Diminished pancreatic beta-cell mass in securin-null mice is caused by beta-cell apoptosis and senescence

Abstract

Pituitary tumor transforming gene (PTTG) encodes a securin protein critical in regulating chromosome separation. PTTG-null (PTTG(-/-)) mice exhibit pancreatic beta-cell hypoplasia and insulinopenic diabetes. We tested whether PTTG deletion causes beta-cell senescence, resulting in diminished beta-cell mass. We examined beta-cell mass, proliferation, apoptosis, neogenesis, cell size, and senescence in PTTG(-/-) and WT mice from embryo to young adulthood before diabetes is evident. The roles of cyclin-dependent kinase inhibitors and DNA damage in the pathogenesis of diabetes in PTTG(-/-) mice were also addressed. Relative beta-cell mass in PTTG(-/-) mice began to decrease at 2-3 wk, whereas beta-cell proliferation rate was initially normal but decreased in PTTG(-/-) mice beginning at 2 months. Apoptosis was also much more evident in PTTG(-/-) mice. At 1 month, beta-cell neogenesis was robust in wild-type mice but was absent in PTTG(-/-) mice. In addition, the size of beta-cells became larger and macronuclei were prominent in PTTG(-/-) animals. Senescence-associated beta-galactosidase was also active in PTTG(-/-) beta-cells at 1 month. Cyclin-dependent kinase inhibitor p21 was progressively up-regulated in PTTG(-/-) islets, and p21 deletion partially rescued PTTG(-/-) mice from development of diabetes. mRNA array showed that DNA damage-associated genes were activated in PTTG(-/-) islets. We conclude that beta-cell apoptosis and senescence contribute to the diminished beta-cell mass in PTTG(-/-) mice, likely secondary to DNA damage. Our results also suggest that ductal progenitor beta-cells are exhausted by excessive neogenesis induced by apoptosis in PTTG(-/-) mice.

Figure 1

β-Cell mass, proliferation, apoptosis, neogenesis, and size. Pancreas from PTTG^−/− mice and their WT littermates (three to six animals per group) from birth to 2 months were fixed and embedded in paraffin and β-cell mass was measured by morphometry, β-cell proliferation by PCNA staining, β-cell apoptosis by TUNEL staining, β-cell neogenesis by percentage of ducts or ductules that have insulin-positive cells in their epithelia, and β-cell size and nucleus size by morphometry. *, P < 0.05; **, P < 0.01.

Figure 2

p21 up-regulation in PTTG^−/− mice. Paraffin sections of pancreas from WT or PTTG^−/− mouse embryo at embryonic d 17 (ED17), newborn (NB), and 1- to 8-wk-old mice were stained with guinea pig antiinsulin (Ins; red) or rabbit antiglucagon (red) and mouse anti-p21 (p21; green), followed with rhodamine-labeled antiguinea IgG or goat antirabbit IgG and FITC-labeled antimouse IgG. Nuclei were counterstained with Hoechst 33342 (blue). Arrow, β-Cells with nuclei that are positive for p21. Please note that red blood cells have autofluorescence but no nuclei.

Figure 3

Cell cycle regulatory protein expression in islets. Left panel, Western blot showing protein levels of p21, phosphorylated Cdk2 (pCdk2), phosphorylated Rb (pRb), and PCNA in WT and PTTG^−/− pancreatic islet cells derived from 3-month-old male mice. Right panel, DNA damage pathway protein expression in islets. Western blot showing protein levels of Rad17, Rad18, Rad51, MSH1, and MutL homolog 1 (MLH1) in WT and PTTG^−/− pancreatic islet cells derived from 3-month-old male mice.

Figure 4

Effects of p21 deletion on diabetes development in PTTG^−/− mice. PTTG^−/−p21^+/+ and PTTG^−/−p21^−/− male mice were tested for fasting blood glucose levels monthly between 5 and 9 months. Graph depicts percentage of animals that developed hyperglycemia (>200 mg/dl).

Figure 5

SA-β-gal activity in islets. Pancreatic cryosections derived from 1-month PTTG^−/− and WT mice stained with X-gal. The blue color reflects SA-β-gal activity.