Fig. 8: The balance of PDX1LOW:PDX1HIGH β-cells influences islet gene expression.
From: PDX1LOW MAFALOW β-cells contribute to islet function and insulin release
a Recombination of RIP7rtTA and TetO/M3C mice allows doxycycline-inducible changes in β-cell Neurog3, Pdx1 and Mafa expression in Tet-MAT but not Tet-NORM (control) islets. b Pdx1, Mafa and Neurog3 expression increases following incubation of Tet-MAT islets with 100 ng/ml doxycycline for 48 h (n = 3 animals; paired t-test). c, d A significant decrease in the number of PDX1LOW β-cells is seen in doxycycline-treated Tet-MAT islets, as shown by representative images (c), and shown also by the loss of cells in the lowest fluorescence intensity bins (d) (n = 6 islets/3 animals; two-way ANOVA, Bonferroni’s multiple comparison) (scale bar = 20 µm) (F = 41368, DF = 20). e–g Mean traces (e) and bar graphs (f and g) showing impaired glucose- and KCl-stimulated Ca2+ rises in Tet-MAT but not Tet-NORM islets (n = 33 islets/4 animals; unpaired t-test). h Volcano plot of differential gene expression between Tet-NORM and Tet-MAT islets. Fold-change (Log2, x-axis) gene expression is plotted against adjusted p-value for differential gene expression (normalized by GLM, -Log10, y-axis). Colored dots represent Ensembl genes that are differentially regulated at an adjusted p-value < 0.05 (n = 5 animals). i Gene ontology analysis of differentially regulated genes in Tet-MAT islets. A set of 83 genes were functionally annotated using DAVID (adjusted p-value of < 0.05). j Gene set enrichment analysis (GSEA) suggests that genes belonging to the gene set “hallmark β-cells” are upregulated in Tet-MAT islets. Normalized enrichment score (NES) and nominal p-value is presented in the top right corner of the graph. k GSEA analysis shows enrichment of genes belonging to glucose and carbohydrate derivative metabolic processes amongst the upregulated genes in Tet-MAT islets. l RT-qPCR analyses confirming upregulation of Ucn3, G6pc2, Cox6a2 and Rgs4 but not Pkib in Tet-MAT islets (n = 3 animals; paired t-test). Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant.
