Type 1 diabetes is an autoimmune disease resulting in the selective destruction of the insulin producing β-cells in the pancreas. Pro-inflammatory cytokines and the free radical nitric oxide (NO) have been implicated in mediating the destruction of β-cells, possibly through activation of the mitogen activated protein kinases (MAPKs) JNK, ERK and p38. In addition to MAPKs, cytokine signaling also results in activation of the transcription factor nuclear factor-kappaB (NF-κB). The upstream signaling events leading to MAPK and NF-κB activation in β-cells are not well known. The work presented in this thesis therefore aims at characterizing the regulation of MAPKs and NF-κB in human islets, with emphasis on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. It was found that MEKK-1 was phosphorylated in response to the nitric oxide donor DETA/NONOate (DETA/NO), the β-cell toxin streptozotocin (STZ) and pro-inflammatory cytokines and that MEKK-1 downstream signaling in response to the same treatments involved activation of JNK but not ERK and p38. MEKK-1 was also found to be essential for cytokine-induced NF-κB activation. MEKK-1 downregulation protected human islet cells from DETA/NO-, STZ, and cytokine-induced cell death. Furthermore, overexpression of the NF-κB subunit c-Rel protected human islet cells from STZ and hydrogen peroxide-induced cell death indicating that NF-κB activity protects against cell death in human islets. In summary, these results support an essential role for MEKK-1 in the activation of JNK and NF-κB, with important consequences for human islet cell death and that strategies preventing human islets death by inhibition of the JNK pathway instead of NF-κB might be suitable.

La Néoplasie Endocrinienne Multiple de type1 (NEM1) est une maladie à transmission autosomique dominante liée à l'inactivation du gène MEN1 codant pour la protéine ménine. Bien que ménine soit exprimée dans tous les tissus testés de l'organisme, elle n'a un effet oncosuppresseur que dans les cellules endocrines. L'hypothèse de mon travail est que ménine interagit avec des fonctions endocrines spécifiques. J'ai ciblé mes études sur une lignée de cellules β pancréatiques INS-1 dans laquelle j'ai étudié la réponse cellulaire au glucose et la régulation du facteur de transcription MAFA en fonction de la variation de l'expression de ménine. Nos résultats ont démontré que l'inhibition de ménine augmente l'incorporation de BrdU en réponse au glucose dans les cellules INS-1, ainsi que l'expression de plusieurs gènes impliqués dans la prolifération de ces cellules. Cette inhibition de ménine est associée avec une réduction dramatique de l'expression de MafA, et celle de certains gènes cibles de MafA. Par ailleurs, la surexpression de la forme sauvage, et non pas des formes mutées de ménine, stimule l'expression de MafA. La variation de l'expression de MafA étant également associée à une variation du taux de prolifération cellulaire. D'autre part, les études in vivo ont montré une bonne corrélation entre le niveau d'expression de ménine et celui de MafA dans les insulinomes du rat et de l'homme. En conclusion, mon travail de thèse a permis de mieux clarifier la fonction biologique de ménine dans les cellules β, et de mettre en évidence l'implication potentielle du facteur MafA dans la tumorigénèse des insulinomes / Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited syndrome caused by mutations of the MEN1 gene coding for the protein menin. Although menin is expressed in all tested tissues, its oncosuppressor effect is limited to the endocrine cells. The assumption of my work was that menin interact with specific endocrine functions. To check out this assumption, we selected the β pancreatic cell line INS-1 in which, we analysed the cellular response to glucose stimulation and the regulation of the transcription factor MAFA according to the variation of menin expression. Our results showed that menin inhibition increased BudU incorporation in response to glucose stimulation in INS-1 cells, as well as the expression of several genes involved in the proliferation of these cells. Menin inhibition was associated with a dramatic reduction of MafA expression level, and some of its targeted genes. Interestingly, wild type menin overexpression, but not mutant forms, stimulated MafA expression. Interestingly, modification of MafA expression modified proliferation rate of INS-1 cells. In addition, the in vivo studies, showed a good correlation between menin and MafA expression levels in both rat and human insulinoma. In conclusion, my thesis work results clarified the biological function of menin in β cells, and highlighted the potential implication of MafA factor in insulinoma tumorigenesis

Local production of reactive oxygen species (ROS) and changes in the redox environment influence the metabolism and function of β cells of the Langerhans islets (LO). Changing the ratio between NAD(P)H / NAD(P)+ redox partners significantly affects sensitive proteins and ROS production. ROS are able to reversibly modify some amino acid residues (eg Cys, Met) of antioxidant enzymes and their interaction partners. Such a signaling cascade allows the transmission of a signal over longer distances and can also interfere with the influence of gene expression. The unique enzyme NADPH oxidase 4 (NOX4) is present on membranes within β cells and constitutively produces H2O2 depending on the presence of NAD(P)H. After glucose stimulation, both NAD(P)H and Nox4 mRNA levels increase. As previously observed in our laboratory, C57BL/6J mice with a specific Nox4 deletion in β cells have a disrupted biphasic insulin release and exhibit insulin resistance in fat and muscle tissue. We found that the absence of NOX4 in C57BL/6J mice affects LO architecture. Wildtype (WT) mice on a normal, predominantly carbohydrate diet (ND) have the majority of small LO with an area of up to 5 000 μm2 (measured on histological sections). High-fat diet (HFD) feeding of WT for 8 weeks leads to the development of diabetic phenotype and...

Glucose homeostasis is fundamental for all living organisms. In vertebrates, the hormone insulin regulates the metabolism of carbohydrates, fats and proteins. In order to sustain the glucose homeostasis, the pancreatic β-cells, which produce and secrete insulin, must coordinate their efforts to secrete the right amounts of insulin required by the organism. In vitro studies, have suggested that a subpopulation of β-cells, referred to as “hub-cells”, coordinate islet Ca2+ dynamics during insulin secretion. However, it is unclear whether the hub-cell model pertains to an in vivo scenario, where the islet is densely vascularized and innervated. In this thesis, we employed the genetically-encoded calcium indicator GCaMP6, confocal imaging and optogenetics, to characterize the Ca2+ dynamics of the zebrafish β-cells in vivo. We found that pancreatic β-cells present endogenous Ca2+ spikes in vivo under basal conditions. These Ca2+ spikes are rapidly suppressed after lowering glucose levels via insulin administration. In addition, the temporal inhibition of blood flow decreases the Ca2+ spikes, suggesting that β-cells are systemically connected. Furthermore, β-cells show a synchronized response to a pericardial glucose injection. Specifically, we found that Ca2+ spikes originate and emanate from a subset of β-cells that are the first to respond to a glucose stimulus. We define these cells as “leader-cells”. We tested if these cells could coordinate the islet in vivo by employing 2-photon laser ablation. Whereas ablation of control cells had no significant effect on the amplitude and duration of the subsequent Ca2+ spikes responses, ablation of leader cells led to a reduction in the Ca2+ response. Furthermore, we developed systems for optogenetic interrogation of β-cells in vivo. We show that the light-gated Cl- ion pump halorhodopsin (NpHR) can be applied to inhibit β-cell depolarization in the zebrafish. We also present the optically orthogonal system of the red Ca2+ indicator K-GECO1 in combination with the blue-shifted channelrhodopsin CheRiff to activate individual β-cell in vivo. Using these new tools, we provide examples where the activation of individual β-cells showed heterogeneous potential to trigger influx of Ca2+ in the rest of the β-cells. Overall, our results led us to propose a hierarchical model of islet coordination. In contrast to the majority of β-cells, which occupy the bottom of the hierarchy since they present low capability to recruit other cells, the leader cells occupy the top levels, being capable to coordinate a majority of the islet’s β-cells.:List of figures xii List of Tables xiii 1. Introduction 1 1.1. Diabetes and insulin 1 1.2. The endocrine pancreas 2 1.3. The diabetes pandemic 4 1.4. β-cell development in zebrafish and mammals 4 1.5. β-cells function and heterogeneity 6 1.6. β-cell coordination 8 1.7. Genetically-encoded calcium indicators 10 1.8. Genetically-encoded optogenetic actuators 13 1.9. Models to study In vivo β-cell coordination 16 2. In vivo β-cell Ca2+ dynamics 19 2.1. β-cells present endogenous Ca2+ spikes in vivo, which are not present ex vivo 19 2.2. Insulin injection reduces endogenous β-cell Ca2+ activity 22 2.3. Pharmacological inhibition of β-cell Ca2+ spikes interferes with glucose control 24 2.4 Transient blood flow interruption decreases β-cell calcium spikes 26 2.5 Glucose bolus leads to a synchronous response of β-cells 29 3. Leader β-cells coordinates Ca2+ dynamics in vivo 32 3.1. High speed 2D and 3D imaging reveals “leader” β-cells 32 3.2. Pan-islet response to glucose is impaired after leader β-cells ablation 41 4. Optically orthogonal toolset for in vivo optogenetics and Ca2+ imaging 46 4.1. Development of optogenetics actuators systems in zebrafish β-cells 46 4.2. Red fluorescent calcium reporters in zebrafish β-cells 47 4.3. In vivo temporal optogenetic silencing of β-cells 50 4.4. In vivo temporal optogenetic silencing of a subset of β-cells can inhibit the islet response 52 4.5. In vivo temporal optogenetic activation of β-cells 55 5. Discussion and future directions 61 5.1. β-cell calcium spikes are systemically influenced 61 5.2. First responder β-cells are present in vivo 64 5.3. Leader β-cells coordinate Ca2+ influx in vivo 66 5.4. β-cell optogenetic interrogation shows heterogeneous potential of individual β-cells for islet coordination 68 6. Materials and methods 75 6.1. Zebrafish strains and husbandry 75 6.2. Transgenic lines generation 76 6.3. Glucose measurements 77 6.4. Pericardial injection of glucose and insulin 77 6.5. Live imaging 77 6.6. Fast whole islet live imaging 78 6.7. Selective two-photon laser ablation of leader cells in the zebrafish islet. 78 6.7. Selective one-photon optogenetic interrogation of β-cells in the zebrafish islet. 79 6.8. Islet blood flow imaging 80 6.9. Mechanical heart stop 80 6.10. Immunostaining 80 6.11. TUNEL assay 81 6.12 Image analysis of GCaMP6s fluorescence intensity from in vivo imaging. 82 6.13 Quantification of GCaMP6s fluorescence intensity 82 6.14 Spatial drift correction images. 83 6.15 Statistical analysis 84 7. References 85 8. Annexes 90 9. Acknowledgments 97 / Die Glukosehomöostase ist für alle lebenden Organismen von grundlegender Bedeutung. Bei Wirbeltieren reguliert das Hormon Insulin den Stoffwechsel von Kohlenhydraten, Fetten und Proteinen. Um die Glukosehomöostase aufrechtzuerhalten, müssen die β-Zellen der Bauchspeicheldrüse, welche Insulin produzieren und absondern, ihre Bemühungen koordinieren, um die richtigen Mengen an Insulin zu sekretieren, die der Organismus benötigt. In-vitro-Studien haben gezeigt, dass eine Subpopulation von β-Zellen, die als „Hub-Zellen“ bezeichnet werden, die Insulinsekretion der Inseln koordiniert. Es ist jedoch unklar, ob sich die Hub-Cell-Theorie auf ein in-vivo-Szenario bezieht, bei dem die Insel dicht vaskularisiert und von Neuronen innerviert ist. In dieser Arbeit verwendeten wir den genetisch kodierten Calcium-Indikator GCaMP6, konfokale Bildgebung und Optogenetik, um die Ca2+-Dynamik der Zebrafisch-β-Zellen in vivo zu charakterisieren. Wir fanden heraus, dass Pankreas-β-Zellen in vivo unter basalen Bedingungen endogene Ca2+-Spitzen aufweisen. Diese Ca2+-Spitzen werden nach Senkung des Glukosespiegels durch Insulinverabreichung schnell unterdrückt. Darüber hinaus verringert die zeitliche Hemmung des Blutflusses die Ca2+-Spitzen, was darauf hindeutet, dass β-Zellen systemisch verbunden sind. Darüber hinaus zeigen β-Zellen eine synchronisierte Reaktion auf die perdikale Glukoseinjektion. Insbesondere fanden wir heraus, dass Ca2+-Spitzen von den β-Zellen hervorgerufen werden, die zuerst auf den Glukosestimulus reagieren. Wir definieren diese Zellen als 'Leader-Zellen'. Wir haben in vivo durch den Einsatz einer 2-Photonen-Laserablation getestet, ob diese Zellen die Insel koordinieren können. Während die Ablation von Kontrollzellen keinen signifikanten Einfluss auf die Amplitude und Dauer der nachfolgenden Ca2+-Spitzenreaktionen hatte, führte die Ablation von Leader-Zellen zu einer signifikanten Verringerung der GCaMP-Reaktion. Darüber hinaus haben wir Systeme für die optogenetische Abfrage von β-Zellen in vivo entwickelt: Wir zeigen, dass die lichtgesteuerte Cl—Ionenpumpe Halorhodopsin (NpHR) angewendet werden kann, um die Depolarisation von β-Zellen in vivo zu hemmen. Wir präsentieren auch das optisch orthogonale System des roten Ca2+-Indikators K-GECO1 in Kombination mit dem blauverschobenen Channelrhodopsin CheRiff, um einzelne β-Zellen in vivo abzufragen. Unter Verwendung dieser neuen Werkzeuge liefern wir Beispiele, bei denen die Aktivierung einzelner β-Zellen ein heterogenes Potenzial für die Auslösung des Ca2+-Einstroms in die übrigen β-Zellen in vivo zeigte. Insgesamt bietet diese Studie Hinweise darauf, dass eine Untergruppe von β-Zellen ein hohes Potenzial zur Koordination der Ca2+-Dynamik der Insel in vivo aufweist.:List of figures xii List of Tables xiii 1. Introduction 1 1.1. Diabetes and insulin 1 1.2. The endocrine pancreas 2 1.3. The diabetes pandemic 4 1.4. β-cell development in zebrafish and mammals 4 1.5. β-cells function and heterogeneity 6 1.6. β-cell coordination 8 1.7. Genetically-encoded calcium indicators 10 1.8. Genetically-encoded optogenetic actuators 13 1.9. Models to study In vivo β-cell coordination 16 2. In vivo β-cell Ca2+ dynamics 19 2.1. β-cells present endogenous Ca2+ spikes in vivo, which are not present ex vivo 19 2.2. Insulin injection reduces endogenous β-cell Ca2+ activity 22 2.3. Pharmacological inhibition of β-cell Ca2+ spikes interferes with glucose control 24 2.4 Transient blood flow interruption decreases β-cell calcium spikes 26 2.5 Glucose bolus leads to a synchronous response of β-cells 29 3. Leader β-cells coordinates Ca2+ dynamics in vivo 32 3.1. High speed 2D and 3D imaging reveals “leader” β-cells 32 3.2. Pan-islet response to glucose is impaired after leader β-cells ablation 41 4. Optically orthogonal toolset for in vivo optogenetics and Ca2+ imaging 46 4.1. Development of optogenetics actuators systems in zebrafish β-cells 46 4.2. Red fluorescent calcium reporters in zebrafish β-cells 47 4.3. In vivo temporal optogenetic silencing of β-cells 50 4.4. In vivo temporal optogenetic silencing of a subset of β-cells can inhibit the islet response 52 4.5. In vivo temporal optogenetic activation of β-cells 55 5. Discussion and future directions 61 5.1. β-cell calcium spikes are systemically influenced 61 5.2. First responder β-cells are present in vivo 64 5.3. Leader β-cells coordinate Ca2+ influx in vivo 66 5.4. β-cell optogenetic interrogation shows heterogeneous potential of individual β-cells for islet coordination 68 6. Materials and methods 75 6.1. Zebrafish strains and husbandry 75 6.2. Transgenic lines generation 76 6.3. Glucose measurements 77 6.4. Pericardial injection of glucose and insulin 77 6.5. Live imaging 77 6.6. Fast whole islet live imaging 78 6.7. Selective two-photon laser ablation of leader cells in the zebrafish islet. 78 6.7. Selective one-photon optogenetic interrogation of β-cells in the zebrafish islet. 79 6.8. Islet blood flow imaging 80 6.9. Mechanical heart stop 80 6.10. Immunostaining 80 6.11. TUNEL assay 81 6.12 Image analysis of GCaMP6s fluorescence intensity from in vivo imaging. 82 6.13 Quantification of GCaMP6s fluorescence intensity 82 6.14 Spatial drift correction images. 83 6.15 Statistical analysis 84 7. References 85 8. Annexes 90 9. Acknowledgments 97

Diabetes ist ein weltweites Problem, das durch den Verlust oder die Dysfunktion der Insulin-produzierenden β-Zellen des Pankreas verursacht wird. In seltenen Fällen entsteht Diabetes durch eine Mutation in einem einzigen Gen. Diese monogenetischen Formen des Diabetes können zur Identifizierung neuer Regulatoren der β-Zellen-Entwicklung und -Funktion beitragen. In der vorliegenden Arbeit habe ich neue putative Diabetes-assoziierte Gene untersucht, die zuvor durch „Next-Generation“ Sequenzierung in einer Gruppe von Kindern und Jugendlichen mit idiopathischem Diabetes festgestellt wurden. Insbesondere analysierte ich neuartige Mutationsvarianten in Genen kodierend für Histone deacetylase 4 (HDAC4), Glioma-associated oncogene homolog 1 (GLI1) und Glioma-associated oncogene homolog 2 (GLI2). Basierend auf den folgenden Kriterien wurden diese Transkriptionsregulatoren zur weiteren funktionellen Analyse priorisiert: Genetische Information, Patientenphänotyp und Expressionsprofil der Kandidaten Gene in Mauspankreas-Vorläuferzellen. Um die Rolle der Varianten während der pankreatischen Zelltypspezifizierung zu untersuchen, nutzte ich die CRISPR-Cas9 Methode in Kombination mit Stammzellendifferenzierung. Im Detail generierte ich diverse Stammzellen mittels CRISPR-Cas9, die die Mutationsvarianten der Patienten trugen und differenzierte diese zu β-ähnlichen Zellen. Weitere in vitro und Transkriptionsanalysen zeigten, dass die Variante c.C4661T in GLI2 die Entwicklung der β-ähnlichen Zellen beeinträchtigte, was für eine genetische Prädisposition zur Entwicklung von Diabetes verantwortlich sein kann. Zusätzlich nutzte ich diese Plattform, um neue extrinsische Faktoren zu untersuchen und zeigte, dass die fördernde Rolle von HC toxin (HDAC Inhibitor) und SLIT3 (ROBO Ligand) konserviert ist. Zusammenfassend habe ich eine Differenzierungsplattform etabliert, um die Rolle von genetischen und extrinsischen Faktoren für die Entwicklung des Pankreas und/oder β-Zellen zu untersuchen. / Diabetes is a worldwide health problem caused by the loss or dysfunction of the insulin-secreting β-cells in the pancreas. Unelucidated forms of monogenic diabetes, arising from rare mutations in one single gene, represent invaluable models for identifying new targets of β-cell development and function. In this study, I focused on putative disease-associated genes for diabetes that have been previously identified by next-generation sequencing of a cohort of patients with puberty-onset diabetes. In particular, I investigated unique mutant variants in genes coding for Histone deacetylase 4 (HDAC4), Glioma-associated oncogene homolog 1 (GLI1) and Glioma-associated oncogene homolog 2 (GLI2). These transcriptional regulators were prioritized for functional analysis based on patient phenotype, expression level in pancreas progenitor cells and available genetic information. To investigate the role of the genetic mutant variants in pancreatic cell fate decisions and cell function, I used the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 genome editing technology in combination with human induced pluripotent stem cell (iPSC)-directed β-cell differentiation. Employing these approaches, I established several patient-like iPSC lines carrying the identified heterozygous missense variants. Specifically, functional experiments and whole transcriptome analysis showed that the variant c.C4661T in GLI2 impairs human β-cell differentiation and β-cell function, which might be responsible for a genetic predisposition to develop diabetes. In addition, I used the same iPSC-based differentiation model system to study novel extrinsic factors, namely the HDAC inhibitor HC toxin and the ROBO ligand SLIT3 and uncovered their conserved role in enhancing human β-cell development. Taking together, I established a human iPSC differentiation platform to study critical genes and extrinsic factors that are necessary for human pancreas development and/or β-cells.

<p>Oscillations in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca²⁺]_i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca²⁺]_i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca²⁺]_i in isolated mouse β-cells into sustained elevation. Increased Ca²⁺ entry promoted the reappearance of the slow [Ca²⁺]_i oscillations. The [Ca²⁺]_i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca²⁺]_i oscillations due to periodic entry of Ca²⁺ as well as with transients evoked by mobilization of intracellular stores. The [Ca²⁺]_i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca²⁺]_i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca²⁺]_i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca²⁺ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca²⁺]_i oscillations. Nevertheless, there was an excessive firing of [Ca²⁺]_i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca²⁺]_i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.</p>

Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

Diabetes mellitus (DM) is a severe and frequent disease with increasing prevalence. It is not possible to achieve long term cure without late complications. Recent advances in cell fate modifications open a pathway to alternative cell therapies for DM cure. My doctoral thesis "Differentiation of pancreatic stem cells into insulin producing β- cells" is focused on the development of a new source of insulin secreting cells for transplantation. Combinatorial testing of numerous potential transcription factors and epigenetic modifiers resulted in a final protocol for the reprogramming pancreatic of exocrine cells into insulin secreting cells. The key transcriptional factors TF (Pdx1, Ngn3 a MafA) were applied in the form of synthetic mRNA. In four independent experiments we applied transcriptional factors in a specific sequence, thus obtaining 14.3 ± 1.9 % insulin positive cells. When challenged in vitro by the glucose levels of 2.5 and 20 mmol/l glucose, respectively, these cells exhibited glucose-sensitivity of insulin secretion (842 ± 72 and 1 157 ± 58 pg insulin/µg DNA/ml, n=5). They also demonstrated a sensitivity of insulin secretion (863 ± 78 and 1 025 ± 66 pg insulin/µg DNA/ml, n=5) to the concentration of depolarization agent KCl applied at 0 and 30 mmol/l, respectively together with 2.5...

Elevated serum levels of trimethylamine N-oxide (TMAO) were first associated with increased risk of cardiovascular disease (CVD) 10 years ago. Research has since defined that serum TMAO accumulation is controlled by the diet-microbiome-liver-kidney axis. Choline related nutrients are consumed in excess during over-nutrition from a Western diet. The resultant elevated serum TMAO is investigated across various chronic metabolic diseases and many tissue types. While TMAO is most clearly linked to CVD mechanisms in vascular tissue, its molecular effects on metabolic tissues are unclear. Here we report the current standing of TMAO research in metabolic disease context across relevant metabolic tissues including liver, kidney, brain, adipose, and muscle tissues. This review explores the variable TMAO effects in healthy and diseased conditions. Since impaired pancreatic β-cell function is a hallmark of metabolic disease pathogenesis which are largely unexplored in TMAO research, the following primary research results investigate TMAO effects on in vitro functional β-cell mass in relation to healthy and type 2 diabetes (T2D) conditions. Although we hypothesized that TMAO would aggravate functional β-cell mass, the data demonstrate that TMAO improves the T2D phenotype by increasing insulin secretion and production and reducing oxidative stress. Therefore, this work provides crucial support for the emerging context dependent molecular effects of TMAO during metabolic disease progression.