Systemic regulation of the age-related decline of pancreatic β-cell replication

Abstract

The frequency of pancreatic β-cell replication declines dramatically with age, potentially contributing to the increased risk of type 2 diabetes in old age. Previous studies have shown the involvement of cell-autonomous factors in this phenomenon, particularly the decline of polycomb genes and accumulation of p16/INK4A. Here, we demonstrate that a systemic factor found in the circulation of young mice is able to increase the proliferation rate of old pancreatic β-cells. Old mice parabiosed to young mice have increased β-cell replication compared with unjoined old mice or old mice parabiosed to old mice. In addition, we demonstrate that old β-cells transplanted into young recipients have increased replication rate compared with cells transplanted into old recipients; conversely, young β-cells transplanted into old mice decrease their replication rate compared with young cells transplanted into young recipients. The expression of p16/INK4A mRNA did not change in heterochronic parabiosis, suggesting the involvement of other pathways. We conclude that systemic factors contribute to the replicative decline of old pancreatic β-cells.

FIG. 1.

Heterochronic parabiosis model. A: One parabiosed partner was injected with BrdU and the joined pair was killed after 3 h. Both the injected mouse and attached mouse were stained for DNA (green) and BrdU (red). B: Parabiosed mice demonstrated a significant decline in body weight (*P < 0.05 for Student t test and ANOVA) in all three groups, after which their weight stabilized. C: Fed blood glucose levels of connected mice (all three groups) decreased significantly after 1 week (*P < 0.05 ANOVA) and remained lower throughout the experiment (N = 5). Het, heterochronic; Iso, isochronic.

FIG. 2.

Heterochronic parabiosis increased β-cell replication of old mice. A: Parabiosis was performed in young mice and old mice to create isochronic and heterochronic groups. After 3 weeks, mice were killed and proliferation was measured using Ki67. Student t test demonstrated a significant increase in replication between old isochronic mice and old heterochronic mice (**P < 0.01, whereas ANOVA analysis of the experiment yielded P < 0.001). Post hoc Tukey honestly significant difference test also showed a significant difference between old isochronic mice and heterochronic mice (*P < 0.005). B: Representative images of the islets from four different groups of parabiosed mice stained for insulin (green), DNA (red), and Ki67 (blue) (N ≥ 4). Het, heterochronic; Iso, isochronic; NS, not significant; Yhet, young heterochronic; Yiso, young isochronic.

FIG. 3.

Heterochronic islet transplants increase proliferation of old β-cells and decrease proliferation of young β-cells. A: Islet grafts were placed under the kidney capsule of young and old mice to create isochronic and heterochronic transplant groups. After 2 weeks, mice were killed and proliferation was measured using Ki67. Heterochronic transplant succeeded in increasing β-cell proliferation of old islets and in decreasing proliferation of young islets using Student t test (*P < 0.05) and ANOVA (P < 0.01). Post hoc Tukey honestly significant difference test also showed a significant difference in both heterochronic transplant groups (P < 0.007; N = 4). B: A representative image of β-cells transplanted under the kidney capsule from four different groups of transplanted mice stained for insulin (green), Nkx6.1 (red), and Ki67 (blue). Ins, insulin; NS, not significant; O, old; Y, young.

FIG. 4.

Serum factors and gene expression in heterochronic parabiosis. A: Fasting insulin was analyzed in young, old, old isochronic, and old heterochronic mice. There was no significant difference between the groups (using both t test and ANOVA). Although IGF-1 levels were significantly higher in old mice (*P < 0.05; t test), no difference was detected between old isochronic mice and old heterochronic mice (no significant difference was found in ANOVA; N = 4). B: Expression of selected cell-cycle genes in islets of young, old, and parabiosed mice. All genes were normalized to the young mouse group except p21 and p16, which were normalized to the old mouse group. Parabiosis to a young partner did not change the expression of any gene in old mice (N = 3). Het, heterochronic; Iso, isochronic; NS, not significant.