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The objective of this thesis was to determine the association of leg length (LL), a marker of early childhood conditions, with metabolic abnormalities underlying type 2 diabetes. Utilizing data from a population at-risk for diabetes, the associations of LL with i) insulin resistance (IR) and beta-cell dysfunction and ii) a continuous metabolic syndrome risk score (MetScore) were analyzed. Results showed that shorter LL was associated with IR and beta-cell dysfunction, and that the combination of short legs and large waist (a marker of adult obesogenic conditions) was associated with the greatest IR. Height, a marker of overall childhood conditions, was found to be inversely associated with the MetScore. Therefore, both adverse childhood conditions and early-late life mismatched conditions may increase the risk for diabetes through differing pathways. Improving childhood conditions (i.e. nutritionally or economically) may be an important strategy to prevent diabetes.

The objective of this thesis was to determine the association of leg length (LL), a marker of early childhood conditions, with metabolic abnormalities underlying type 2 diabetes. Utilizing data from a population at-risk for diabetes, the associations of LL with i) insulin resistance (IR) and beta-cell dysfunction and ii) a continuous metabolic syndrome risk score (MetScore) were analyzed. Results showed that shorter LL was associated with IR and beta-cell dysfunction, and that the combination of short legs and large waist (a marker of adult obesogenic conditions) was associated with the greatest IR. Height, a marker of overall childhood conditions, was found to be inversely associated with the MetScore. Therefore, both adverse childhood conditions and early-late life mismatched conditions may increase the risk for diabetes through differing pathways. Improving childhood conditions (i.e. nutritionally or economically) may be an important strategy to prevent diabetes.

The biological, technical and computational aspects of this project collectively focused on using electron tomography (ET) for the high-resolution (10-20 nm) 3D reconstruction of entire insulin-secreting beta cells within islets of Langerhans isolated from mouse pancreata. Islets were cultured overnight to represent either steady-state (non-stimulated) or elevated glucose (stimulated) conditions, prior to fast-freezing, freeze-substitution, plastic embedment and cutting into 250-400 nm thick sections for tomographic imaging using intermediate voltage electron microscopy (EM). 3D images (tomograms) of each section were used to evaluate the performance of the new technical and computational approaches developed, and make biological comparisons of intercellular structure-function. Analysis focused on key compartments/organelles of the insulin-secretory pathway - Golgi apparatus, mitochondria, insulin secretory granules and multi-granular bodies. To allow the application of ET to entire mammalian cells, several technical limitations were addressed. Since segmenting (delimiting compartments of interest) tomograms manually, represented the major ërate-limiting stepí of ET, an interactive approach for 3D segmentation using novel interpolation algorithms (crude smooth, pointwise smooth and spherical interpolation) to iteratively predict the shape of 3D surfaces between user-drawn contours was developed. The performance of these tools in segmenting a range of compartment types was examined, and found to significantly enhance the speed and accuracy of manual segmentation. To better compensate for the physical collapse of plastic sections in the EM, a novel method was developed for estimating section collapse by analyzing approximately spherical organelles. Using this method on mature insulin granules in high-resolution datasets, coupled with measurements from the whole cell reconstructions, section collapse was found to be substantially less (~25%) than the value (40%) previously used to re-scale 3D models. Other new approaches developed to further improve the accuracy and quality of tomograms, included interactive tools for fiducial tracking, and the use of larger gold particles, a ëreduced second axisí to account for the missing wedge problem, and deformation grids to account for anisotropic deformation. As well as affording more efficient and precise mapping of cell ultrastructure in 3D for subsequent quantitative analysis, these developments provided new insights for future automated (hybrid) segmentation pipelines and new computational approaches for improving quality and isotropic accuracy of volumetric image data. The Interpolator and DrawingTools for segmentation, AnalysisTools for estimating section collapse and BeadHelper for tracking fiducial particles, written as plug-ins for the IMOD software package distributed by the University of Colorado, are now being used by the wider ET community with significant positive feedback. Using the novel approaches developed, four insulin-secreting beta cells - two from the periphery of an islet frozen 1 hr after stimulation with 11 mM glucose, and two from the periphery of another islet under steady-state 5.6 mM glucose conditions - were reconstructed in their entirety in 3D. Quantitative data on the key compartments/organelles provided new information regarding global changes in cellular organization, and enabled robust comparisons of each pair of functionally equivalent cells at unprecedented spatial resolution. Relative differences in the number, dimensions, architecture and distribution of organelles per cubic micron of cellular volume (including mitochondrial branching) reflected differences in the cellsí individual capacity/readiness to respond to secretagogue stimulation. In the two stimulated cells this was reflected by inverse relationships between the number/size of mature granules versus immature granules, the number/size of mitochondria, and the volume of the trans-Golgi network relative to the entire Golgi ribbon. Complementary stereological analysis of whole islets indicated which cells were the most representative under stimulated versus non-stimulated conditions, and revealed a marked natural heterogeneity between cells both within and between individual islets. Overall, this project led to significant improvements in efficiency and accuracy for segmenting cellular compartments/organelles, and in image quality and accuracy for tomogram computation and reconstruction through use of the newly developed techniques. The improved 3D reconstruction and analysis of pancreatic beta cells in toto in native tissue provided a powerful approach for quantitatively mapping the organelles involved in insulin synthesis/secretion at unprecedented detail, and afforded a level of insight into the complex 3D organization of mammalian cells not previously achieved by any other analytical technique or imaging method.

La prévalence de l’obésité et du diabète de type 1 (DT1) s’accroit dans le monde à une vitesse alarmante. L’augmentation du poids corporel, de la quantité d’acides gras libres (AGL) circulant et de la résistance à l’insuline peut induire un stress du réticulum endoplasmique (RE) dans les cellules beta du pancréas, ce qui pourrait favoriser l’inflammation. Afin de tester cette hypothèse, nous avons exposé des cellules beta à un léger stress chronique du RE induit par de l’acide ciclopiazonique (ACP) ou l’AGL palmitate et les avons ensuite traitées avec une faible dose d’interleukine 1β (IL-1β) ou de facteur de nécrose tumorale (TNF-α). L’addition d’IL- 1β, mais pas de TNF-α, a conduit à l’augmentation de la production de marqueurs pro-inflammatoires et de chimiokines. Cette différence de résultat en fonction de l’exposition à l’IL-1β ou au TNF-α peut-être au moins partiellement expliquée par un usage différentiel du complexe de la kinase IκB (IKK) et de la voie du facteur nucléaire κ-B (NF-κB), menant à terme à une activation plus élevée par l’IL-1β en comparaison à TNF-α. L’analyse des branches de la réponse de la protéine dépliée impliquées a révélé que la voie de l’Inositol-requiring enzyme 1 (IRE1) / X-box binding protein 1 spliced (XBP1s) est le médiateur clé dans le couplage avec la réponse inflammatoire déclenchée par l’IL-1β. En effet, le knockdown d’IRE1 ou de XBP1 a permis d’éviter l’exacerbation de l’activité du promoteur NF-κB et l’expression des gènes cibles de NF-κB. Les mécanismes impliqués dans la régulation XBP1-dépendante de la réponse pro-inflammatoire sont partiellement dépendant de la modulation de la Forkhead box O1 (FoxO1). Le stress du RE et l’IL-1β participent aussi à un autre évènement crucial dans le développement du DT1, à savoir la mort progressive des cellules beta, qui est également exacerbée par la combinaison ACP + IL-1β. Contrairement à l’inflammation, la voie IRE1/XBP1 n’est pas impliquée dans l’apoptose cellulaire induite par la combinaison d’ACP et d’IL-1β. Dans ce contexte, nous suggérons que le devenir de la cellule est décidé par la balance entre les protéines Bcl-2 anti-apoptotiques et pro-apoptotiques. Ainsi, nous avons montré que le gène A1 associé à Bcl-2 (A1) est négativement régulé par le stress du RE tandis que l’IL-1β active la protéine BH3-only Bim, aboutissant à une apoptose accrue des cellules beta. En conclusion, nos données suggèrent que le stress du RE est un facteur sensibilisation pour l’induction de l’inflammation des ilots pancréatiques et de l’apoptose des cellules beta. Ceci pourrait fournir une explication mécanistique à l’augmentation parallèle observée de l’obésité infantile et de la fréquence du DT1.\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Le diabète de type 2, défini par une hyperglycémie chronique, résulte d'un déficit de la sécrétion d'insuline et d'une insulinorésistance. Durant le prédiabète qui précède la maladie, la cellule ß pancréatique est capable d'établir une hyperactivité sécrétoire compensatrice de l'insulinorésistance. Les NO synthases neuronales (nNOS) pancréatique et musculaire contrôlent respectivement la sécrétion d'insuline induite par le glucose dans la cellule ß et la force contractile, la captation et l'utilisation du glucose dans les myocytes. Dans le modèle génétique du rat obèse Zucker fa/fa mimant l'état prédiabétique associant un hyperinsulinisme et une insulinorésistance, nous avons retrouvé au niveau de la cellule ß une forte augmentation du complexe entre la nNOS et son inhibiteur endogène PIN (Protein Inhibitor of Neuronal NOS) au niveau des granules de sécrétion d'insuline. Ce complexe, grâce à une interaction accrue avec la myosine V, participe à l'hyperactivité sécrétoire de la cellule ß pancréatique. En effet, des molécules inhibant spécifiquement l'interaction nNOS-PIN permettent de rétablir, chez le rat fa/fa, une sécrétion d'insuline normale. Au niveau musculaire, nous avons observé, dans ce modèle animal, une diminution d'expression de la nNOS sans variation du taux d'ARNm, traduisant une protéolyse accrue de la protéine. L'inhibition de la dégradation protéasomale permet de restaurer l'expression et l'activité catalytique de la nNOS dans le muscle squelettique. Cette perte de fonctionnalité de l'enzyme participerait à l'installation de l'insulinorésistance. Ces travaux ont permis de valider la nNOS comme une cible potentielle pour la prévention du diabète de type 2. / Type 2 diabetes is a chronic disorder defined by chronic hyperglycemia resulting from a deficiency of insulin secretion and an insulin resistance in peripheral tissues and liver. A long lasting silent phase, called prediabetes, precedes the disease and in which pancreatic ß cell is able to improve insulin secretion to compensate for the insulin resistance. The pancreatic and muscular neuronal nitric oxide synthases (nNOS) control respectively glucose-induced insulin secretion in pancreatic ß cell and glucose uptake and utilization in myocytes. In the genetic model of obese Zucker fa/fa rat mimicking the prediabetic state characterized by hyperinsulinemia and insulin resistance, we found a high increase in the amount of the complex between nNOS and its endogenous inhibitor PIN (Protein Inhibitor of Neuronal NOS) at the level of insulin secretory granules within the ß cell. This complex, through an increased interaction with myosin V, participates in the secretory hyperactivity of the pancreatic ß cell, observed in this model of prediabetes. Indeed, molecules that specifically inhibit nNOS-PIN interaction allow to restore a normal insulin secretion in fa/fa rat. In skeletal muscle of this model, we observed a decreased expression of nNOS protein with no change in mRNA levels, suggesting an increased proteolysis of the protein. Inhibition of proteasomal degradation restores the expression and the catalytic activity of nNOS in skeletal muscle. Thus, this loss of functionality of the enzyme could participate in the installation of insulin resistance. This work therefore validated nNOS as a potential target for the prevention of type 2 diabetes.

Diabetes Mellitus is a group of complex and heterogeneous metabolic disorders characterized by hyperglycemia. Even though the condition has been extensively studied, its causes and complex pathologies are still not fully understood. The occurring damage to the pancreatic islets is strikingly heterogeneous. In type 1 diabetes, the insulin producing beta cells are all destroyed within some islets, and similarly in type 2 diabetes, some islets may be severely affected by amyloid. At the same time other islets, in the near vicinity of the ones that are affected by disease, may appear fully normal in both diseases. Little is known about this heterogeneity in susceptibility to disease between pancreatic islets. This thesis examines the physiological and pathophysiological characteristics of islet subpopulations. Two subpopulations of islets were studied; one constituting highly vascularized islets with superior beta cell functionality, and one of low-oxygenated islets with low metabolic activity. The highly functional islets were found to be more susceptible to cellular stress both in vitro and in vivo, and developed more islet amyloid when metabolically challenged. Highly functional islets preferentially had a direct venous drainage, facilitating the distribution of islet hormones to the peripheral tissues. Further, these islets had an increased capacity for insulin secretion at low glucose levels, a response that was observed abolished in patients with recent onset type 1 diabetes.  The second investigated islet subpopulation, low-oxygenated islets, was found to be an over time stable subpopulation of islets with low vascular density and beta cell proliferation. In summary, two subpopulations of islets can be identified in the pancreas based on dissimilarities in vascular support and blood flow. These subpopulations appear to have different physiological functions of importance for the maintenance of glucose homeostasis. However, they also seem to differ in vulnerability, and a preferential death of the highly functional islets may accelerate the progression of both type 1 and type 2 diabetes.

The dynamics of β-cell mass are at the focus of an extensive international effort to develop β-cell replacement therapies for type 1 diabetes. During normal fetal development endocrine cells emerge from a pool of PDX1+/SOX9+ multipotent progenitors that transiently express the proendocrine gene NGN3. These cells become hormone-positive and are seen to bud from the ductal structures and aggregate into islet clusters. Congenital hyperinsulinism in its diffuse form (CHI-D) is characterised by an increase in hormone-positive cells associated with ducts and diffuse patterns of insulin expression. CHI-D arises from mutations inactivating the KATP channel and is diagnosed following persistent episodes of hypoglycaemia caused by an inappropriate secretion of insulin. Whilst existing knowledge has focused on the β-cell, we have explored the histology of CHI-D across multiple pancreatic cell lineages. The starting hypothesis considered CHI-D as an over-exuberance of endocrine differentiation with a progenitor population underlying this process. We suggest CHI-D is not simply an excessive proliferation of pre-existing β-cells. Expression of many transcription factors involved in endocrine differentiation were unchanged in CHI-D, NKX2.2 was increased and persisted in δ-cells. The incidence of nucleomegaly was also confirmed in CHI-D samples, predominantly in the β- and δ-cell lineages. Whilst increases in endocrine cell proliferation were subtle, the ductal and acinar cell lineages had significantly elevated proliferation correlating with changes in cell cycle regulation. The expression of NGN3 was profiled in a range of human fetal samples to determine whether a competence window for endocrine differentiation exists during development. Peak expression was observed between 10-17 wpc whilst protein and transcript expression were both reduced by birth and postnatally. Combined with the data in CHI-D and postnatal controls, it is likely that endocrine commitment ceases in human towards the end of gestation and that further increases in β-cell mass rely on proliferation or NGN3-independent pathways. These data provide new clues for the pathological mechanisms of CHI-D and the establishment and maintenance of the β-cell mass in the human pancreas. We have shown an altered potential for cell proliferation in CHI-D in previously unappreciated ways and provide a rationale for studying molecular components of the β-cell to help unlock β-cell proliferation as a therapeutic option in diabetes.

Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP).

In this project I sought to analyse the effects of different free fatty acids (FFAs) on INS-1E β-cells. The saturated fatty acid palmitate is considered toxic whereas the monounsaturated fatty acid oleate is harmless. In my working hypothesis I assumed an additional protective effect of oleate when used in combination with palmitate. Furthermore I aimed to explore in detail the possible causes and signalling pathways responsible for apoptosis or sustained cell survival. I examined the Endoplasmic Reticulum (ER) stress response, called unfolded protein response (UPR), as one essential criterion deciding about cell death or life. Analysis of viability and apoptosis confirmed the deleterious effect of palmitate on INS-1E β-cells after 24h of incubation. Oleate proved not to be harmful and even reversed the toxicity of palmitate. When the main components of the UPR were assessed using Western blot analyses and quantitative PCR was performed I found positive proof that palmitate activated the UPR and ultimately led to apoptosis. By contrast, oleate completely prevented UPR signalling. I conclude that oleate rescues INS-1E β-cells by inhibiting ER stress and its signalling.