Δ40 Isoform of p53 controls β-cell proliferation and glucose homeostasis in mice

Abstract

Objective: Investigating the dynamics of pancreatic β-cell mass is critical for developing strategies to treat both type 1 and type 2 diabetes. p53, a key regulator of the cell cycle and apoptosis, has mostly been a focus of investigation as a tumor suppressor. Although p53 alternative transcripts can modulate p53 activity, their functions are not fully understood. We hypothesized that β-cell proliferation and glucose homeostasis were controlled by Δ40p53, a p53 isoform lacking the transactivation domain of the full-length protein that modulates total p53 activity and regulates organ size and life span in mice.

Research design and methods: We phenotyped metabolic parameters in Δ40p53 transgenic (p44tg) mice and used quantitative RT-PCR, Western blotting, and immunohistochemistry to examine β-cell proliferation.

Results: Transgenic mice with an ectopic p53 gene encoding Δ40p53 developed hypoinsulinemia and glucose intolerance by 3 months of age, which worsened in older mice and led to overt diabetes and premature death from ∼14 months of age. Consistent with a dramatic decrease in β-cell mass and reduced β-cell proliferation, lower expression of cyclin D2 and pancreatic duodenal homeobox-1, two key regulators of proliferation, was observed, whereas expression of the cell cycle inhibitor p21, a p53 target gene, was increased.

Conclusions: These data indicate a significant and novel role for Δ40p53 in β-cell proliferation with implications for the development of age-dependent diabetes.

FIG. 1.

p44tg mice exhibit hypoinsulinemia and hyperglycemia with age. Body weight (A), blood glucose (B), plasma insulin (C), and plasma glucagon (D) levels were measured at 3, 10, and 12–14 months of age from random-fed control (□) and p44tg (■) (n = 5–9) mice. *P < 0.05 for p44tg vs. control mice. #P < 0.05 for control mice vs. different ages.

FIG. 2.

p44tg mice exhibit age-dependent glucose intolerance and normal insulin sensitivity. Glucose tolerance tests performed at 2 months (A), 3–5 months (B), and 10–14 months (C) of age (n = 6–8). Blood glucose was measured at 0, 10, 20, 30, 60, and 120 min after intraperitoneal injection of glucose. Insulin tolerance tests were performed in each group at 2 months (D), 3–4 months (E), and 15–16 months (F) of age (n = 6–8). Glucose was measured at 0, 15, 30, 45, and 60 min after intraperitoneal injection of insulin. IP, intraperitoneal; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test. *P < 0.05 for p44tg vs. control mice.

FIG. 3.

Age-dependent alterations in β-cell size in p44tg mice. A: Pancreatic weight expressed as a percentage of the total body weight (n = 3). B: β-Cell mass was assessed as described in research design and methods. C and D: β-Cell size was assessed by coimmunostaining for β-catenin (green) and insulin (red) with DAPI (blue) in pancreas sections from control (n = 5) and p44tg (n = 5) mice at 3 and 12–14 months of age as described in research design and methods. A representative islet for each group at magnification 40× is presented with the quantification of relative β-cell size (mean ± SEM from n ≥200 cells counted per mouse). E: Real-time RT-PCR on RNA extracted from islets of 3- and 10- to 12-month-old control (n = 3–8) and p44tg (n = 4) mice. Results are normalized to TATA-binding protein (TBP) and expressed relative to control mice. *P < 0.05 for p44tg vs. control mice. #P < 0.05 for 3- vs. 12- to 14-month-old control mice. §P < 0.05 for 3- vs. 12- to 14-month-old p44tg mice. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Reduction in insulin-positive cells and increase in somatostatin-positive cells in islets from p44tg mice. A: Triple coimmunostaining for insulin (blue), somatostatin (green), and glucagon (red) on pancreas sections from control or p44tg mice at 3 and 12–14 months of age as described in research design and methods (n = 5). Two representative islets for each group at magnification 40× are presented. B: The number of insulin-positive cells was counted in at least 10 islets randomly selected from pancreas sections from each mouse (n = 3 mice at each age per group). C: Real-time RT-PCR on RNA extracted from islets of 3- and 10- to 12-month-old control (n = 3–8) and p44tg (n = 4) mice. Results are normalized to TBP and expressed relative to controls. D: In vivo GSIS. Insulin was measured in plasma samples extracted from blood collected at 0, 2, and 5 min after intraperitoneal injection of glucose (3 g/kg body wt) from 4–5-month-old control and p44tg mice. E: In vitro GSIS. Islets isolated from 4–5-month-old control and p44tg mice were incubated for 1 h with 3 or 16.7 mmol/L glucose as described in research design and methods. Bar graph depicts data obtained from three to five individual batches of size-matched islets from at least three independent mice per group. *P < 0.05 for p44tg vs. control mice. #P < 0.05 for 3- vs. 12- to 14-month-old control mice. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Reduced β-cell proliferation in p44tg mice. β-Cell proliferation was assessed by coimmunostaining for BrdU (A, green) and pHH3 (B, green) with insulin (red) and DAPI (blue) in pancreas sections from control and p44tg mice at 3 and 12–14 months of age as described in research design and methods. A representative islet for each group at magnification 40× is presented (n = 5). Quantification is shown on the right. *P < 0.05 for p44tg vs. control mice. #P < 0.05 for 3- vs. 12- to 14-month-old control mice. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Regulation of cyclin D2 expression in p44tg islets. A: Real time RT-PCR was performed on RNA extracted from islets of 3- and 10- to 12-month-old control (n = 3–8) and p44tg (n = 4) mice. Results are normalized to TBP and expressed relative to controls. *P < 0.05 for p44tg vs. control mice. B: Western blotting for cyclin D2 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age. *P < 0.05 for p44tg vs. control mice.

FIG. 7.

Regulation of PDX-1 expression in p44tg islets. A: Real-time RT-PCR was performed on RNA extracted from islets of 3- and 10- to 12-month-old control (n = 3–8) and p44tg (n = 4) mice. Results are normalized to TBP and expressed relative to control mice. *P < 0.05 for p44tg vs. control mice. B: Western blotting for FoxO1, PDX-1, and p27 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age. *P < 0.05 for p44tg vs. control mice.

FIG. 8.

Increased p21 expression in p44tg islets. A: Real-time RT-PCR was performed on RNA extracted from islets of 3- and 10- to 12-month-old control (n = 3–8) and p44tg (n = 4) mice. Results are normalized to TBP and expressed relative to control mice. *P < 0.05 for p44tg vs. control mice. B: Western blotting for p21 and quantification normalized to actin in islets from control and p44tg at 3 and 10 months of age. *P < 0.05 for p44tg vs. control mice. C: Quantification of p21-positive β-cell nuclei in pancreas sections from control and p44tg mice at 3 and 12–14 months of age (n = 5). *P < 0.05 for p44tg vs. control mice (at least 8–10 islets from five mice per group).