites, was prevailing. However, it was rapidly reversed at both implantation sites, in contrast to when islets were transplanted intraportally. At the intramuscular site hypoxia was further relieved by co-transplantation of an oxygen carrier, polymerized hemoglobin, which also improved the functional outcome. The complement system was activated after islet transplantation to muscle, but did not hamper graft function. Both mouse and human islets transplanted to omentum become well re-vascularized and have a functional blood flow and oxygenation comparable with that of endogenous islets. Animals transplanted with islets to the omentum had a superior graft function compared with animals receiving intraportal islet grafts. Alloxan-diabetic animals were cured with a low number of islets both when the islets were implanted in the omentum and muscle. The islet grafts responded adequately to both glucose and insulin and displayed a favorable mRNA gene expression profile. A challenge in diabetes research and in islet transplantation is that there are no established techniques for quantifying beta-cell mass in vivo. By using radiolabeled Exendin-4, a GLP-1 receptor agonist, beta-cell mass after transplantation to muscle of mice was quantified. The results may well be translated to the clinical setting. By comparing the pancreatic accumulation of [11C]5-hydroxy tryptophan ([11C]5-HTP) as detected by positron emission tomography (PET) in T1D patients with that of healthy controls, a 66% decrease was observed. This may in fact represent the loss of beta-cells, taking into account that other cells within the islets of Langerhans are largely unaffected in T1D.  In conclusion, the data presented support the use of alternative implantation sites for islet transplantation. In addition to improving the functional outcome this may enable more transplantations since the number of transplanted islets may be reduced. The techniques investigated for quantifying transplanted and endogenous beta-cell mass may greatly improve our knowledge of the pathophysiology of T1D and become a valuable tool for evaluation of beta-cell mass.

Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet β cells. However, the role and sequence of β cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of β cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, β cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained β cell volume further declines. These results indicate that dysfunction of persisting β cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.

Numerous epidemiological studies show a strong association between low birth weight and later life hypertension and metabolic disease. Excessive in utero exposure to glucocorticoids (‘stress hormones’) has been hypothesized to be important in such developmental ‘programming’, acting via crucial physiological, gene expression or structural changes in the developing fetus. Normally, the fetus is protected from the high levels of maternal glucocorticoids by an enzymic placental barrier, 11 betahydroxysteroid dehydrogenase type 2 (11β-HSD2). In the placenta, 11β-HSD2 efficiently converts active maternal glucocorticoids (cortisol in humans; corticosterone in rodents) to physiologically inactive 11-keto forms. In previous studies in rats, maternal administration of dexamethasone, a synthetic glucocorticoid which is minimally metabolized by 11β-HSD2, or carbenoxolone, a potent inhibitor of 11 β-hydroxysteroid dehydrogenase, increased glucocorticoid load to the fetus. This resulted in lower offspring birthweight and later life hypertension and hyperglycemia — important components of the metabolic syndrome. These programming effects were seen when dexamethasone was administered selectively during the third week of gestation. We have used this well-validated model of programming to dissect the molecular mechanisms that mediate the programming of hypertension. In accord with previous observations, administration of dexamethasone (100μg/kg/day) to pregnant rats during the last week of pregnancy significantly reduced offspring birthweight by 10%. Moreover, the 9 month-old adult offspring had systolic hypertension (9% rise) accompanied by significant hypokalemia (10% fall K+). The coexistence of hypertension and hypokalemia suggested that prenatal overexposure to dexamethasone might increase mineralocorticoid activity in the kidney. Intriguingly, although offspring of dexamethasone-treated dams had 46% lower plasma renin concentrations (consistent with intravascular fluid volume expansion), 24-hour total urinary aldosterone levels were significantly reduced compared to controls (reduction of 56%). Maternal dexamethasone treatment was associated with a permanent decrease in 11β- HSD2 mRNA and activity in the kidney of the offspring (45% and 36% respectively). 11β-HSD2 plays an important role in regulation of renal sodium reabsorption (and thereby blood pressure) by acting as a pre-receptor barrier to MR access, preventing glucocorticoids from activating MR in the distal nephron. Thus, the decrease in renal 11β-HSD2 activity would allow greater endogenous glucocorticoids to activate MR, likely accounting for the low-renin, low-aldosterone hypokalemic hypertensive phenotype observed in these offspring. Other components of mineralocorticoid or glucocorticoid signaling pathways, including mineralocorticoid receptor (MR), glucocorticoid receptor (GR) and 11-beta hydroxysteroid dehydrogenase type 1 (11β-HSD1) were not altered in the offspring kidney by prenatal glucocorticoid exposure. Dexamethasone-programmed offspring also showed exaggerated mineralocorticoid activity with increased kalliuresis in response to exogenously administered corticosterone, suggesting that the decrease in renal 11β-HSD2 is functionally important. In this respect, our rat model resembles the syndrome of apparent mineralocorticoid excess where reduced 11β-HSD2 allows illicit activation of MR by glucocorticoids, resulting in excessive sodium reabsorption, hypertension and hypokalemia. We also studied the effects of maternal dexamethasone on offspring erythropoietin expression in the kidney. This followed from previous observations that identified the hepatocyte nuclear factor 4 alpha (HNF4α) as a key gene up-regulated in dexamethasone-programmed offspring liver, where it might be involved in mediating hyperglycemia. HNF4α is also expressed in the kidney. The role of HNF4α in the kidney is not fully understood, but has been implicated in regulation of erythropoietin synthesis. As in the liver, prenatal exposure to dexamethasone caused a significant increase (64% increase) in renal HNF4α expression. The increase in renal HNF4α mRNA was observed early (in one week old offspring) and persisted into adulthood. This was associated with significantly elevated levels of erythropoietin in circulation (110% increase). Moreover, animals that were exposed to prenatal dexamethasone had significantly increased red blood cell mass (7% increase), presumably as a result of upregulation of erythropoietin.

As proteína quinases C (PKC) pertencem à família das serina/treonina quinases, que vem sendo apontadas como importantes enzimas para os processos de proliferação e diferenciação das células tronco embrionárias (CTE), todavia, a função exata de cada isoforma dessa família ainda não está clara. Dados anteriores do nosso laboratório indicam que dentre as PKCs expressas em CTE, formas cataliticamente ativas da PKCβI são altamente expressas no núcleo das CTE murinas. Estas ao se diferenciarem expressam essa quinase no seu citoplasma ou deixam de expressar a mesma, e que a maioria dos alvos da PKCβI em CTE indiferenciada estão envolvidos em processos de regulação da transcrição de proteínas envolvidas em processos de proliferação/ diferenciação. Dando continuidade aos resultados anteriores do laboratório, no presente trabalho, com técnicas de proteômica e fosfoproteômica identificamos outros alvos nucleares da PKCβI em CTE indiferenciadas. Vimos que de fato inibindo-se a PKCβI diminuiu-se a fostorilação de fatores envolvidos com a indiferenciação das CTE. Dentre os alvos da PKCβI encontramos a proteína adaptadora, TIF1 que recruta proteínas remodeladoras de cromatina. Essa proteína é essencial para a manutenção do estado indiferenciado das CTE. In vitro a PKCβI foi capaz de fosforilar a TIF1β e inibindo-se a PKCβI por RNAi vimos uma diminuição na expressão da TIF1β e no fator de indiferenciação Nanog cuja expressão já foi demonstrada ser regulada pela TIF1β. Além disso vimos que inibindo-se a PKCβI com o peptídeo inibidor da PKCβI aumentou a expressão de proteínas reguladas pelo c-Myc. E que o RNAi para a PKCβI aumentou a expressão de proteínas que regulam a expressão do c-Myc. Não vimos nenhum efeito na fosforilação ou expressão do c-Myc após a inibição da PKCβI o que sugere que a PKCβI ative proteínas repressoras do c-Myc. Nossos estudos sugerem que a PKCβI regula a manutenção do estado indiferenciado das CTE regulando a expressão e atividade da Tif1β um possível alvo direto da PKCβI. Levando a modificações da cromatina e regulação da expressão de genes que mantém as CTE indiferenciadas. Outro ponto de regulação da PKCβI parece ser a nibição da atividade de c-Myc o que seria importante para a manutenção do estado indiferenciado visto que o c-Myc é um amplificador das vias de sinalização que mantém as células proliferando. Desta forma a PKCβI parece ter um papel central na regulação da expressão gênica de CTE à nível de modificações epigenéticas e a nível transcricional mantendo as CTE indiferenciadas.
The Protein kinase C (PKC) family of serine/treonine kinases, are being described as important enzymes for proliferation and diferentiation of embryonic stem cells (ESC), however, the exact function of the different isoenzymes of this family still is unclear. Previous data from our laboratory indicates that amongst the PKCs expressed in ESC, catalytically active forms of PKCβI are highly expressed in nucleus of murine ESC. When these cells differentiate this kinase can be found in the cytoplasm or not expressed at all, and that the majority of PKCβI targets in undifferentiated ESC are involved in the regulation of proteins involved in transcription of proteins involved in proliferation/ diferentiation. Continuing our previous work herewith using proteomics and phosphoproteomics techniques we identified other nuclear PKCβI targets in undifferentiated ESC. We indeed saw that inhibiting PKCβI decreased the phosphorylation of factors involved with maintainance of the undifferentiated state of ESC. Amongst the targets of PKCβI we found the adaptor protein, TIF1βI, that recruits cromatin remodeling proteins. This protein is essential for the maintenance of the undifferentiated state of ESC. In vitro PKCβI phosphorylated TIF1β and inhibiting PKCβI with RNAi decreased the expression of TIF1β and of the undifferentiation factor Nanog whose expression has been shown to be regulated by TIF1β. We also saw that inhibiting PKCβI with a peptide inhibitor increased the expression of proteins regulated by c-Myc, and that RNAi for PKCβI increased the expression of proteins that regulate the expression of c-Myc. We did not see any effect on the phosphorylation or expression of c-Myc after inhibition of PKCβI suggesting that PKCβI activates c-Myc repressor proteins. Our studies sugest that PKCβI regulates the maintenance of the undiferentiated state of ESC regulating the expression and activity of Tif1β a possibly a direct target of PKCβI, leading to chromatin modifications and regulation of genes that maintain ESC undiferentiated. Another form of regulation of PKCβI seems to be by inhibiting the activity of c-Myc which is importante to maintain ESC undifferentiated since c-Myc is na an amplifyer of signaling patheways that maintain ESC proliferating. Together PKCβI has a central role in the regulation of the gene expression of ESC at the level of epigenetic modifications and transcriptional regulation

Les cellules β pancréatiques synthétisent et sécrètent l'insuline, seule hormone hypoglycémiante de l'organisme. Dans le cas du diabète de type 2, du diabète de type 1 et suite à une greffe d'îlots de Langherans, on observe une diminution drastique de cette masse fonctionnelle β. L'hyperglycémie chronique et la libération de cytokines proinflammatoires jouent un rôle cytotoxique prépondérant dans ces phénomènes. Dans le but de préserver ou de restaurer cette masse fonctionnelle β chez les patients diabétiques, notre objectif était d'identifier des outils permettant de protéger des effets délétères de l'hyperglycémie chronique et des cytokines proinflammatoires, en s'intéressant à 3 cibles potentielles. Nous montrons tout d'abord que les activités du système ubiquitine protéasome (UPS), impliqué dans la dégradation de protéines, sont altérées en condition d'hyperglycémie chronique. Ces altérations sont corrélées à l'émergence d'un programme apoptotique au sein des cellules β. L'activation du récepteur du GLP-1 (Glucagon-Like Peptide-1), stratégie thérapeutique majeure dans le diabète de type 2, protège l'UPS des effets délétères de l'hyperglycémie chronique. Le facteur de transcription CREB (cAMP Response Element Binding Protein), essentiel pour la survie et la fonction des cellules β, est dégradé par l'hyperglycémie chronique et l'inflammation. Nous montrons que la prévention de sa dégradation prévient les effets de l'hyperglycémie chronique, mais pas de l'inflammation. Ces observations nous ont amenés à étudier la MAP3 kinase Tpl2 (Tumor progression locus 2), impliquée, notamment via l'activation de ERK1/2 (Extra-cellular Regulated Kinases 1/2), dans les processus inflammatoires d'autres types cellulaires. Nous montrons que Tpl2 est exprimé dans la lignée cellulaire β INS-1E, et dans les îlots murins et humains, et qu'elle gouverne spécifiquement l'activation des kinases ERK1/2 induite par les cytokines proinflammatoires IL-1β, TNFα et IFNγ. Cette protéine est surexprimée dans des conditions d'inflammation (in vitro et modèle de diabète murin). L'inhibition de Tpl2 protège contre l'apoptose induite par les cytokines, dans les INS-1E et les îlots de souris et restaure la capacité sécrétrice d'insuline des ilots de souris altérée suite à une exposition aux cytokines. En combinaison avec un analogue du GLP-1, l'inhibition pharmacologique de cette kinase protège totalement contre les effets délétères des cytokines sur la fonction et la survie des îlots humains. Ces données suggèrent que l'inhibition pharmacologique de la kinase Tpl2, seule ou en combinaison avec un analogue du GLP-1, pourrait constituer de nouvelles stratégies thérapeutiques pour protéger contre l'altération de la masse fonctionnelle β pouvant survenir chez des patients diabétiques de type 2 ou après la transplantation d'îlots
Pancreatic β cells synthesize and secrete insulin, the sole hormone of the organism able to reduce glycemia. In the course of type 2 and type 1 diabetes, and after islet transplantation, there is a drastic loss of function and mass of these cells. Among the common origins of this decrease, chronic hyperglycemia and the release of proinflammatory cytokines play major roles. With the aim to preserve or to restore this functional β cell mass in diabetic patients, our objective was to identify tools able to protect against deleterious effects of these two phenomenons, interesting in three potential targets. We first demonstrated that the ubiquitin-proteasome system (UPS) activities, that degrade proteins, are altered in β cells exposed to chronic hyperglycemia, and correlated with apoptosis. Activation of the GLP-1 (Glucagon-Like Peptide-1) receptor, a key therapeutic strategy in type 2 diabetes, protects UPS from deleterious effects of chronic hyperglycemia. The transcription factor CREB (cAMP Response Element Binding Protein), crucial for β cell survival and function, is involved in deleterious effects of chronic hyperglycemia and inflammation. We demonstrated that prevention of CREB degradation protects β cells from chronic hyperglycemia, but not from the deleterious effects of the proinflammatory cytokines. These observations prompted us to study the MAP3 kinase Tpl2 (Tumor progression locus 2), known to be implicated in inflammatory process in other cell types, through the activation of the kinases ERK1/2 (Extra-cellular Regulated Kinases 1/2). We showed that Tpl2 is expressed in INS-1E clonal β cells and in mouse and human islets, and that it governs specifically the activation of ERK1/2 in response to proinflammatory cytokines IL-1β, TNFα and IFNγ. This protein is overexpressed by inflammatory conditions and in a rat type 2 diabetes model. Inhibition of Tpl2 protects against cytokine-induced apoptosis in INS-1E and in mouse islets. Furthermore, the capacity of mouse islets to secrete insulin in response to glucose, that is altered by a chronic exposure to cytokines, is restored by Tpl2 inhibitor. Finally, we showed that in combination with GLP-1 analog (Exendin-4), Tpl2 inhibitor can entirely restore the survival and function in human islets cultured in pro-inflammatory conditions. These results suggest that pharmacological inhibition of Tpl2, alone or in combination with Exendin-4, may be novel therapeutic strategies to alleviate β-cell failure observed in Type 2 diabetes and islets transplantation

O transplante de ilhotas pancreáticas humanas representa uma estratégia promissora para a cura do diabetes mellitus tipo 1 (DM1), mas a aplicação a todos os pacientes diabéticos ainda é impraticável devido à limitada disponibilidade de ilhotas ou células β e à necessidade de utilização de drogas imunossupressoras pelo paciente transplantado. O tratamento com imunossupressores após o transplante de ilhotas pode ser abolido quando se realiza o microencapsulamento das ilhotas pancreáticas. Neste trabalho investigou-se um novo biomaterial, Biodritina® (alginato/sulfato de condroitina) adequado ao microencapsulamento que gelifica na presença de íons de cálcio ou bário. A biocompatibilidade das microcápsulas tem sido avaliada segundo o grau de pureza do alginato utilizado na sua confecção. Amostras de alginato comercial purificado foram analisadas, comprovando-se a presença de impurezas (polifenóis, endotoxinas, proteínas) em níveis elevados, que impedem sua aplicação clínica. Optou-se, portanto pela utilização do alginato comercial ultrapurificado nos experimentos descritos neste trabalho. Das formulações de biomateriais avaliadas, as microcápsulas de bário-Biodritina apresentaram o melhor desempenho em testes de estabilidade físico-química. Estas microcápsulas mantiveram sua morfologia e estabilidade estrutural após permanecerem 30 dias na cavidade peritoneal de camundongos, conforme demonstrado por microscopia eletrônica de varredura (MEV). Análises histológicas mostraram que microcápsulas de bário-Biodritina explantadas, não possuíam adesão celular em sua superfície. Estudos de permeabilidade demonstraram que o tamanho médio dos poros das microcápsulas de bário-Biodritina permite passagem de proteínas de até 70 kDa, enquanto os poros daquelas de cálcio-Biodritina comportam proteínas de até 100 kDa. Experimentos de coResumo | x cultivo de macrófagos peritoneais com ilhotas de rato microencapsuladas demonstraram uma capacidade imunoprotetora maior das microcápsulas de bário-Biodritina em relação às de cálcio- Biodritina, sendo que as primeiras não ativaram os macrófagos. A manutenção da viabilidade e função de ilhotas humanas microencapsuladas com bário-Biodritina foi confirmada através de ensaio funcional in vitro, no qual ilhotas microencapsuladas apresentaram níveis de secreção de insulina idênticos aos de ilhotas nuas. A prova de conceito do biomaterial foi realizada através do implante de ilhotas humanas microencapsuladas em bário-Biodritina em camundongos com DM1 induzido por estreptozotocina. A hiperglicemia desses animais foi corrigida pelo implante por um período superior a 60 dias, durante os quais o teste oral de tolerância à glicose mostrou-se normal, demonstrando completa funcionalidade e eficiência das ilhotas microencapsuladas com bário-Biodritina. Partindo de observações de que animais inoculados com a peçonha do escorpião Tityus serrulatus apresentam nesidioblastose, foi realizado o fracionamento do veneno por HPLC de fase reversa e 24 frações obtidas foram submetidas a ensaios de proliferação celular através da incorporação de 3H-timidina em células de insulinoma de rato RINm5F. Uma dessas frações foi capaz de induzir a proliferação das células RINm5F e quando aplicada a ilhotas humanas isoladas, elevou o índice de secreção de insulina e induziu um aumento da expressão dos mRNAs de insulina e PCNA. Portanto, demonstrou-se que o biomaterial bário-Biodritina possui as características necessárias para microencapsular células/ilhotas com eficiência e que a \"fração ativa\" do veneno do escorpião T. serrulatus induz proliferação de células RINm5F e melhora a secreção de insulina de ilhotas humanas.
Islet transplantation has been proposed as a promising therapeutic strategy for the cure of type 1 diabetes mellitus (DM), however, its application to all diabetic patients is still not possible due to the limited source of islets or β cells and to the need of an immunosuppressive treatment of the recipient to avoid graft rejection. The use of immunosupressors may be abolished when pancreatic islets are microencapsulated prior to transplantation. Here, we investigated the use of a new biomaterial suitable for cell microencapsulation, namely, Biodritin®, composed of alginate and chondroitin sulphate, which is capable of gelation in the presence of barium or calcium ions. Microcapsules biocompatibility has been evaluated according to the purity of the alginate used in its production. Samples of purified commercial alginate were analyzed, but the high levels of contaminants (proteins, endotoxins and polyphenols) detected prevented its use in clinical applications. On the other hand, also commercially available ultrapure alginate fulfills the requirements for this application, therefore, this biomaterial was chosen for our experiments. Among the different biomaterial formulations evaluated, barium-Biodritin microcapsules displayed the best performance in the physico-chemical tests. Scanning electronic microscopy revealed that barium-Biodritin microcapsules maintained their morphology and structural stability after being implanted for 30 days in the peritoneal cavity of mice. No cellular adhesion was detected on the surface of explanted barium-Biodritin microcapsules by histological analysis. Permeability studies determined the medium pore size of barium-Biodritin microcapsules, which allows proteins of up to 70 kDa to pass through the biomaterial, while calcium-Biodritin pores accomodate proteins of up to 100 kDa. Co-culture of peritoneal macrophages with microencapsulated rat islets, revealed a superior immunoprotective capacity of barium-Biodritin microcapsules, which were capable of protecting the islets with no macrophage activation. Microencapsulated and naked human islets presented identical insulin secretion levels upon stimulation with glucose in vitro, confirming that barium-Biodritin microencapsulation maintains the function and viability of human islets. Proof-of-concept experiments in which barium-Biodritin microencapsulated human islets were implanted into chemically-induced diabetic mice, showed that these animals maintained normal blood glucose levels for more than 60 days, during which oral glucose tolerance tests were normal, demonstrating the complete functionality and efficiency of barium-Biodritin microencapsulated human islets. From the observation that animals inoculated with the venom of the scorpion Tityus serrulatus presented nesidioblastosis, we decided to fractionate the venom to isolate the active principle. The venom was fractionated by reversed phase HPLC and 24 fractions were obtained and submitted to cellular proliferation assays, in which rat insulinoma RINm5F cells evaluated for 3H-timidina incorporation. One of these fractions was capable of inducing cell proliferation and was also applied to isolated human islets. Treated islets presented a higher insulin secretion index and an increase in insulin and PCNA mRNA expression. In conclusion, we demonstrated that the barium-Biodritin biomaterial possesses all characteristics required for efficient cell/islet microencapsulation and that the active fraction of Tityus serrulatus venom induces the proliferation of RINm5F cells and improves insulin secretion in human islets.

Proghrelin produces three proghrelin derived peptides (PGDP) known for their roles in glucose homeostasis: acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob). Only the receptor for AG, growth hormone secretagogue receptor 1a (GHS R1a), is known, however there is evidence for additional receptors for AG, UAG and Ob. Our aim was to determine the actions of PGDP on two beta cell lines, MIN6 and INS-1, which we have shown to contain and lack GHS R1a respectively. PGDP increased proliferation in INS-1 but not MIN6 cells, measured by BrdU incorporation. AG decreased apopotosis in both cell lines, measured by decreased levels of activated caspase 3. Insulin secretion was investigated in INS-1 cells, where PGDP modulated insulin release in a glucose dependent manner. Our results indicate that PGDP modulate beta cell mass in the presence and absence of GHS R1a, and present a detailed anaylsis of insulin secretion in INS-1 cells.

PI3K signaling in pancreatic β cells has been shown to be important in modulating β cell mass and function under basal condition. Evidence suggests that a specific group of insulin promoter-expressing neurons also modulates glucose metabolism and energy homeostasis through their PI3K signaling. Thus we hypothesize that PI3K activation via PTEN deletion under the control of rat insulin promoter (RIP) in pancreatic β cells and RIP-expressing neurons will protect against hyperglycemia and diabetes in experimentally induced mouse models of type 2 diabetes. In Chapter IV, we showed that RIP-mediated PTEN deletion in pancreatic β cells led to PI3K activation and subsequent increased β cell mass and function, thus protected the mice from high fat diet (HFD)-induced diabetes. Furthermore in the absence of global leptin signaling, β cell-specific PTEN deletion maintained β cell function in the setting of severe insulin resistance, therefore prevented diabetes development. Interestingly, RIP-mediated PTEN deletion also resulted in increased peripheral insulin sensitivity due to PI3K activation in central nervous system. In Chapter V, we showed this increased insulin sensitivity was maintained after HFD feeding, which also contributed to the protection against diabetes. These mice also showed increased visceral adipogenesis and subcutaneous adiposity on HFD, which were dramatically attenuated in the absence of leptin signaling, indicated the essential role of peripheral leptin action in mediating the insulin sensitive phenotype from neuronal RIP PTEN deletion. Finally, we demonstrated that the insulin sensitizing phenotype in these mice was not mediated through ventromedial hypothalamic nuclei (VMH), such that VMH-specific PTEN deletion did not alter energy homeostasis or glucose metabolism. Together, the data from this thesis points to an inhibitory role of PTEN in both central nervous system and pancreatic β cells in glycemic control. Therefore, PTEN may represent a potential target for diabetes prevention and treatment.

Pui Lan Wong.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1993.
Includes bibliographical references (Leaves 109-122).
Abstract --- p.i
Acknowledgements --- p.iii
Chapter Chapter1 --- Introduction
Chapter 1.1 --- A general introduction on mast cells --- p.1
Chapter 1.2 --- Activation of mast cells --- p.6
Chapter 1.3 --- Mediators of mast cells --- p.18
Chapter 1.4 --- Usage of β-adrenoceptor agonists in asthma therapy --- p.26
Chapter 1.5 --- Aim of this study --- p.32
Chapter Chapter2 --- Materials and methods
Chapter 2.1 --- Chemicals --- p.42
Chapter 2.2 --- Buffers and stock solutions --- p.43
Chapter 2.3 --- Source of mast cells --- p.45
Chapter 2.4 --- Animal sensitization --- p.45
Chapter 2.5 --- Isolation of mast cells --- p.46
Chapter 2.6 --- Procedure for the investigation of the effects of adrenoceptor agonists on histamine release from mast cells --- p.48
Chapter 2.7 --- Procedure for the investigation of propranolol antagonism --- p.49
Chapter 2.8 --- Histamine assay --- p.50
Chapter 2.9 --- Data analysis --- p.50
Chapter Chapter3 --- Results
Chapter 3.1 --- Establishment of experimental conditions --- p.53
Chapter 3.2 --- The effects of β-agonists on immunologically induced histamine release from guinea pig lung mast cells --- p.54
Chapter 3.3 --- The effects of β-agonists and two anti-allergic drugs on immunologically induced histamine release from guinea pig lung mast cells --- p.56
Chapter 3.4 --- The effects of β2-agonists on histamine release induced by non-immunological agents from guinea pig lung mast cells --- p.56
Chapter 3.5 --- Antagonism by propranolol on the effects of β2-agonists on histamine release from guinea pig lung mast cells --- p.57
Chapter 3.6 --- The effects of β2-agonists on immunologically induced histamine release from rat peritoneal mast cells --- p.58
Chapter 3.7 --- The effects of β2-agonists on immunologically induced histamine release from human lung mast cells --- p.58
Chapter 3.8 --- "Comparison of the effects of β2-agonists on immunologically induced histamine release from mast cells isolated from the rat peritoneum, the guinea pig lung and the human lung" --- p.59
Chapter Chapter4 --- Discussion
Chapter 4.1 --- The effects of β-agonists on immunologically induced histamine release from guinea pig lung mast cells --- p.89
Chapter 4.2 --- The effects of β2-agonists and two anti-allergic drugs on immunologically induced histamine release from guinea pig lung mast cells --- p.97
Chapter 4.3 --- The effects of novel β2-agonists on histamine release induced by non-immunological agents from guinea pig lung mast cells --- p.99
Chapter 4.4 --- The study of propranolol --- p.100
Chapter 4.5 --- The heterogeneity of mast cells --- p.103
Chapter Chapter5 --- General conclusion --- p.107
References --- p.109

Indiana University-Purdue University Indianapolis (IUPUI)
Stem cell factor (SCF) mediated c-Kit signaling, and downstream activation of Phosphatidylinositol-3 Kinase (PI-3K) is critical for multiple biological effects mediated by mast cells. Mast cells express multiple regulatory subunits of PI-3Kinase, including p85α, p85β, p50α and p55α. In the present study, we have examined the relationship between p85α and p85β subunit in mast cell development and show that loss of p85α in mast cell progenitors impairs their growth, maturation and survival whereas loss of p85β enhances this process. To further delineate the mechanism (s) by which p85α provides specificity to mast cell biology, we compared the amino acid sequences between p85α and p85β subunits. The two isoforms share significant structural homology in the two SH2 domains, but show significant differences in the N-terminal SH3 domain as well as the BCR homology domain. To determine whether the c-Kit induced reduction in growth of mast cells is contributed via the N-terminal SH3 or the BCR homology domain, we cloned and expressed the shorter splice variant p50α, and various truncated mutant versions of p85α in p85α deficient mast cells. We demonstrate both invitro and invivo that while the SH3 and the BH domains of p85 are dispensable for mast cell maturation; they are essential for normal growth and survival. In contrary to existing dogma on redundant functional role of PI-3K regulatory subunits, this study proves that p85α and p85β regulatory subunits of PI-3K have unique roles in mast cell development. We prove that p85α deficiency impairs the expression of multiple growth, survival and maturation related genes whereas p85β deficiency inhibits c-Kit receptor internalization and degradation. This novel finding on negative role of p85β in mast cell development has significant clinical implication, as this knowledge could be used to develop treatments for mast-cell-associated leukemia and mastocytosis.