Unknown,Transcriptomics,Genomics,Proteomics

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Glis3 regulates pancreatic beta cells development and maturation

ABSTRACT: Glis3 is expressed in pancreatic beta and PP cells. To identify down stream target genes of Glis3, we performed microarray analysis using pancreas total RNAs from 1 week-old WT and Glis3KO2 mice. insulin and pancreatic polypeptide (Ppy) was significantly decreased together with several other Î² cell markers, Glut2 and MafA by microarray analysis. Immunohistochemistry, QRT-PCR, and transmission electron microscopy indicated that postnatal Glis3KO2 pancreas still contains a large population of Î² cells that express Pdx-1, Nkx6.1, and Isl-1; however, insulin production and secretory granules were greatly reduced in these cells. In addition, chromogranin A (ChgA) and Urocortin 3, which are associated with mature Î² cells, was dramatically decreased in Glis3KO2 pancreas. These observations suggest that Glis3 plays a critical role in the maturation of pancreatic Î² cell phenotype. Pancreatic total RNAs were purified from 4 WT and 4 Glis3KO2 at 1 week old age. Then the samples were applied to Agilent mouse genome chip.

ORGANISM(S): Mus musculus

SUBMITTER: NIEHS Microarray Core

PROVIDER: E-GEOD-67238 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet Î²-cell development, but its effects on adult Î²-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-Î²GAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are Î² cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating Î²-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of Î²-cell mass in treatment of both major forms of diabetes. Keywords: Insulin secretion, islet biology, transcription factor, cell cycle regulation, diabetes We utilized a âsample x referenceâ experimental design strategy in which RNA extracted from rat pancreatic islets was hybridized to the microarray slide in the presence of labeled rat reference RNA (RRR, Stratagene, LaJolla, CA). Cultures were treated with adenoviruses expressing either the hamster form of Nkx6.1 or the beta-galactosidase enzyme. 5 biological replicates each representing independent islet isolations were used for microarray analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5. Labeled subject cRNA was co-hybridized to Operon rat 27K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled RRR. Slides were hybridized, washed, and scanned on a Gene Pix 5000 microarray scanner.

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Glis3 regulates pancreatic beta cells development and maturation

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