Fig. 6: A proportional increase in PDX1^LOW/MAFA^LOW β-cells impairs islet function.

a shPdx1 increases the proportion of β-cells in the islet with low levels of PDX1 and MAFA (β-cell immature; B-IMMAT) (scale bar = 60 µm). b Quantification of PDX1 and MAFA expression intensity shows an increase in β-cells occupying the bottom 15 percentile in B-IMMAT islets (n = 13–14 islets/3 animals; two-way ANOVA, Bonferroni’s multiple comparison) (PDX1: F = 2.38, DF = 20) (MAFA: F = 3.20, DF = 20). c RT-qPCR showing a decrease in Pdx1 expression levels in B-IMMAT islets (n = 5; paired t-test). d Induction of homogenous β-cell immaturity does not alter the α- to β-cell ratio (scale bar = 42.5 µm) (n = 18 islets/ 2–3 animals; unpaired t-test). e–g B-IMMAT islets display decreased insulin content (e), increased basal insulin release and absence of significant glucose-stimulated insulin secretion (f and g) (n = 10–12 replicates/4 animals; paired t-test and one-way ANOVA, Sidak’s multiple comparison) (G3, 3 mM glucose; G16.7, 16.7 mM glucose; Ex4, 20 nM Exendin-4). h–j Ca²⁺ traces (h) and bar graphs (i and j) showing impaired responses to glucose and glucose + KCl in B-IMMAT islets (n = 49–51 islets/4–5 animals; unpaired t-test) (representative images shown above bar graph, scale bar = 75 µm). k mRNA for the L-type VDCC subunits Cacnb2 and Cacna1d are significantly downregulated in B-IMMAT islets (n = 5–6; paired t-test). l Schematic showing the proposed changes in B-IMMAT islets. Color scale shows Ca²⁺ as min (0%) to max (100%) value. Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant. shPdx1- short hairpin RNA against Pdx1; VDCC-voltage-dependent Ca²⁺ channels.