Neue molekulare Regulatoren für die Differenzierung und den Funktionserhalt des endokrinen Pankreas

Abstract Considering the globally increasing rate of incidence, Type 2 diabetes mellitus belongs to the most frequent endocrine metabolic disorders today. In addition to insulin resistance of peripheral tissues, this disease is the result of dysfunction of the endocrine pancreas and in particular of the functional failure of β-cells. The progress of therapeutical strategies is based on the research of the underlying mechanisms. The aim of the present dissertation was the analysis of new molecular regulators which might improve our underdstanding of the differentiation of pancreatic islets and the functional maintenance of adult β-cells. The first part of this work concerned the role of the transcription factor Pax6 and especially the role of its transactivation domain (TA) and of its two DNA binding domains, the paired domain (PD) and the homeodomain (HD), in differentiation of pancreatic endocrine cells. By analyzing four different mouse lines with specific mutations in one of these three domains, we found that the PD of Pax6 is essential for differentiation of glucagon producing α-cells. Inactivation of this domain resulted in a phenotype similar to that of Pax6 knockout mice (Pax6-/-) with a near complete absence of glucagon positive α-cells, a markedly reduced number of insulin producing β-cells, and a disorganized islet structure. Mutations of HD or TA showed a less severe pancreatic phenotype. Islets either exhibited no morphological changes or they showed a reduction of α- and β-cells. Intraperitoneal glucose tolerance tests demonstrated the utmost importance of the transcription factor Pax6 for maintenance of normal pancreatic endocrine function in adult animals. In the second part of this study we identified new genes and proteins, respectively, which could play a regulatory role in normal function of β-cells. In particular it was possible to show that Eny2, hitherto a protein only described in yeast or invertebrates like drosophila, is involved in the regulation of insulin secreting vertebrate cells. si-RNA mediated knockdown of Eny2 resulted in markedly increased glucose and incretin-induced insulin secretion. This could be at least in part attributed to a higher glucose-dependent cellular metabolism and an enhanced signal transduction via protein kinase A and is accompanied by elevated levels of intracellular calcium. Taken together, these results indicate that Eny2 functions as a negative regulator of glucose-stimulated and incretin-mediated insulin secretion, at least in vitro. However, a gap of knowledge still remains between the established nuclear functions of Eny2 and the cellular phenotype we observed upon its suppression. Nevertheless, the effects of an Eny2-knockdown are glucose dependent and additive to the incretin signaling. This feature makes this model attractive to obtain new insights in how insulin secretion of β- cells proceeds and how to find new therapeutical strategies to treat type 2 diabetes mellitus

Distribution of serotonergic and dopaminergic nerve fibers in the salivary gland complex of the cockroach Periplaneta americana

BACKGROUND: The cockroach salivary gland consists of secretory acini with peripheral ion-transporting cells and central protein-producing cells, an extensive duct system, and a pair of reservoirs. Salivation is controled by serotonergic and dopaminergic innervation. Serotonin stimulates the secretion of a protein-rich saliva, dopamine causes the production of a saliva without proteins. These findings suggest a model in which serotonin acts on the central cells and possibly other cell types, and dopamine acts selectively on the ion-transporting cells. To examine this model, we have analyzed the spatial relationship of dopaminergic and serotonergic nerve fibers to the various cell types. RESULTS: The acinar tissue is entangled in a meshwork of serotonergic and dopaminergic varicose fibers. Dopaminergic fibers reside only at the surface of the acini next to the peripheral cells. Serotonergic fibers invade the acini and form a dense network between central cells. Salivary duct segments close to the acini are locally associated with dopaminergic and serotonergic fibers, whereas duct segments further downstream have only dopaminergic fibers on their surface and within the epithelium. In addition, the reservoirs have both a dopaminergic and a serotonergic innervation. CONCLUSION: Our results suggest that dopamine is released on the acinar surface, close to peripheral cells, and along the entire duct system. Serotonin is probably released close to peripheral and central cells, and at initial segments of the duct system. Moreover, the presence of serotonergic and dopaminergic fiber terminals on the reservoir indicates that the functions of this structure are also regulated by dopamine and serotonin

Relative roles of the different Pax6 domains for pancreatic alpha cell development

<p>Abstract</p> <p>Background</p> <p>The transcription factor Pax6 functions in the specification and maintenance of the differentiated cell lineages in the endocrine pancreas. It has two DNA binding domains, the paired domain and the homeodomain, in addition to a C-terminal transactivation domain. The phenotype of Pax6^-/-knockout mice suggests non-redundant functions of the transcription factor in the development of glucagon-expressing α-cells as this cell type is absent in the mutants. We ask the question of how the differentiation of pancreatic endocrine cells, in particular that of α-cells, is affected by selective inactivation of either one of the three major domains of Pax6.</p> <p>Results</p> <p>The Pax6^Aey18mutant mouse line, in which the paired domain is inactivated, showed a phenotype similar to that of Pax6^-/-knockout mice with a near complete absence of glucagon-positive α-cells (0-4 cells/section; ≤1% of wt), reduced β-cell area (74% of wt) and disorganized islets. The proportion of ghrelin-positive ε-cells was expanded. In Pax6^Sey-Neumutants, which lack the transactivation domain, α-and β-cells where reduced to 25 and 40% of wt, respectively. We also studied two mouse lines with mutations in the homeodomain, Pax6^4Neuand Pax6^132-14Neu. Neighboring amino acids are affected in the two lines and both point mutations abolish DNA binding of the classical P3 homeodomain target sequence. The pancreatic phenotype of the two mutants however was divergent. While Pax6^4Neuhomozygotes showed a reduction of α- and β-cells to 59 and 61%, respectively, pancreatic endocrine development was unaltered in the Pax6^132-14Neumutant strain.</p> <p>Conclusions</p> <p>We show that inactivation of the Pax6 paired domain leads to a more severe phenotype with regards to the differentiation of pancreatic α-cells than the loss of the transactivation domain. The analysis of two different homeodomain mutants suggests that the binding of Pax6 to P3 homeodomain consensus sequences is not required for α-cell development. It rather seems that the homeodomain has a modulating role in Pax6 function, possibly by facilitating a PH0-like binding confirmation on paired domain target genes like proglucagon. This function is differentially affected by the two homeodomain mutations analyzed in this study.</p

Nonviral gene transfer into the lungs of mice: Influence of application method, molecular biological factors and magnetic drug targeting mechanisms

Eine mögliche Strategie des Gentransfers in die Lunge ist die Verwendung von PEI-pDNA Komplexen, die als Aerosol oder via intranasaler Instillation appliziert werden können. In dieser Arbeit wurden mehrere Ansätze verfolgt, um den nichtviralen Gentransfer in Lungengewebe zu verbessern und die Kinetik der Genexpression sowie die Auswirkung auf die Lungenfunktion zu bestimmen. Nach intranasaler Instillation von PEI-pDNA Komplexen in Mäuse war die pDNA- Clearance aus dem Lungengewebe um das 3,5-fache höher als nach Aerosolapplikation. Eine Luziferaseexpression als Zeichen eines erfolgreichen Gentransfers konnte nach intranasaler Instillation nur kurzfristig 24 h nach der Behandlung detektiert werden. Nach Aerosolapplikation war die Luziferaseexpression nach 72 h am höchsten und blieb bis zum letzten gemessenen Zeitpunkt (7 Tage) konstant. Dieses Ergebnis steht im Einklang mit der geringeren Clearance nach Aerosolapplikation. Die Luziferaseexpression konnte nach Aerosolapplikation von CpG-freier pDNA weiter gesteigert werden. Bezüglich der Lungenfunktion fand sich sowohl nach Aerosolapplikation als auch nach intranasaler Instillation von PEI-pDNA z.B. eine erniedrigte Compliance der Lunge, was möglicherweise auf eine durch die Behandlung ausgelöste Entzündungsreaktion zurückzuführen ist. Diese zeigte sich auch in histologischen Untersuchungen der Mauslungen. Wurde statt der CpG-haltigen pDNA CpG-freie pDNA vernebelt, war die Lungenfunktion weniger eingeschränkt und es zeigte sich keine Veränderung der Lungenhistologie. In dieser Arbeit wurde außerdem untersucht, ob die pDNA mittels magnetischer Ablenkung gezielt an den Ort einer möglichen Erkrankung in der Lunge gelenkt werden kann. Dazu wurden PEI-pDNA Komplexe zusammen mit SPION formuliert. Es konnte gezeigt werden, dass die magnetische Ablenkung von SPION in der Mauslunge funktioniert und zu einer gezielten, erhöhten Deposition von pDNA führt. Die Lungenfunktion wird durch die Verwendung von SPION dabei nicht zusätzlich eingeschränkt. Eine neue Methode zur Steigerung der Gentransfereffizienz wurde etabliert, die auf der Verwendung des Glucocorticoidrezeptors (GR) als Transkriptionsfaktor beruht. Wird GR durch Dexamethason aktiviert, führt dies zu einem Transport des GR aus dem Zytoplasma in den Zellkern einer Zelle. Wird vorher pDNA, die Glucocorticoidrezeptor-responsive Elemente enthält (GRE), an GR gebunden, kann der Transfer von pDNA aus dem Zytoplasma in den Zellkern verbessert werden. In in vivo Versuchen, in denen PEI-pDNA mittels Aerosol im Mausmodel appliziert wurde, konnte eine verbesserte Genexpression (4,7-fach) nach Gabe von pEGFPLucGRE2 im Vergleich zu pEGFPLuc nachgewiesen werden, wenn 2 h nach der Aerosolapplikation Dexamethason intraperitoneal appliziert wurde. Eine Herausforderung in der Zukunft wird sein, die Methode der Aerosolapplikation von Gentransferkomplexen auf ein Großtiermodel und letztendlich auf Patienten zu übertragen und zu optimieren. Insbesondere sollte dabei CpG-freie DNA verwendet werden, da sich dadurch die Verträglichkeit und die Gentransfereffizienz verbessern lassen.One strategy of gene transfer to the lung is to use PEI-pDNA complexes, which can be applied via aerosol or intranasal instillation. In this work different strategies were followed to enhance nonviral gene transfer into mice lungs and to improve the kinetic of gene expression. Additionally, the influence of gene transfer on lung function of mice was determined. The pDNA-clearance out of lungs was 3.5-times higher after intranasal instillation of PEI-pDNA complexes to mice compared to the clearance after aerosol application. Luciferase expression as a sign for effective gene transfer was observed only 24 h after intranasal instillation. After aerosol application, the highest expression rate was detected after 72 h and remained at high levels up to day seven. This result is consistent with the lower clearance rate after aerosol application. Luciferase expression could be further enhanced in experiments, which used CpG-depleted pDNA for aerosol gene delivery. Concerning lung function parameters, compliance of lungs was reduced after intranasal instillation as well as after aerosol application. This may be due to an inflammatory effect caused by the treatment with PEI-pDNA complexes. This was also observed in histological examinations and may be caused by CpG motives in the pDNA used for the treatment. After aerosol application of CpG-depleted PEI-pDNA complexes, lung function of mice was less impaired and histology of mice lungs was not changed compared to untreated control mice. Hence, the usage of CpG- depleted pDNA also resulted in improved lung function parameters, beside increased gene expression levels. To further improve gene delivery into the lung the targeted aerosol delivery to the lung with aerosol droplets comprising superparamagnetic iron oxid nanoparticles (SPION) – so called nanomagnetosols – in combination with a target directed magnetic gradient field was examined. PEI-pDNA complexes were formulated together with SPION. It was shown, that magnetic deflection of SPION in mice lungs was possible and a targeted and increased deposition of pDNA was achieved. Lung function was not deteriorated additionally by the usage of SPION. Furthermore, in this work a new method to increase gene transfer efficiency was established, which is based on the usage of the glucocorticoid receptor (GR) as transcription factor. After activation of GR by dexamethasone, GR is transported from the cytoplasm into the nucleus of a cell. pDNA containing a glucocorticoid responsive element (GRE) can bind to GR in the cytoplasm. Therefore, binding of the GR to its GRE and activation by its ligand (dexamethasone) can be used for the active transport of pDNA from the cytoplasm into the nucleus. In in vivo experiments PEI-pDNA complexes were applied to mice via aerosol. After application of pEGFPLucGRE2 gene expression increased about 4.7-times compared to pEGFPLuc, if dexamethasone was injected intraperitoneally 2 h after aerosol application. One challenge in the future will be to transfer the method of aerosol application of gene transfer complexes to a big animal model and finally to patients and to optimize these methods. In particular, CpG-depleted pDNA should be used because tolerance and gene transfer efficiency are increased

No non-redundant function of suppressor of cytokine signaling 2 in insulin producing β-cells

The members of the Suppressor of Cytokine Signaling (SOCS) protein family mainly modulate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. SOCS-1 and SOCS-3 have already been shown to influence growth and apoptosis of pancreatic β-cells. We hypothesized that SOCS-2, which is expressed in pancreatic islets, also contributes to β-cell physiology. We tested this hypothesis in vivo in SOCS-2−/− knockout mice and in vitro in Ins-1E rat insulinoma cells. We found that SOCS-2−/− mice have normal islet insulin secretion and unchanged glucose and insulin tolerance compared to wildtype controls. SOCS-2−/− are bigger than wildtype mice but body weight-corrected β-cell mass and islet morphology were normal. Growth hormone-induced proliferation of Ins-1E cells was not affected by either siRNA-mediated SOCS-2 knockdown or stable SOCS-2 overexpression. Interleukin-1β mediated cell death in vitro was unchanged after SOCS-2 knockdown. Similarly, autoimmune destruction of β-cells in vivo after multiple low-dose injections of streptozotocin (STZ) was not altered in SOCS-2−/− mice

The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC) via SGK-1 and MAPKs.

In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc)) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-α. This was in part due to up-regulation of β- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of β- and γ-ENaC-subunits along with induction of sgk-1

ENaC Dysregulation Through Activation of MEK1/2 Contributes to Impaired Na+ Absorption in Lymphocytic Colitis

Background: Lymphocytic colitis (LC) causes watery diarrhea. We aimed to identify mechanisms of altered Na+ absorption and regulatory inputs in patients with LC by examining the epithelial Na+ channel (ENaC) function as the predominant Na+ transport system in human distal colon. Methods: Epithelial Na+ channel function and regulation was analyzed in biopsies from sigmoid colon of patients with LC and in rat distal colon in Ussing chambers. ENaC-subunit expression was measured by real-time PCR and RNA sequencing. Correction factors for subepithelial resistance contributions were determined by impedance spectroscopy. Upstream regulators in LC were determined by RNA sequencing. Results: Epithelial Na+ channel-mediated electrogenic Na+ transport was inhibited despite aldosterone stimulation in human sigmoid colon of patients with LC. The increase in gamma-ENaC mRNA expression in response to aldosterone was MEK1/2-dependently reduced in LC, since it could be restored toward normal by MEK1/2 inhibition through U0126. Parallel experiments for identification of signaling in rat distal colon established MEK1/2 to be activated by a cytokine cocktail of TNF alpha, IFN gamma, and IL-15, which were identified as the most important regulators in the upstream regulator analysis in LC. Conclusions: In the sigmoid colon of patients with LC, the key effector cytokines TNF alpha, IFN gamma, and IL-15 inhibited gamma-ENaC upregulation in response to aldosterone through a MEK1/2-mediated pathway. This prevents ENaC to reach its maximum transport capacity and results in Na+ malabsorption which contributes to diarrhea