Glucose and aging control the quiescence period that follows pancreatic beta cell replication

Abstract

Pancreatic beta cell proliferation has emerged as the principal mechanism for homeostatic maintenance of beta cell mass during adult life. This underscores the importance of understanding the mechanisms of beta cell replication and suggests novel approaches for regenerative therapy to treat diabetes. Here we use an in vivo pulse-chase labeling assay to investigate the replication dynamics of adult mouse beta cells. We find that replicated beta cells are able to re-enter the cell division cycle shortly after mitosis and regain their normal proliferative potential after a short quiescence period of several days. This quiescence period is lengthened with advanced age, but shortened during injury-driven beta cell regeneration and following treatment with a pharmacological activator of glucokinase, providing strong evidence that metabolic demand is a key determinant of cell cycle re-entry. Lastly, we show that cyclin D2, a crucial factor in beta cell replication, is downregulated during cell division, and is slowly upregulated post-mitosis by a glucose-sensitive mechanism. These results demonstrate that beta cells quickly regain their capacity to re-enter the cell cycle post-mitosis and implicate glucose control of cyclin D2 expression in the regulation of this process.

Fig. 1.

An assay for measuring the beta cell post-replication quiescence period. (A) Outline of the pulse-chase experiment for analyzing the return of beta cells to the cell cycle. BrdU was injected a total of six times (three times every day for 2 days) and mice were sacrificed at the indicated chase periods. (B) A computer-generated model shows possible beta cell behaviors after replication. Green shading represents the normal population, in which beta cell replication declines with age. Black line represents a similar behavior of post-replicating beta cells and hence no quiescence/refractory period. Red line represents a short refractory period and the blue line represents a long refractory period, as proposed by Teta et al. (Teta et al., 2007). (C) In our assay, the percentage of BrdU⁺ Ki67⁺ out of the total BrdU⁺ population (the division rate of replicated cells) represents the likelihood of a replicated cell to divide again. When the replicated cell division rate immediately equals the normal rate (black, represented at 100%) there is no refractory period, when it recovers quickly there is a short refractory period (red), and when there is a long lag there is a long refractory period (blue). (D) After mitosis, dividing cells retain BrdU but lose expression of Ki67. Beta cells were pulsed with a single injection of BrdU. At 4 hours all BrdU⁺ beta cells expressed Ki67, but by 48 hours only 1% co-expressed BrdU and Ki67. (E) The percentage of BrdU⁺ cells in the total population approximately doubles between 4 and 48 hours, consistent with productive cytokinesis of cells that incorporated BrdU. (F) Representative images of beta cells 4 and 48 hours after injection of BrdU, stained for insulin (green), BrdU (red) and Ki67 (blue). The boxed regions indicate BrdU⁺ cells and their Ki67 expression **, P<0.01; NS, not significant.

Fig. 2.

Beta cells can re-enter the cell cycle shortly after mitosis and proliferate normally after 1 week. (A) Beta cells were pulsed with six injections of BrdU and the percentage of BrdU⁺ beta cells was analyzed at each time point. (B) The percentage of Ki67⁺ beta cells in the general population (blue bars) and in the BrdU⁺ pulse-labeled population (red bars). Whereas at 2 and 5 days there was a significant difference in these two populations, by 7 days and onwards there was no difference. (C) The percentage BrdU⁺ Ki67⁺/BrdU⁺ population was divided by the percentage Ki67⁺ at each time point. Whereas on day 2 a replicated cell was only 15% as likely to replicate as the general population, by day 7 it was 70% as likely to replicate, and by day 12 it was equally as likely. (D) Representative images demonstrating beta cells pulse chased with BrdU and stained for insulin (green), BrdU (red) and Ki67 (blue) at 0, 2 and 7 days. Red boxes indicate Ki67⁺ BrdU⁺ cells; blue boxes point to BrdU⁺ cells that are Ki67⁻ (i.e. that were quiescent at the time of sacrifice). The boxed regions indicate BrdU⁺ cells and their Ki67 expression. *, P<0.05; **, P<0.01; NS, not significant.

Fig. 3.

Clonal analysis using RipCreER-MADM mice supports multiple divisions of the same beta cell and a short post-replication quiescence period. (A) Outline of the pulse-chase labeling experiment. Mice were activated with Tamoxifen between 4 and 8 weeks and chased for 1 and 2 months. (B) A model of short- and long-term quiescence periods. Short quiescence periods should quickly yield numerous labeled cells. (C) Distribution of clonal sizes at 1 and 2 months. At both time points there were large clones (more than 10 cells), strongly suggesting that beta cells can undergo several sequential divisions.

Fig. 4.

Older beta cells have a longer quiescence period. (A) Three-month-old mice were pulsed with six injections of BrdU and analyzed at the same time points as 1-month-old mice. (B) The percentage of Ki67⁺ beta cells in the general population (blue bars) and in the BrdU⁺ pulse-labeled population (red bars). In contrast to 1-month-old mice, in which at 2 and 5 days there was a significant difference between these two populations but after 7 days there was not, in 3-month-old mice there was a significant difference between the normal population and replicated population at all time points. (C) The percentage BrdU⁺ Ki67⁺/BrdU⁺ population was divided by the percentage Ki67⁺ at each time point for 1- and 3-month-old mice. Whereas replicated cells in young mice were 70% as likely to replicate on day 7 and equally as likely by day 12, replicated beta cells in older mice were 25% as likely to replicate on day 7 and 40% as likely to replicate on day 12. *, P<0.05; NS, not significant.

Fig. 5.

Beta cell regeneration and glucose stimulation shorten the beta cell quiescence period. (A) The percentage of beta cells expressing Ki67 in the general population was compared with the percentage of Ki67⁺ beta cells among the BrdU⁺ population at each time point. In beta cells of Insulin-rtTA; TET-DTA mice, there was no evidence for a quiescence period at 2 or 5 days after division. (B)The percentage BrdU⁺ Ki67⁺/BrdU⁺ population was divided by the percentage Ki67⁺ at both time points, demonstrating that in regenerating mice a replicated beta cell regains its normal proliferation rate already after 2 days. (C) Representative images from islets of Insulin-rtTA; TET-DTA mice pulsed with BrdU and chased for 2 days. Insulin (green), BrdU (red), Ki67 (blue). The boxed regions indicate BrdU⁺ cells and their Ki67 expression. (D) Glucokinase activator (GKA) transiently reduces blood glucose levels due to enhanced glycolysis in beta cells and increases insulin secretion. (E) The percentage of Ki67⁺ beta cells among the BrdU⁺ population increases after GKA injection. (F) GKA injection restores the ability to re-enter the cell cycle in beta cells that completed mitosis. **, P<0.01; NS, not significant.

Fig. 6.

Cyclin D2 is downregulated during the S/G2 phases and slowly returns post-mitosis via a glucose-regulated pathway. (A) Pancreas sections stained for insulin (green), cyclin D2 (red) and BrdU (blue) after a 2-hour pulse with BrdU. The boxed regions indicate BrdU⁺ cells and their cyclin D2 expression. (B) Beta cells were pulsed with a single injection of BrdU and the percentage of cyclin D2⁺ cells among BrdU⁺ beta cells was measured at each time point. (C) Replicated (BrdU⁺) beta cells in Insulin-rtTA; TET-DTA mice and in GKA-treated mice regain cyclin D2 expression faster than BrdU⁺ beta cells in control mice. (D) Replicated (BrdU⁺) beta cells in 1- and 3-month-old mice have similar dynamics of cyclin D2 expression. **, P<0.01; NS, not significant.