Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes (2021)
Figure 5

Figure 5. Diabetes in Rb∆K7 mice is associated with failure of pancreatic β-cells to re-enter the cell cycle

A. Insulin Tolerance Test (ITT) demonstrating that hyperglycemic Rb∆K7 mice are insulin-sensitive. Blood glucose in fasting Rb∆K7 mice (n=6) vs. control littermates (n=6) following insulin injection. Error bars represent SD.

B. Glucose tolerance Test (GTT) demonstrating progressive defect in clearance of blood glucose in Rb∆K7 mice. 6-7 week (left) or 6 month (right) old fasting Rb∆K7 mice and control littermates (n=5-6 each per group) were injected with glucose, and blood glucose was determined at indicated intervals. Error bars represent SD.

C. Glucose-stimulated insulin secretion (GSIS) analysis demonstrating near-normal insulin secretion in response to glucose injection in young Rb∆K7 mice. Serum insulin levels in 5-6-week old fasting Rb∆K7 vs. control mice (n=4 each) after glucose injection determined by ELISA. P values were calculated by two-tailed unpaired student's t-test. Bars represent mean ±SD.

D. Rb∆K7 mice exhibit a progressive loss of pancreatic islet mass. Left to right, representative images of pancreatic islets of 1-year old Rb∆K7 vs. control mice stained for insulin (green). Note the enlarged, irregular shape and reduced number of pancreatic insulin+ β−cells in Rb∆K7 islets; Ratio of β−cell area to pancreatic area in 1 month and 1 year-old Rb∆K7 vs. control mice determined by immunostaining for insulin followed by image J analysis; relative length of pancreatic insulin+ β−cells in 9-month old Rb∆K7 islets (n=5) vs control (n=4) using >8 determination per biological replicate, normalized for control length. P values calculated by two-tailed unpaired student's t-test. Scale bar, 100 μm.

E. Reduced levels of fasting circulating insulin in 6-10 month old diabetic Rb∆K7 mice compared with control wild type littermates (0.13 vs 0.2 ng/ml; n=8 for each group). P value was calculated by one-tailed unpaired student's t-test. Bars represent mean ±SE. Average levels of blood glucose are indicated (17 mM in Rb∆K7 compared with 3.2 in control mice; P=0.0001 by two-tailed unpaired student's t-test).

F. Loss of pancreatic islet area in Rb∆K7 mice correlates with reduced proliferation and H3K36me3 histone marks, but not with increased cell death. TUNEL analysis showing apoptosis in 10 day-old Rb∆K7 islets is even lower than in control littermates. Arrow points to a rare apoptotic cell in control islet. Serine800/804 phosphorylation of pRb in β-cells from 10 day-old control but not Rb∆K7 islets with a Ser-to-Ala substitution at this site. BrdU incorporation and PH3 staining showing reduced S-phase and mitotic cells, respectively, in 2- and 3-week old Rb∆K7 vs control islets. Reduced H3K36me3 staining in pancreatic islets from 3-week old Rb∆K7 mice compared to control littermates (n>3 for each experiment). Scale bar, 100 μm.

G. Quantification of BrdU incorporation, PH3 and H3K36me3 staining shown in panel E. Mean ±SD, P values calculated by two-tailed unpaired student's t-test

H. Reduced ability of Rb∆K7 β-cell to re-enter the cell cycle in response to pregnancy (top) or mitogenic (bottom) signals. Top, cell proliferation in P14.5 pregnant Rb∆K7 islet β-cell vs. control was determined by double immunostaining with BrdU and insulin (left); quantification (right). Results were normalized for the number of BrdU/Insulin positive cells in control islets. An independent experiment using ki67 to quantify cell cycle re-entry is shown in Fig. S6A, top. Bottom, cell proliferation in Rb∆K7 β-cell vs. control islets following Exendin4 treatment was determined by immunostaining for Ki67; results were normalized for the number of positive cells in control islets. Mean ±SD, P values by two-tailed unpaired student's t-test. Scale bar, 100 μm.

I. Representative immunostaining showing induction of PDX1 (islet), SOX9 (pancreatic epithelium) and MCM4 (islet) expression in Rb∆K7 vs control mice. See also Fig. S5. Scale bar, 100 μm.