Figure 1.
STZ-mediated β-cell destruction.
Adult C57BL/6 mice were treated with STZ at a dose of 130 mg/kg body weight via i.p. injection, monitored for the development of hyperglycemia, and sacrificed at multiple time points post-STZ. Shown here are representative data. A: Anti-Insulin staining of the mouse pancreases showing β-cell loss following STZ treatment. B: Quantitative distribution of β-cells in each islet section. The number of β-cells in each islet was counted and plotted. Each circle represents one islet. For each group (normal, and 2, 4, 6, 8, and 10 days post-STZ), 26–33 representative islets from 4–5 mice were counted. The red line marks the mean value for each group. C: the statistical summary of β-cell distribution in islets from normal mice and STZ-treated mice. D: Blood glucose levels of the mice at different day post-STZ. Note: the blood glucose levels during the first 2 days were not included because STZ-induced hypoglycemia at early hours requiring a glucose injection to overcome hypoglycemia (see methods). E: Serum insulin concentrations in normal mice and those treated with STZ.
Table 1.
Composition of α-, β- and δ-cells in islets from normal and STZ-treated mice.
Figure 2.
α-cell expansion following STZ-mediated β-cell destruction.
A: Immunofluorescence staining showing α-cell expansion following STZ treatment. The pancreatic islets from normal and STZ-treated mice were co-stained with anti-Insulin (green) and anti-glucagon (red) antibodies. Hoechst 33342 was used to label nuclei (blue). B: Glucagon levels in the serum of normal and STZ-treated mice. C: Quantitative distribution of α-cells in each islet section. The number of α-cells in each islet was counted and plotted in the same way as described in Fig. 1B. 22–34 representative islets from 4–5 mice were counted. The red line marks the mean value for each group. D: the statistical summary of α-cell distribution in islets from normal mice and STZ-treated mice. *** indicates p<0.001; ns: not significant.
Figure 3.
The distribution of α-cells and δ-cells following STZ treatment.
The pancreatic islets from normal and STZ-treated mice were co-stained with anti-glucagon (green) and anti-somatostatin (red) antibodies. A: representative images showing δ-cells expanded in a similar pattern to α-cells following STZ treatment. B: Quantitative distribution of δ-cells in each islet group. The quantification of δ-cells was obtained the same way as described for α-cells and β-cells in Figures 1 and 2. The redlines represent the mean values for each islet group. C: the statistical summary of δ-cell distribution in islets from normal mice and STZ-treated mice. The mark ** indicates p<0.005, and *** indicates p<0.0005; ns: not significant.
Figure 4.
Islet architectural changes following STZ-mediated β-cell loss and non-β cell regeneration.
A: Distribution of α-, β- and δ-cells as assessed by triple staining with anti-insulin (blue), anti-glucagon (green) and anti-somatostatin (red) antibodies. The three cell types gradually lost their normal mantle-to-core zone organization, and became intermingled throughout the islets. B: Distribution of α-, β- and PP cells as assessed by anti-insulin (blue), anti-glucagon (green)) and anti-PPP (red) antibodies. Note: most regenerated PPP+ cells appeared to co-express glucagon (white arrows) following STZ treatment. This did not seem to be attributable to antibody cross-reaction because there were many individually stained α-cells and PP-cells (pink arrows).
Figure 5.
BrdU incorporation demonstrating that cell proliferation was involved in non-β-cell regeneration.
Following STZ injection, 1 mg/ml of BrdU was added into the mice's drinking water. Normal mice were included as the control. At different day post-STZ, the pancreases were processed for immunofluorescence staining with anti-BrdU (red, A and C) and anti-glucagon (green, A) or anti-somatostatin (green, C). The arrows mark BrdU+ α-cells (A) and BrdU+ δ-cells (C) in the representative islets. The percentage of BrdU+ α-cells (B) and BrdU+ δ-cells (D) were calculated by dividing the number of these cells by total α- and δ-cells in each islet, respectively. Unpaired t-tests were performed to compare whether the percentage at each time point following STZ treatment was significantly different from that in normal mice. *: p<0.05, **: p<0.005, ***: p<0.0005, #: p>0.05 (not significant).
Figure 6.
Ki67 staining highlighted proliferating α-cells and δ-cells.
A and C: double staining of the islets with the proliferating cell marker Ki67 (red) with anti-glucagon (green, A) or with anti-somatostatin (green, C). The white arrows mark the proliferating α-cells and δ-cells, respectively. B and D: the percentage of Ki67+ α-cells/total α-cells (B) and Ki67+ δ-cells/total δ-cells (D) in each islet was quantified. Unpaired t-tests were performed to compare whether the data at each time point post-STZ was significantly different from that in normal mice. *: p<0.05, **: p<0.005, #: p>0.05 (not significant).
Figure 7.
Pdx1 expression in STZ-treated mouse islets.
The pancreatic islets from normal and STZ-treated mice were co-stained with anti-Pdx1 (red in A, B, and C) and islet cell markers including anti-insulin (A, green), anti-glucagon (B, green) and anti-somatostatin (C, green). In the normal islets, Pdx1 co-localized with insulin-expressing β-cells. Following STZ-mediated β-cell destruction, Pdx1 expression diminished, but became detectable in more and more non-β cells (A, arrows). Co-staining of Pdx1 with glucagon or somatostatin antibodies showed presence of Pdx1+/glucagon+ (B, arrows) and Pdx1+/somatostatin+ (C, arrows) cells following STZ-treatment, suggesting these cells are multipotent progenitor cells in transition to α- and δ-cells respectively.