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P Serup

Publications and source records attributed to P Serup.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterization of beta cells developed in vitro from rat embryonic pancreatic epithelium.

The present study evaluates the development and functional properties of beta cells differentiated in vitro. The authors have previously demonstrated that when E12.5 rat pancreatic rudiments are cultured in vitro in the absence of mesenchyme, the majority of the epithelial cells differentiate into endocrine beta cells. Thus, depletion of the mesenchyme provokes the expansion of endocrine tissue at the expense of exocrine tissue. The potential use of this procedure for the production of beta cells led the authors to characterize the beta cells differentiated in this model and to compare their properties with those of the endocrine cells of the embryonic and adult pancreas. This study shows that the beta cells that differentiate in vitro in the absence of mesenchyme express the homeodomain protein Nkx6.1, a transcription factor that is characteristic of adult mature beta cells. Further, electron microscopy analysis shows that these beta cells are highly granulated, and the ultrastructural analysis of the granules shows that they are characteristic of mature beta cells. The maturity of these granules was confirmed by a double-immunofluorescence study that demonstrated that Rab3A and SNAP-25, two proteins associated with the secretory pathway of insulin, are strongly expressed. Finally, the maturity of the differentiated beta cells in this model was confirmed when the cells responded to stimulation with 16 mM glucose by a 5-fold increase in insulin release. The authors conclude that the beta cells differentiated in vitro from rat embryonic pancreatic rudiments devoid of mesenchyme are mature beta cells.

Animals↗
PubMed1999

Beta-cell maturation leads to in vitro sensitivity to cytotoxins.

Pancreatic beta-cells are more sensitive to several toxins (e.g., streptozotocin, alloxan, cytokines) than the other three endocrine cell types in the islets of Langerhans. Cytokine-induced free radicals in beta-cells may be involved in beta-cell-specific destruction in type 1 diabetes. To investigate if this sensitivity represents an acquired trait during beta-cell maturation, we used two in vitro cultured cell systems: 1) a pluripotent glucagon-positive pre-beta-cell phenotype (NHI-glu) that, after in vivo passage, matures into an insulin-producing beta-cell phenotype (NHI-ins) and 2) a glucagonoma cell-type (AN-glu) that, after stable transfection with pancreatic duodenal homeobox factor-1 (PDX-1), acquires the ability to produce insulin (AN-ins). After exposure to interleukin (IL)-1beta, both of the insulin-producing phenotypes were significantly more susceptible to toxic effects than their glucagon-producing counterparts. Nitric oxide (NO) production was induced in both NHI phenotypes, and inhibition with 0.5 mmol/l N(G)-monomethyl-L-arginine (NMMA) fully protected the cells. In addition, maturation into the NHI-ins phenotype was associated with an acquired dose-dependent sensitivity to the toxic effect of streptozotocin. Our results support the hypothesis that the exquisite sensitivity of beta-cells to IL-1beta and streptozotocin is an acquired trait during beta-cell maturation. These two cell systems will be useful tools for identification of molecular mechanisms involved in beta-cell maturation and sensitivity to toxins in relation to type 1 diabetes.

Animals↗
PubMed1998

Glucose stimulates the activation domain potential of the PDX-1 homeodomain transcription factor.

Glucose-stimulated expression of the insulin gene in beta cells is mediated by the PDX-1 transcription factor. In this report, we show that stimulation results from effects on activation and DNA-binding potential. Thus, glucose specifically stimulated expression in MIN6 beta cells from chimeras of PDX-1 and the GAL4 DNA-binding domain which spanned the N-terminal PDX-1 activation domain located between amino acids 1 to 79. GAL4:PDX activity was induced over physiological glucose concentrations and was also regulated by effectors of this response. The level of endogenous PDX-1 binding and phosphorylation were also induced under these conditions. We discuss how changes in PDX-1 phosphorylation may influence activity in glucose-treated beta cells.

Base Sequence↗
PubMed1998

Rat endocrine pancreatic development in relation to two homeobox gene products (Pdx-1 and Nkx 6.1).

We studied the distribution of the homeodomain proteins Pdx-1 and Nkx 6.1 in the developing rat pancreas. During early development, nuclear staining for both Pdx-1 and Nkx 6.1 occurred in most epithelial cells of the pancreatic anlage. Subsequently, Nkx 6.1 became more beta-cell-restricted, and Pdx-1 also occurred in other islet cell types and in the duodenal epithelium. During early pancreatic development, cells co-storing insulin and glucagon were regularly detected. The vast majority of these did not possess nuclear staining for either Pdx-1 or Nkx 6.1. Subsequently, cells storing insulin only appeared. Such cells displayed strongly Pdx-1- and Nkx 6.1-positive nuclei. Therefore, Nkx 6.1, like Pdx-1, may be an important factor in pancreatic development and in mature insulin cell function.

Animals↗
PubMed1997

Transcription factors contributing to the pancreatic beta-cell phenotype.

Insulin promoter factor-1 (IPF1) (renamed to pancreatic-duodenal homeobox factor-1, PDX1) was originally cloned and characterized as an islet beta-cell specific insulin gene transcription factor (1) and later shown to be essential for the formation of the mature pancreas (2, 3). In the adult normal pancreas PDX1 is almost exclusively expressed in the beta-cell compartment and generally absent from the alpha-cell while it is widely expressed in the pancreatic epithelium during development. Using pluripotent rat islet tumor cultures and derived insulinomas and glucagonomas we have analyzed differential expression of a large number of genes including the transcription factors PDX1, Nkx6.1, Pax6, and NeuroD. While NeuroD and Pax6 expression was detectable among all phenotypes, PDX1 was expressed in the pluripotent culture and maintained in the insulinoma, while Nkx6.1 was selectively co-induced with insulin during insulinoma formation. Both factors were not detectable in the glucagonoma. Nkx6.1 proved to have a highly beta-cell restricted expression in the adult rat. Forced expression of recombinant PDX1 in the glucagonoma resulted in efficient transcriptional activation of the endogenous insulin and IAPP genes, but did not affect glucagon gene activity. In this hybrid alpha/beta-cell phenotype the endogenous Nkx6.1 gene remained silent. We conclude that PDX1 in synergy with NeuroD specifies part of the beta-cell phenotype including transcriptional activation of insulin and IAPP genes, but that other factors such as Nkx6.1 and Pax6 are required for additional features of the fully mature beta-cell phenotype.

Animals↗
PubMed1996

Pancreatic development and maturation of the islet B cell. Studies of pluripotent islet cultures.

Pancreas organogenesis is a highly regulated process, in which two anlage evaginate from the primitive gut. They later fuse, and, under the influence of the surrounding mesenchyme, the mature organ develops, being mainly composed of ductal, exocrine and endocrine compartments. Early buds are characterized by a branching morphogenesis of the ductal epithelium from which endocrine and exocrine precursor cells bud to eventually form the two other compartments. The three compartments are thought to be of common endodermal origin; in contrast to earlier hypotheses, which suggested that the endocrine compartment was of neuroectodermal origin. It is thus generally believed that the pancreatic endocrine-lineage possesses the ability to mature along a differentiation pathway that shares many characteristics with those of neuronal differentiation. During recent years, studies of insulin-gene regulation and, in particular, the tissue-specific transcriptional control of insulin-gene activity have provided information on pancreas development in general. The present review summarizes these findings, with a special focus on our own studies on pluripotent endocrine cultures of rat pancreas.

Amyloid↗
PubMed1996

Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1.

Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371, 606-609]. In adults, IPF1 expression is restricted to the beta-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of beta-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet alpha-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta 2/NeuroD, which is expressed in both alpha- and beta-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-beta islet cells, which suggests an important role of IPF1 in beta-cell maturation.

Amyloid↗
PubMed1996

mRNA profiling of rat islet tumors reveals nkx 6.1 as a beta-cell-specific homeodomain transcription factor.

Development of a high capacity multiplex reverse transcriptase-polymerase chain reaction protocol has allowed us to screen lineage related rat islet tumors classified as alpha-, beta-, and delta-like as judged by their hormone profile for differential expression of more than 50 selected genes. We find that in addition to insulin the insulinoma express the normal beta-cell markers Pdx-1, IAPP, and Glut-2, and that these markers are absent from the glucagonoma: a reflection of the normal alpha-cell. Furthermore, this study suggests that the GLP-1, glucagon, GIP, IGF-1, and insulin receptors as well as E-cadherin, R-cadherin, Id-1, and Id-2 are differentially expressed within the islet of Langerhans. Importantly, insulinoma-specific expression of the recently cloned homeodomain protein Nkx 6.1 predicted beta-cell-specific expression in the normal islet. Immunohistochemistry using antibodies raised against recombinant Nkx 6.1 did indeed localize Nkx 6.1 expression exclusively to the nuclei of normal islet beta-cells. Apart from pancreatic islets only the antral part of the stomach contained Nkx 6.1 mRNA. We conclude that multiplex reverse transcriptase-polymerase chain reaction-based mRNA profiling is a powerful tool to identify differentially expressed genes within phenotypically related cells and propose that Nkx 6.1 is involved in specifying the unique characteristics of the beta-cell.

Adenoma, Islet Cell↗
PubMed1996

Pancreatic-duodenal homeobox 1 -role in gastric endocrine patterning.

The gastrointestinal tract is subdivided into regions with different roles in digestion and absorption. How this patterning is established is unknown. We now report that the pancreatic-duodenal homeobox 1 gene (pdx1) is also expressed in cells of the distal stomach. Positive cells include subpopulations of the three main endocrine (gastrin, somatostatin and serotonin) cell types of this region. Pdx1 deficient mice were virtually devoid of gastrin cells, had normal numbers of somatostatin cells and increased numbers of serotonin cells. Pdx1 is thus important for development of the gastrin cells of the antropyloric mucosa of the stomach and probably acts by controlling the fate of gastrin/serotonin precursor cells.

Animals↗
PubMed1996

Identification of a growth hormone-responsive STAT5-binding element in the rat insulin 1 gene.

GH and PRL stimulate both proliferation and insulin production in pancreatic beta-cells as well as in the rat insulinoma cell line RIN-5AH, We report here that human GH increases insulin mRNA levels in RIN-5AH cells via both somatogenic and lactogenic receptors. GH stimulated the rat insulin 1 promoter activity 2-fold, and this stimulation was abolished by introduction of a block mutation in a gamma-interferon-activated sequence (GAS)-like element (GLE) with the sequence 5'-TTCTGGGAA-3' located in the rat insulin 1 enhancer at position -330 to -322. This element, termed Ins-GLE, was able to confer GH responsiveness to a heterologous promoter. GH induced the binding of two protein complexes to the Ins-GLE. An antibody directed against the transcription factor STAT5 (signal transducer and activator of transcription) supershifted the GH-induced complexes. Furthermore, in COS7 cells transiently transfected with STAT5 and GH receptor cDNAs, it was found that expression of STAT5 was necessary for GH induction of these two DNA-binding complexes. These results suggest that GH stimulates insulin 1 promoter activity by inducing the binding of STAT5 to Ins-GLE.

Animals↗
PubMed1995

The homeodomain protein IPF-1/STF-1 is expressed in a subset of islet cells and promotes rat insulin 1 gene expression dependent on an intact E1 helix-loop-helix factor binding site.

The mouse homeodomain protein insulin promoter factor-1 (IPF-1) and the rat homologue somatostatin transactivating factor-1 (STF-1) are involved in early pancreatic development and have been implicated in the cell-specific regulation of insulin- and somatostatin-gene expression in mature islet beta- and delta-cells. The cell specificity of IPF-1/STF-1 expression in mature islets is, however, still unclear. Using antisera against recombinant IPF-1 and STF-1 in combination with antisera against islet hormones we find that all beta-cells in monolayers of newborn rat islet cells express STF-1, as do a fraction of the delta-cells. In adult rat and mouse pancreas we find a similar distribution. IPF-1/STF-1 expression was not detected in glucagon-producing alpha-cells. In islet cell tumour models we found that a glucagon/islet amyloid polypeptide (IAPP)-producing pluripotent rat islet cell line (NHI-6F-GLU) expresses STF-1 in all cells prior to insulin gene activation induced by in vivo culture. In contrast, a mouse alpha-cell line (alpha TC1) exclusively expressed IPF-1 in a small subset of insulin-producing cells while an insulin-negative subclone (alpha TC1.9) was negative for IPF-1. In transfection experiments using alpha TC1.9 cells STF-1 activated a rat insulin 1 reporter gene dependent not only on both STF-1-binding sites, but also on the E1-binding site for the helix-loop-helix factor IEF-1. However, the endogenous mouse insulin genes remained inactive in these cells. These results suggest that the insulin promoter acquires its very high, yet cell-specific, activity at least partly through the action of IPF-1/STF-1. This action is dependent on helix-loop-helix factors bound to the E1 element.

Animals↗
PubMed1994

Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes.

Insulin gene transcription is a unique feature of the pancreatic beta cells and is increased in response to glucose. The recent cloning of insulin promoter factor 1 (IPF1) and somatostatin transcription factor 1 (STF1) unexpectedly revealed that these are mouse and rat homologues of the same protein mediating transactivation through binding of CT box-like elements in rat insulin 1 and somatostatin promoter/enhancer regions, respectively. By using oligonucleotides representing each of the three CT boxes of the human insulin (HI) gene enhancer and nuclear extracts from the mouse islet tumor cell lines beta TC3 and alpha TC1, we have identified a beta-cell-specific binding activity as reported for IPF1, which has maximal affinity toward the CT2 box. However, in pluripotent, HI-transfected rat islet tumor cells, NHI-6F, this binding activity is present prior to induction of (human) insulin gene transcription. Its migration is identical to that of in vitro translated STF1 in electrophoretic mobility-shift assays; it is specifically recognized by anti-STF1 antibodies and has an apparent molecular mass of 46 kDa. Mutation of the CT2 box decreases transcriptional activity of a HI reporter plasmid by approximately 65% in beta TC3 cells and blocks the glucose response in isolated newborn rat islet cells. Furthermore, cotransfection with STF1 cDNA into the glucagon-producing alpha TC1 cells increases the activity of the HI enhancer 4- to 5-fold, suggesting that STF1/IPF1 can confer on alpha TC1 cells the ability to transcribe the HI gene. We conclude that STF1/IPF1 is a necessary but not sufficient key regulator of insulin gene activity, possibly also involved in glucose-regulated transcription.

Animals↗
PubMed1992

The LIM family transcription factor Isl-1 requires cAMP response element binding protein to promote somatostatin expression in pancreatic islet cells.

Many eukaryotic genes are regulated by cAMP through a conserved cAMP response element (CRE). Here we show that, in the pancreatic islet cell line Tu6, a well-characterized CRE in the somatostatin gene does not provide cAMP responsiveness but functions as an essential element for its basal activity. DNA-binding and functional analyses indicate that the cAMP-responsive factor CREB regulates somatostatin expression in these cells without requirement for phosphorylation at the protein kinase A-regulated Ser-133 phosphorylation site. In addition to the CRE site, cell-specific expression of the somatostatin gene requires a second promoter element, which binds the recently characterized LIM family protein Isl-1. Thus, Isl-1 and CREB appear to synergize on the somatostatin promoter to stimulate high-level expression in Tu6 cells. The ability of CREB to function in a phosphorylation-independent manner suggests a mechanism by which this protein can regulate gene transcription.

Base Sequence↗
PubMed1992

Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene.

Synthetic peptides representing unique sequences in rat proinsulin C-peptide I and II were used to generate highly specific antisera, which, when applied on sections of normal rat pancreas, confirm a homogeneous coexpression of the two C-peptides in all islet beta-cells. Insulin gene expression is induced in the transformed heterogeneous rat islet cell clone, NHI-6F, by transient in vivo passage. During this process a transfected human insulin gene is coactivated with the endogenous nonallelic rat insulin I and II genes. Newly established cultures from NHI-6F insulinomas having a high frequency of insulin-producing cells showed highly differential expression at the cellular level of the three proinsulin C-peptide immunoreactivities, as follows: C-peptide I greater than human C-peptide greater than C-peptide II. The fractions of cells expressing human C-peptide and C-peptide II decreased in time and were absent after more than 50 successive passages, while a C-peptide I-producing population was still present. Double-labeling experiments revealed a heterogeneous distribution of the three different C-peptides. Surprisingly, in the early passages a large fraction of cells would express only a single species of proinsulin-C-peptide immunoreactivity but still at high levels. However, rat C-peptide II and human C-peptide were often colocalized, even in later passages. In situ hybridization studies combined with the immunocytochemical data suggest that the differential expression occurs at the level of transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells.

The stimulation of insulin biosynthesis in the pancreatic insulinoma cell line RIN5-AH by growth hormone (GH) is initiated by GH binding to specific receptors. To determine whether the recently cloned rat hepatic GH receptor is able to mediate the insulinotropic effect of GH, we have transfected a GH receptor cDNA under the transcriptional control of the human metallothionein promoter into RIN5-AH cells. The transfected cells were found to exhibit an increased expression of GH receptors and to contain a specific GH receptor mRNA that was not expressed in the parent cell line. The expression of GH receptors in one clone (1.24) selected for detailed analysis was increased 2.6-fold compared to untransfected cells. The increased GH receptor expression was accompanied by an increased responsiveness to GH. Thus, the maximal GH-stimulated increase of insulin biosynthesis was 4.1-fold in 1.24 cells compared to 1.9-fold in the nontransfected RIN5-AH cells. The expression of the transfected receptor was stimulated 1.6- and 2.3-fold when cells were cultured in the presence of 25 or 50 microM Zn2+, respectively. The increased expression of the GH receptor by Zn2+ was associated with an increased magnitude of GH-stimulated insulin biosynthesis. A close stoichiometric relationship between the level of receptor expression and the level of GH-stimulated insulin biosynthesis was observed. We conclude from these results that the hepatic GH receptor is able to mediate the effect of GH on insulin biosynthesis in RIN5-AH cells.

Animals↗