<p>The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied <i>in vitro</i> in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound.</p><p>It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which <i>in vivo</i> may increase a virus’s ability to persist in islets.</p><p>Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect.</p><p>All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis <i>in vivo</i>.</p><p>Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study.</p><p>To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells <i>in vivo</i>, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction <i>in vivo</i>.</p>

The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied in vitro in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound. It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which in vivo may increase a virus’s ability to persist in islets. Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect. All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis in vivo. Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study. To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells in vivo, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction in vivo.

Mesenchymal-epithelial interactions are pivotal for proper pancreatic growth and development. The pancreatic progenitor cells present in the early pancreatic anlagen proliferate and eventually give rise to all pancreatic cell types. The Fibroblast Growth Factor 2b (FGFR2b) high-affinity ligand Fibroblast Growth Factor 10 (FGF10) has been linked to pancreatic epithelial cell proliferation and we have previously shown that Notch signalling controls pancreatic cell differentiation via lateral inhibition. By overexpressing FGF10 under the control of the Ipf1/Pdx1 promoter in mice, we have shown that persistent FGF10 activation in the embryonic pancreas of transgenic mice perturbs pancreatic epithelial cell proliferation and also inhibits pancreatic cell differentiation by maintaining Notch activation. In the Ipf1/Fgf10 transgenic mice, the pancreatic epithelial cells are ‘locked’ in an undifferentiated progenitor-like state with sustained proliferative capacity. Collectively, our data suggest a key role for FGFR2b/FGF10 signalling in the regulation of pancreatic growth and differentiation and that FGFR2b/FGF10 signalling interact with the Notch signalling pathway. Glucose homeostasis in mammals is critically dependent on co-ordinated glucose uptake, oxidative metabolism and insulin secretion in β-cells. Although, several key genes controlling various aspects of glucose sensing, glucose metabolism, insulin expression and secretion have been identified, we know relatively little about the molecular mechanisms that induce and maintain the expression of genes required for glucose-stimulated insulin secretion (GSIS) in β-cells. Attenuation of FGFR1c signalling leads to diabetes in mice. Overexpression of FGF2, a high-affinity FGFR1c ligand, under the control of the Ipf1/Pdx1 promoter also leads to diabetes in mice. The Ipf1/Fgf2 mice present with normal endocrine and exocrine differentiation but display impaired glucose-stimulated insulin secretion (GSIS), perturbed expression of genes required for glucose sensing uptake together with oxidative metabolism and increased expression of the FGF-signalling inhibitors Spry-2 and Pyst1/MKP3 in β-cells. Thus, stringent control of FGF signalling activation appears crucial for the maintenance of the regulatory circuit that ensures proper GSIS in pancreatic β-cells and hence normoglycaemia. The Wnt family of ligands via their receptors Frizzled (Frz) have been shown to mediate mesenchymal-epithelial interactions and cell proliferation in a variety of different systems. Expression of a plethora of Wnt ligands and Frz receptors has been previously reported in the pancreas and mice missexpressing Wnt1 and Wnt5a under the Ipf1/Pdx1 promoter display severely perturbed development. Here, we show the temporal and spatial expression of Wnt4, Wnt7b and Frz3 at different stages of pancreas development. To elucidate the role of Wnt signalling in the pancreas, we overexpressed a dominant negative form of mouse Frz8 under the Ipf1/Pdx1 promoter in mice. The Ipf1/Frz8CRD mice display severe pancreatic hypoplasia demonstrating that attenuation of Wnt signalling in the pancreas leads to perturbed pancreatic growth. Nevertheless, the transgenic mice present with normal endocrine and exocrine differentiation and remain normoglycaemic. The maintenance of normoglycaemia in these mice appears to be the consequence of a relative increase in endocrine cell number per pancreatic area combined with enhanced insulin biosynthesis and insulin secretion. Collectively our data provide evidence that Wnt signalling is required pancreatic growth but not adult β-cell function.

O diabetes mellitus tipo 1 (DM1) é uma síndrome autoimune órgão-específica caracterizada pela destruição seletiva de células β que leva à morte celular. A DM1 é a causa de mais de 5% do total de mortes na população por ano e são poucas as estratégias de tratamento disponíveis. As linhagens celulares, são amplamente utilizadas nos estudos da DM1 como um modelo in vitro. A linhagem celular derivada de insulinoma murino, chamada de MIN6, assemelha-se as ilhotas de camundongo. Sendo assim, pesquisadores comprovaram que estas células apresentam características funcionais semelhantes às células β-pancreáticas em resposta a glicose e a outros secretagogos. Através de agregação espontânea e crescimento tridimensional, estas células formam as pseudoilhotas (PIs). A frutose-1,6-bisfosfato (FBP) é um açúcar bifosforilado, que apresenta duas estruturas anoméricas denominadas α e β furanose. Já são conhecidas algumas ações importantes da FBP, como: ação citoprotetora, antioxidante e anti-inflamatória. Com base no descrito, o objetivo deste estudo foi avaliar a possível ação citoprotetora da FBP sobre células MIN6, cultivadas em monocadas e como pseudoilhotas. Para tal, foram avaliados os efeitos do tratamento com diferentes concentrações (0,30 mM, 0,62 mM e 1,25 mM) de FBP sobre a viabilidade, proliferação e síntese de insulina em células MIN6. Também foram determinados os efeitos da FBP na formação, crescimento e viabilidade das PIs. MIN6 Os resultados mostraram, que em qualquer das doses testadas, a FBP aumentou a adesão das células MIN6. No entanto, os tratamentos com 0,62 e 1,25 mM de FBP provocaram uma inibição significativa da proliferação. A sintese de insulina foi detectada por imunocitoquímica e sua secreção basal estimulada por glicose determinada por ELISA. Como esperado, a formação e o crescimento de PIs é tempo dependente, tanto nas culturas controle como nas tratadas com FBP. Nas culturas tratadas com FBP, a formação de PIs maiores (150-300 μm) foi menor que nas culturas controle. A incorporação de iodeto de propídeo mostrou um aumento da porcentagem de células inviáveis nas culturas de PIs tratadas com 1,25 mM de FBP, sendo citotóxica nessa concentração. Embora ainda sejam necessários mais estudos para aprofundar o conhecimento da atividade citoprotetora da FBP em outros modelos celulares e em ilhotas pancreáticas, nossos resultados mostram que em monocamadas e PIs, da linhagem celular MIN6 utilizadas nos experimentos, que a FBP não apresentou atividade citoprotetora. / Diabetes mellitus type 1 (DM 1) is an organ-specific autoimmune syndrome characterized by the selective destruction of β cells that leads to cell death.The DM1 is the cause of more than 5% of all deaths in the population per year and there are few treatment strategies available. Cell lines are widely used in studies of DM1 in vitro model. The cell line derived from a mouse β insulinoma MIN6 is similar to the mouse islets. Studies of these cells exhibit functional characteristic similar to pancreatic β-cells in response to glucose and other secretagogues. Spontaneous aggregation and tridimensional growth those cells form the pseudoislets (PIs). Fructose-1,6-bisphosphate (FBP) is a biphosphorylated sugar with two anomeric forms designated α and β furanoses. Important actions of FBP as antioxidant, cytoprotective and anti-inflammatory. Based on the described, in this work, our objective is evaluate the cytoprotective effect of FBP on MIN6 cells in monolayer and pseudoislets agregate cells. The effects of FBP with different concentrations (0.30 mM, 0.62 mM and 1.25 mM) in MIN6 cells on the proliferation, viability and insulin synthesis. We also determined the effects of FBP in the formation, growth and viability of PIs. The results showed that all doses of FBP tested increased adhesion on MIN6 cells. However, the treatment with 0.62 and 1.25 mM of FBP causes a significant decrease in cell proliferation. Insulin synthesis detected by immunocytochemistry, basal and glucose-stimulated insulin secretion was determined by ELISA. As expected, PIs formation and growth is time dependent in the control cultures and treated with FBP. In cultures treated with FBP the formation of larger (150-300 μm) PIs was lower than in control cultures. Propidium iodide incorporation showed an increasing percentage of nonviable PIs cells in cultures treated with 1.25 mM of FBP, including cytotoxic. More studies are required to deepen understanding of the role of cytoprotective activity of FBP in other cell types and in pseudoislets, but our results indicate in cell line MIN6, monolayers and pseudoislets, that FBP did not show cytoprotective activity.

O diabetes mellitus tipo 1 (DM1) é uma síndrome autoimune órgão-específica caracterizada pela destruição seletiva de células β que leva à morte celular. A DM1 é a causa de mais de 5% do total de mortes na população por ano e são poucas as estratégias de tratamento disponíveis. As linhagens celulares, são amplamente utilizadas nos estudos da DM1 como um modelo in vitro. A linhagem celular derivada de insulinoma murino, chamada de MIN6, assemelha-se as ilhotas de camundongo. Sendo assim, pesquisadores comprovaram que estas células apresentam características funcionais semelhantes às células β-pancreáticas em resposta a glicose e a outros secretagogos. Através de agregação espontânea e crescimento tridimensional, estas células formam as pseudoilhotas (PIs). A frutose-1,6-bisfosfato (FBP) é um açúcar bifosforilado, que apresenta duas estruturas anoméricas denominadas α e β furanose. Já são conhecidas algumas ações importantes da FBP, como: ação citoprotetora, antioxidante e anti-inflamatória. Com base no descrito, o objetivo deste estudo foi avaliar a possível ação citoprotetora da FBP sobre células MIN6, cultivadas em monocadas e como pseudoilhotas. Para tal, foram avaliados os efeitos do tratamento com diferentes concentrações (0,30 mM, 0,62 mM e 1,25 mM) de FBP sobre a viabilidade, proliferação e síntese de insulina em células MIN6. Também foram determinados os efeitos da FBP na formação, crescimento e viabilidade das PIs. MIN6 Os resultados mostraram, que em qualquer das doses testadas, a FBP aumentou a adesão das células MIN6. No entanto, os tratamentos com 0,62 e 1,25 mM de FBP provocaram uma inibição significativa da proliferação. A sintese de insulina foi detectada por imunocitoquímica e sua secreção basal estimulada por glicose determinada por ELISA. Como esperado, a formação e o crescimento de PIs é tempo dependente, tanto nas culturas controle como nas tratadas com FBP. Nas culturas tratadas com FBP, a formação de PIs maiores (150-300 μm) foi menor que nas culturas controle. A incorporação de iodeto de propídeo mostrou um aumento da porcentagem de células inviáveis nas culturas de PIs tratadas com 1,25 mM de FBP, sendo citotóxica nessa concentração. Embora ainda sejam necessários mais estudos para aprofundar o conhecimento da atividade citoprotetora da FBP em outros modelos celulares e em ilhotas pancreáticas, nossos resultados mostram que em monocamadas e PIs, da linhagem celular MIN6 utilizadas nos experimentos, que a FBP não apresentou atividade citoprotetora. / Diabetes mellitus type 1 (DM 1) is an organ-specific autoimmune syndrome characterized by the selective destruction of β cells that leads to cell death.The DM1 is the cause of more than 5% of all deaths in the population per year and there are few treatment strategies available. Cell lines are widely used in studies of DM1 in vitro model. The cell line derived from a mouse β insulinoma MIN6 is similar to the mouse islets. Studies of these cells exhibit functional characteristic similar to pancreatic β-cells in response to glucose and other secretagogues. Spontaneous aggregation and tridimensional growth those cells form the pseudoislets (PIs). Fructose-1,6-bisphosphate (FBP) is a biphosphorylated sugar with two anomeric forms designated α and β furanoses. Important actions of FBP as antioxidant, cytoprotective and anti-inflammatory. Based on the described, in this work, our objective is evaluate the cytoprotective effect of FBP on MIN6 cells in monolayer and pseudoislets agregate cells. The effects of FBP with different concentrations (0.30 mM, 0.62 mM and 1.25 mM) in MIN6 cells on the proliferation, viability and insulin synthesis. We also determined the effects of FBP in the formation, growth and viability of PIs. The results showed that all doses of FBP tested increased adhesion on MIN6 cells. However, the treatment with 0.62 and 1.25 mM of FBP causes a significant decrease in cell proliferation. Insulin synthesis detected by immunocytochemistry, basal and glucose-stimulated insulin secretion was determined by ELISA. As expected, PIs formation and growth is time dependent in the control cultures and treated with FBP. In cultures treated with FBP the formation of larger (150-300 μm) PIs was lower than in control cultures. Propidium iodide incorporation showed an increasing percentage of nonviable PIs cells in cultures treated with 1.25 mM of FBP, including cytotoxic. More studies are required to deepen understanding of the role of cytoprotective activity of FBP in other cell types and in pseudoislets, but our results indicate in cell line MIN6, monolayers and pseudoislets, that FBP did not show cytoprotective activity.

O diabetes mellitus tipo 1 (DM1) é uma síndrome autoimune órgão-específica caracterizada pela destruição seletiva de células β que leva à morte celular. A DM1 é a causa de mais de 5% do total de mortes na população por ano e são poucas as estratégias de tratamento disponíveis. As linhagens celulares, são amplamente utilizadas nos estudos da DM1 como um modelo in vitro. A linhagem celular derivada de insulinoma murino, chamada de MIN6, assemelha-se as ilhotas de camundongo. Sendo assim, pesquisadores comprovaram que estas células apresentam características funcionais semelhantes às células β-pancreáticas em resposta a glicose e a outros secretagogos. Através de agregação espontânea e crescimento tridimensional, estas células formam as pseudoilhotas (PIs). A frutose-1,6-bisfosfato (FBP) é um açúcar bifosforilado, que apresenta duas estruturas anoméricas denominadas α e β furanose. Já são conhecidas algumas ações importantes da FBP, como: ação citoprotetora, antioxidante e anti-inflamatória. Com base no descrito, o objetivo deste estudo foi avaliar a possível ação citoprotetora da FBP sobre células MIN6, cultivadas em monocadas e como pseudoilhotas. Para tal, foram avaliados os efeitos do tratamento com diferentes concentrações (0,30 mM, 0,62 mM e 1,25 mM) de FBP sobre a viabilidade, proliferação e síntese de insulina em células MIN6. Também foram determinados os efeitos da FBP na formação, crescimento e viabilidade das PIs. MIN6 Os resultados mostraram, que em qualquer das doses testadas, a FBP aumentou a adesão das células MIN6. No entanto, os tratamentos com 0,62 e 1,25 mM de FBP provocaram uma inibição significativa da proliferação. A sintese de insulina foi detectada por imunocitoquímica e sua secreção basal estimulada por glicose determinada por ELISA. Como esperado, a formação e o crescimento de PIs é tempo dependente, tanto nas culturas controle como nas tratadas com FBP. Nas culturas tratadas com FBP, a formação de PIs maiores (150-300 μm) foi menor que nas culturas controle. A incorporação de iodeto de propídeo mostrou um aumento da porcentagem de células inviáveis nas culturas de PIs tratadas com 1,25 mM de FBP, sendo citotóxica nessa concentração. Embora ainda sejam necessários mais estudos para aprofundar o conhecimento da atividade citoprotetora da FBP em outros modelos celulares e em ilhotas pancreáticas, nossos resultados mostram que em monocamadas e PIs, da linhagem celular MIN6 utilizadas nos experimentos, que a FBP não apresentou atividade citoprotetora. / Diabetes mellitus type 1 (DM 1) is an organ-specific autoimmune syndrome characterized by the selective destruction of β cells that leads to cell death.The DM1 is the cause of more than 5% of all deaths in the population per year and there are few treatment strategies available. Cell lines are widely used in studies of DM1 in vitro model. The cell line derived from a mouse β insulinoma MIN6 is similar to the mouse islets. Studies of these cells exhibit functional characteristic similar to pancreatic β-cells in response to glucose and other secretagogues. Spontaneous aggregation and tridimensional growth those cells form the pseudoislets (PIs). Fructose-1,6-bisphosphate (FBP) is a biphosphorylated sugar with two anomeric forms designated α and β furanoses. Important actions of FBP as antioxidant, cytoprotective and anti-inflammatory. Based on the described, in this work, our objective is evaluate the cytoprotective effect of FBP on MIN6 cells in monolayer and pseudoislets agregate cells. The effects of FBP with different concentrations (0.30 mM, 0.62 mM and 1.25 mM) in MIN6 cells on the proliferation, viability and insulin synthesis. We also determined the effects of FBP in the formation, growth and viability of PIs. The results showed that all doses of FBP tested increased adhesion on MIN6 cells. However, the treatment with 0.62 and 1.25 mM of FBP causes a significant decrease in cell proliferation. Insulin synthesis detected by immunocytochemistry, basal and glucose-stimulated insulin secretion was determined by ELISA. As expected, PIs formation and growth is time dependent in the control cultures and treated with FBP. In cultures treated with FBP the formation of larger (150-300 μm) PIs was lower than in control cultures. Propidium iodide incorporation showed an increasing percentage of nonviable PIs cells in cultures treated with 1.25 mM of FBP, including cytotoxic. More studies are required to deepen understanding of the role of cytoprotective activity of FBP in other cell types and in pseudoislets, but our results indicate in cell line MIN6, monolayers and pseudoislets, that FBP did not show cytoprotective activity.

Increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-cells triggers insulin exocytosis. Among the Ca2+ channels present in the plasma membrane, the transient receptor potential (TRP) channels receptors are currently of great interest. The mechanisms by which the extracellular adenosine diphosphate ribose (ADPr) increases the [Ca2+]i is unknown. Our aims were to study the roles of the TRP channels in the tolbutamide induced [Ca2+]i increase and to identify the surface receptor that is activated by ADPr. We used S5 cells, a highly differentiated rat insulinoma cell line, as a model for β-cells. Single cell ratiometric microfluorometry was used to measure the [Ca2+]i changes in the Fura-2 loaded cells. Tolbutamide increased [Ca2+]i in the form of oscillations. After tolbutamide increased [Ca2+]i,capsazepine, a potent blocker of the transient receptor potential vanilloid subtype 1 (TRPV1) channel was added to the β-cells, which reduced the tolbutamide-induced [Ca2+]i increase. capsazepine, N-(p-Amylcinnamoyl) anthranilic acid (ACA),  TRPM2 channel blocker, and triphenyl phosphine oxide (TPPO), TRPM5 channel blocker were tested for their effect on potassium chloride (KCl) induced [Ca2+]i response. These blockers did not inhibit the KCl induced [Ca2+]i increase.   Adenosine diphosphate ribose (ADPr) increased [Ca2+]i in the form of initial transient peak followed by an elevated plateau. Application of ADPr shortly after a prior application and washout of Adenosine diphosphate (ADP) elicited only small [Ca2+]i increase  indicating desensitization of the receptor involved. 2´deoxy-N6-methyladenosine 3´5´bis-phosphate (MRS2179), and chloro N6-methyl-(N)-methanocarba 2´deoxyadenosine 3´5´ bis-phosphate (MRS2279), two selective inhibitors of P2Y1 receptor, abolished the ADPr-induced [Ca2+]i increase. Tolbutamide closes ATP sensitive potassium (KATP) channels. Our results demonstrate that besides the closure of the KATP channels, inward cation currents carried by Ca2+through the TRPV1 channel are necessary for depolarization to the threshold for the activation of the voltage gated calcium channels (VGCC) to increase the [Ca2+]i. Our results also show that ADPr increases [Ca2+]i by activating the P2Y1 receptor.

Les cellules β des îlots de Langerhans jouent un rôle central dans l’homéostasie glucidique car elles seules sécrètent l’insuline, unique hormone hypoglycémiante de l’organisme. La cellule β est un détecteur du glucose qui couple sa réponse sécrétoire et son expression génique aux niveaux ambiants de glucose. Le couplage entre le métabolisme du glucose et l’exocytose des granules d’insuline implique la génération d’une activité électrique. Son étude est importante pour déchiffrer la façon dont la cellule β encode la demande en insuline de l’organisme. Afin de contourner les limites des approches électrophysiologiques classiques incompatibles avec les études à long-terme, les enregistrements extracellulaires par matrice de microélectrodes (MEA) ont été mis en place.L’objectif de ma thèse était de mieux comprendre les signaux complexes enregistrés par MEAs. Cette étude a révélé l’existence d’une nouvelle signature électrique des cellules des îlots, les slow potentials (SP), qui reflète la fonction de couplage des cellules β. Les SP jouent un rôle important dans l’homéostasie du glucose et représentent un biomarqueur de la fonction normale des îlots. La réponse en hystérèse des îlots au glucose suggère l’existence d’un algorithme d’encodage de la demande en insuline intégrée au niveau du micro-organe. De plus, ce nouveau signal a été exploité pour le phénotypage d’îlots de souris invalidées pour le gène GluK2, que nous avons utilisées comme modèle d’interaction entre les cellules α et β. La caractérisation de ce nouveau type de signal constitue aussi une avancée importante pour le développement d’un biocapteur destiné à être intégré dans le futur à un pancréas artificiel. / Pancreatic β cells are central to glucose homeostasis because they are the only cell that secretes insulin, the sole hypoglycemic hormone in the organism. The β cell is a glucose sensor that regulates its secretory response and gene expression according to ambient glucose levels. The coupling between glucose metabolism and insulin granule exocytosis involves the generation of electrical activity. An investigation of this activity is important to decipher how β cells encode the organism’s insulin demand. In order to overcome the limits of classically used electrophysiological approaches that are not compatible with long-term studies, extracellular recordings using multielectrode arrays (MEA) have been set-up.My thesis aim was to better understand the complex signals recorded with MEA. This study revealed the existence of a new electrical signature of islet cells: slow potentials (SP) that reflect the coupling function of β cells. SP play an important role in glucose homeostasis and represent a biomarker of normal functioning of islets. The observed hysteretic response of islets to glucose suggests the existence of an algorithm encoding the insulin demand embedded at the microorgan level. Moreover, this new signal was used for the phenotyping of GluK2 deficient mouse islets that were employed as an α-to-β cell interaction model. The characterization of this new signal is an important progress in the development of a biosensor intended to be integrated in an artificial pancreas in the future.

<p>Protein tyrosine kinases play an important role in the regulation of various cellular processes such as</p><p>growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27^KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.</p>

Protein tyrosine kinases play an important role in the regulation of various cellular processes such as growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.