Activation of FoxM1 Revitalizes the Replicative Potential of Aged β-Cells in Male Mice and Enhances Insulin Secretion

Abstract

Type 2 diabetes incidence increases with age, while β-cell replication declines. The transcription factor FoxM1 is required for β-cell replication in various situations, and its expression declines with age. We hypothesized that increased FoxM1 activity in aged β-cells would rejuvenate proliferation. Induction of an activated form of FoxM1 was sufficient to increase β-cell mass and proliferation in 12-month-old male mice after just 2 weeks. Unexpectedly, at 2 months of age, induction of activated FoxM1 in male mice improved glucose homeostasis with unchanged β-cell mass. Cells expressing activated FoxM1 demonstrated enhanced glucose-stimulated Ca2+ influx, which resulted in improved glucose tolerance through enhanced β-cell function. Conversely, our laboratory has previously demonstrated that mice lacking FoxM1 in the pancreas display glucose intolerance or diabetes with only a 60% reduction in β-cell mass, suggesting that the loss of FoxM1 is detrimental to β-cell function. Ex vivo insulin secretion was therefore examined in size-matched islets from young mice lacking FoxM1 in β-cells. Foxm1-deficient islets indeed displayed reduced insulin secretion. Our studies reveal that activated FoxM1 increases β-cell replication while simultaneously enhancing insulin secretion and improving glucose homeostasis, making FoxM1 an attractive therapeutic target for diabetes.

Figure 1

Quantitative real-time PCR reveals that expression of Foxm1 and its direct targets declines with age. RU, relative units. *P < 0.05 vs. 2-month islets; **P < 0.01 vs. 2-month islets.

Figure 2

β-Cell mass in young mice does not increase in response to increased FoxM1 activity. Insulin labeling and eosin (EOS) staining on P8 RIP-rtTA (A) and β-FoxM1* (B) pancreas sections. C: Quantification of P8 β-cell mass. D: Ad libitum–fed glucose in P8 mice. Insulin labeling and eosin staining on 2-month old RIP-rtTA (E) and β-FoxM1* (F) pancreas sections. Quantification of β-cell mass (G) and proliferation (H) in 2-month-old mice.

Figure 3

β-Cell mass and proliferation increase in aged mice expressing activated FoxM1. A: β-Cell mass in aging mice. B: β-Cell proliferation in 12-month-old mice. C: Quantification of Ki67 in activated FoxM1-expressing (HA⁺) or activated FoxM1-absent (HA⁻) β-cells at 12 months of age. D–G: Representative images for data quantified in A and B. Insulin (Ins) labeling and eosin (EOS) staining (D and E) and insulin and Ki67 labeling (F and G) on 12-month-old RIP-rtTA (D and F) and β-FoxM1* (E and G) pancreas sections. The arrows indicate positive cells. Images in F and G were acquired at original magnification ×200. Inset in G shows a higher-resolution Ki67⁺ β-cell. H: β-Cell size in 12-month-old mice. I: Proliferation in size-sorted islets. J and K: Representative image of data quantified in C with all three immunolabels (J) or with only insulin and HA immunolabels (K). *P < 0.05.

Figure 4

FoxM1 decreases γ-H2AX with no change in p16. A: Insulin (Ins), HA, and p16 labeling on 12-month-old and β-FoxM1* pancreas sections. B: Single-channel labeling of p16 from image in A. The black arrows indicate β-cells positive for p16 and activated FoxM1. Images were acquired at original magnification ×200. Insulin and γ-H2AX labeling on RIP-rtTA (C) and β-FoxM1* (D) pancreas sections. The white arrow indicates a cell positive for insulin and γ-H2AX. E: Quantification of γ-H2AX⁺ β-cells. Scale bar: 20 µm. *P < 0.05.

Figure 5

FoxM1 regulates β-cell function. A and B: IP-GTT on 2-month-old RIP-rtTA and β-FoxM1* mice before and after 2 weeks of Dox administration (n = 7–10). *P < 0.05. C: Ad libitum–fed glucose in 2-month-old mice. D: Insulin tolerance test in 2-month-old RIP-rtTA and β-FoxM1* mice after 2 weeks of Dox treatment (n = 4–6). E and F: IP-GTT on 12-month-old RIP-rtTA and β-FoxM1* mice before and after 2 weeks of Dox administration (n = 6–7). AUC, area under the curve. *P < 0.05, **P < 0.01. G: Ad libitum–fed glucose in 12-month-old mice. H: Perifusion of isolated islets from 9-week-old RIP-Cre, Foxm1^β+/−, and Foxm1^Δβ mice. G5.6, glucose concentration of 5.6 mmol/L; G16.7, glucose concentration of 16.7 mmol/L; IEQ, islet equivalents. *P < 0.05 and **P < 0.01 for Foxm1^Δβ mice vs. RIP-Cre mice; †P < 0.5 for Foxm1^β+/− mice vs. RIP-Cre mice; ‡P < 0.05 for Foxm1^Δβ mice vs. Foxm1^β+/− mice.

Figure 6

FoxM1 acts cell autonomously to enhance calcium influx upstream of insulin secretion. A: Average calcium measurements from β-FoxM1*¹⁴ β-cells from mice expressing or lacking activated FoxM1. Dispersed β-cells were maintained in 2 mmol/L glucose until 180 s, when they were treated with 14 mmol/L glucose. B: Representative calcium traces from individual cells. C: Area under the curve (AUC) for HA⁺ and HA⁻ cells for indicated time period (n = 2 mice, 24–98 cells). *P < 0.0005.

Figure 7

FoxM1 is more highly expressed in proliferating β-cells. FoxM1 immunohistochemistry on E15.5 WT (A and C) and Foxm1-null (B and D) hearts (A and B) and pancreas (C and D). E–H: FoxM1 immunohistochemistry on Lep^ob/ob pancreas sections. The lines denote boundaries between islet and exocrine tissue. E: The arrowheads indicate some FoxM1^LO islet cells; the arrow indicates a FoxM1^HI islet cell (original magnification ×200). F: The arrows indicate FoxM1^HI cells (original magnification ×100). FoxM1 and Ki67 double immunohistochemistry on Lep^ob/ob pancreas sections showing proliferating cells in an islet (G) and in acinar tissue (H) (original magnification ×200). The arrows indicate double-positive cells. I: Model depicts FoxM1 regulation of optimal β-cell function and proliferation.