Gene expression in 832/13 INS1 beta-cells transduced with lentiviral vector conferring expression of GFP or Pax5
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE211310 | GEO | 2023/01/11
REPOSITORIES: GEO
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Project description:Purpose: The goal of this study is to investigate how NIK regulates islet β-cell function. Methods: mRNA profiles of β-Gal-overexpressing, NIK-overexpressing, and NIK(KA)-overexpressing INS-1 832/13 cells were generated by deep sequencing using an Illumina Hiseq platform. Paired-end clean reads were aligned to the rat reference genome (ftp://ftp.ensembl.org/pub/release-90/fasta/rattus_norvegicus/dna/) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1). Conclusion: Our study represents the first detailed analysis of mRNA profiles in NIK-overexpressing and NIK(KA)-overexpressing INS-1 832/13 cells, generated by RNA-seq technology. Our results show that 471 genes were upregulated and 249 genes were downregulated following NIK overexpression. GO analysis indicated that the upregulated genes were primarily related to the immune response. KEGG pathway enrichment analysis showed that immune signaling pathways, including the TNF, NF-κB, antigen processing and presentation signaling pathways, were significantly activated by NIK overexpression in INS-1 832/13 cells, whereas genes related to insulin secretion and regulation of secretion were significantly decreased. NIK(KA), the dominant-negative mutant of NIK, does not induce the expression of genes related to immune response in INS-1 832/13 cells.
2020-07-28 | GSE130121 | GEO
Project description:Purpose: Test the role of ChREBPbeta in pancreatic beta cells using tools that distinguish ChREBPalpha and ChREBPbeta and loss and gain of function experiments, confirming its role in glucose toxicity and finding mitigation strategies. Methods: INS-1 832/13 cells cultured in 2 mM or 11 mM glucose were transduced with adenoviruses expressing GFP as control or ChREBPbeta. After 48 h, RNA was isolated and amplified via the NuGEN Ovation RNA-Seq System V2 prior to RNA sequencing. Human islets from non-diabetic and T2D subjects were transduced with adenovirus ZSGreen and cell sorted. RNA was isolated and subjected to RNAseq. Results: Overexpression of ChREBPbeta led to increased expression of cell cycle and apoptotic genes and a decrease in beta cell maturity genes. T2D human beta cells expressed an increased ratio of ChREBPbeta to ChREBPalpha compared to non-diabetics. Conclusions: ChREBPbeta is necessary for adaptive beta cell expansion, but drives glucose toxicity when overexpressed, mimicking the graded effect of Myc overexpression in beta cells.
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Project description:Pancreatic β-cells have the key homeostatic function of releasing insulin to keep blood glucose in the normal range. It is not fully understood how β-cell mass is determined. Ablation of a nuclear receptor, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), in mouse pancreatic β-cells results in glucose intolerance and 50% fewer β-cells during the postnatal period. By testing islets isolated from these mice and cultured β-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces β-catenin thus upregulating target genes like cyclin D1. Beta-cell expansion triggered by glucagon-like peptide 1 (GLP-1) is known to be mediated by β-catenin controlling cyclin D1. We show that in the absence of COUP-TFII, exendin-4, an agonist of the GLP-1 receptor, does not fully induce cyclin D1 expression, while overexpression of β-catenin induces cyclin D1. A marked decrease in cyclin D1 expression is detected in islets isolated from transgenic mice lacking both GLP-1 receptor and gastric inhibitory peptide receptor. COUP-TFII is therefore required for GLP-1 activation of the β-catenin-dependent pathway in pancreatic β-cells to increase β-cell number. To understand the molecular mechanisms by which COUP-TFII controls β-cell mass, we determined the RNA profile of COUP-TFII knockdown β-cells using Affymetrix oligonucleotide microarrays. Computational analysis identified clusters of genes shared by different gene regulatory Networks that are up or down regulated namely genes involved in Wnt/β-catenin signaling, insulin signaling and lipid/carbohydrate metabolism. We determined the RNA profile of COUP-TFII knockdown β-cells (832/13 INS-1 cells transfected with COUP-TFII specific siRNA) with respect to control cells (832/13 INS-1 cells transfected with scrambled siRNA) using Affymetrix expression analysis technical manual 701025Rev.5 (Affymetrix, Santa Clara, California, USA). Briefly, 1 mg of total cellular mRNA was reverse transcribed into cDNA (SuperScript Choice System Invitrogen, Carlsbad, CA) using oligo-dT primers and a T7 RNA polymerase promoter site. The cDNA was in vitro transcribed and biotin-labeled for microarray analysis using the Affymetrix IVT labeling kit. The concentration of labelled cRNA was measured using a NanoDrop ND-1000 spectrophotometer. Labeled cRNA was fragmented in a fragmentation buffer for 35 min at 94°C. The quality of labeled and fragmented cRNA was analyzed using the Agilent bioanalyzer 2100 (Van Lommel et al, 2006). Fragmented cRNA was hybridized to the rat 230 2.0 array (Affymetrix) during 16h at 45°C. Arrays were washed and stained in a fluidics station (Affymetrix) and scanned using the Affymetrix 3000 GeneScanner.
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Project description:The goal of this study was to identify genes which are differentiatlly expresesd upon induced inactivation of Rfx6 in beta cell in adult mice Rfx6fl/fl; Ins1-CreERT2 (mut) and Rfx6fl/fl (ctrl) 8 weeks old mice were injected subcutaneously with tamoxifen daily during 3 days. Pancreatic islets were isolated 5 days after the first injection and RNA purified.
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Project description:13 ATAC-Seq datasets of human pancreatic islets from 13 donors
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Project description:We find that GLP-1 signaling-activated PKA directly phosphorylates menin at serine 487 residue, relieving menin mediated suppression of insulin expression in beta cells and islets of diabetic GK rat. Mechanistically, GLP-1-induced PKA activation leads to menin Ser487 phosphorylation, and phosphorylated menin gains increased binding affinity to cytoskeleton proteins such as beta actin and myosin. Sequestration of Ser487 phosphorylated menin from the Ins1 gene promoter leads to reduced binding of menin-associating repressive epigenetic regulators such as SUV39H1 and HDAC1 at the locus, resulting in increased Ins1 transcription. Our results have linked GLP-1 signaling to physiologically suppression of menin function in repressing insulin expression, and uncovered a potential step to modulate menin phosphorylation to improve T2D therapy.
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Project description:Cocaine and amphetamine-regulated transcript (CART) is expressed in pancreatic islet cells and neuronal elements. We have previously established insulinotropic actions of CART in human and rodent islets. The receptor for CART in the pancreatic beta cells is unidentified. We used RNA sequencing of Cartpt knock down (KD) INS-1 832/13 cells and identified GPR162 as the most Cartpt-regulated receptor. We therefore tested if GPR162 mediates the effects of CART in beta cells. Binding of CART to GPR162 was established using proximity ligation assay, radioactive binding and co-immunoprecipitation, and KD of Gpr162 mRNA caused reduced binding. Gpr162 KD cells had blunted CARTp-induced exocytosis, and reduced CARTp-induced insulin secretion. Furthermore, we identified a hitherto undescribed GPR162-dependent role of CART as a regulator of cytoskeletal arrangement. Thus, our findings provide mechanistic insight into the effect of CART on insulin secretion and show that GPR162 is the CART receptor in beta cells.
2023-10-20 | GSE245796 | GEO
Project description:Cocaine and amphetamine-regulated transcript (CART) is expressed in pancreatic islet cells and neuronal elements. We have previously established insulinotropic actions of CART in human and rodent islets. The receptor for CART in the pancreatic beta cells is unidentified. We used RNA sequencing of Cartpt knock down (KD) INS-1 832/13 cells and identified GPR162 as the most Cartpt-regulated receptor. We therefore tested if GPR162 mediates the effects of CART in beta cells. Binding of CART to GPR162 was established using proximity ligation assay, radioactive binding and co-immunoprecipitation, and KD of Gpr162 mRNA caused reduced binding. Gpr162 KD cells had blunted CARTp-induced exocytosis, and reduced CARTp-induced insulin secretion. Furthermore, we identified a hitherto undescribed GPR162-dependent role of CART as a regulator of cytoskeletal arrangement. Thus, our findings provide mechanistic insight into the effect of CART on insulin secretion and show that GPR162 is the CART receptor in beta cells.
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Project description:PIEZO1 is a mechanosensitive ion channel involved in the regulation of a diverse range of physiological responses. We examined the role of the mechanosensor ion channel PIEZO1 in glucose-induced insulin secretion in pancreatic β-cells. PIEZO1 expression is elevated in β-cells from human donors with type-2 diabetes (T2D) and a rodent T2D model (db/db 48 mouse). Silencing of Piezo1 inhibits glucose-induced Ca2+ signaling and insulin secretion. PIEZO1 translocates from the cytosol and plasmalemma into the nucleus in cells cultured at high glucose, experimental conditions emulating diabetes. The translocation of PIEZO1 into the nucleus in response to hyperglycemia suggests that PIEZO1 might be involved in transcriptional control in addition to serving as a mechanosensor in the plasma membrane. To address this, we performed mRNA sequencing in INS-1 832/13 cells to determine which genes are regulated by Piezo channels. Overall, we found 3292, 1656, and 1920 genes were significantly differentially expressed after silencing Piezo1, Piezo2, or both genes (adj.p-value < 0.05). Among these, the expression of the gene encoding cocaine- and amphetamine-regulated transcript (Cartpt) was increased >15-fold. We confirmed this effect by qPCR, which indicated a corresponding stimulation of expression. In insulin-secreting INS-1 832/13 cells, Cart has been reported to influence the expression and release of insulin. Thus, the impact of PIEZO1 on β-cell function may not be limited to electrical excitability but these changes are likely to operate on a slower timescale than the acute/electrical effects.
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