Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes (2021)
Figure 6

Figure 6. Diabetes in Rb∆K7 mice involves a DDR and senescence/SASP, and is attenuated by vitamin C diet

A. Induction of SASP pathways in Rb∆K7 islet cells. Pathway analysis by GSEA comparing Rb∆K7 and control islets using expression data for all genes. Nodes (circles) represent pathways significance at FDR <0.01 with normalized enrichment scores of >1.4; red indicates pathways enriched in up-regulated genes in Rb∆K7 and blue indicates pathways enriched in control for array 1 (node center) and array 2 (node border). Green line width between nodes corresponds to the number of shared genes. Murine SASP genes (yellow triangle) are connected to enriched-pathways (yellow node center) by gray lines; line thickness indicates significance level (proportional to log(p value), P<0.05). Red font corresponds to pathways in common with the g:Profiler analysis in panel (B).

B. g:Profiler analysis showing significantly enriched pathways (FDR<0.05) using 228 genes that are significantly regulated in Rb∆K7 vs. control islets (>2 fold; P<0.05; see panel (A) for details). In panels A and B, murine SASP genes (yellow triangle) are connected to enriched-pathways (yellow node center) by gray lines when the gene overlap size estimated by a Fisher's exact test is significant at P<0.05; line thickness is proportional to significance level. Red label font corresponds to pathways in common between the GSEA analysis (panel A) and the g:Profiler analysis (panel B).

C. Fold induction (log) of SASP genes in Rb∆K7 vs control littermates. Genes that overlap between the GSEA and G:profiler are highlighted in green. Yellow indicates genes that are transcriptionally regulated in epithelial SASP. Cxcl12, chemokine (C-X-C motif) ligand 12, has not been previously implicated in SASP in epithelial cells or fibroblasts, but is induced in Rb∆K7 islets. Log ratio is the difference between the mean of RbΔK7 samples and the mean of control samples of the log2 normalized values.

D. Representative image of a pancreatic islet from a 9-month old Rb∆K7 mouse immunestained for the SASP marker IL-6 (red) and insulin (green). DAPI was used to stain nuclei (blue). Arrows point to IL-6+, insulin+ pancreatic β-cells. Scale bar, 20 μm. See Fig. S6B for wildtype control, MMP-2 and CXCL12 staining.

E. Representative immunostaining for the DDR marker γ-H2A in islets from 3-week old Rb∆K7 and control mice (left); statistical analysis (right; n=5 each). Arrows point to pancreatic γ-H2A-positive Rb∆K7 islet cells. Error bars represent SD; P value calculated by one-way ANOVA, Tukey's multiple comparisons test. Scale bar, 100 μm.

F. A schematic outline for the vitamin C experiment in vivo. RbK7/+ heterozygous mice fed on regular or vitamin C diet were intercrossed. Progenies were genotyped to identify Rb+/+ and Rb∆K7 male mice, continuously maintained on the same diet, and monitored for blood glucose.

G. Vitamin C-supplemental diet (1%) significantly delays diabetes in Rb∆K7 mice. Shown are two independent experiments with 8 mice per group at indicated time points (left), and 28 and 34 Rb∆K7 mice or 37 and 20 control littermates in each group fed on regular or vitamin C diet, respectively (right). Blood glucose was determined in fasting mice at indicated months (left) or at ~9 months (right). Bars denote mean ±SD. Adjusted P values were calculated using one-way ANOVA, Tukey's multiple comparisons test.

H. Vitamin C diet increases 5hmC expression and improves β-cell morphology and islet size in Rb∆K7 mice. Scale bar, 100 μm.

I. Vitamin C diet reduces the difference in γ−H2A+ β-cells in Rb∆K7 vs control pancreatic islets relative to regular diet by 3.74 folds. Bars indicate mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test.

J. Vitamin C diet increases pancreatic islet cell cycle re-entry during pregnancy of Rb∆K7 mice relative to control pregnant mice (no statistical difference, n.s.) or relative to pregnant Rb∆K7 vs control mice fed on regular diet (P<0.0001) by 3.8 folds. Bars represent mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test.