DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 3205
Development 137, 3205-3213 (2010) doi:10.1242/dev.054304
© 2010. Published by The Company of Biologists Ltd
Glucose and aging control the quiescence period that
follows pancreatic beta cell replication
Seth J. Salpeter1, Allon M. Klein2,3, Danwei Huangfu4,*, Joseph Grimsby5 and Yuval Dor1,†
SUMMARY
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.
INTRODUCTION
The maintenance of adult tissue mass can be controlled by the
differentiation of adult stem cells or by the replication of
differentiated cells in the tissue. In the case of pancreatic beta cells,
recent studies have shown that tissue homeostasis relies on the
replication of differentiated, insulin-expressing beta cells, rather
than stem cells (Brennand et al., 2007; Dor et al., 2004; Georgia
and Bhushan, 2004; Meier et al., 2008; Nir et al., 2007; Teta et al.,
2007). Furthermore, it has been shown that the rate of beta cell
proliferation responds to certain physiological conditions, such as
pregnancy and the destruction of most beta cells (Cano et al., 2008;
Gupta et al., 2007; Nir et al., 2007; Parsons et al., 1992). Despite
these findings, it remains unclear what factors govern adult beta
cell proliferation and to what extent beta cell mass can be expanded
in vitro and in vivo.
Understanding the processes that regulate beta cell replication
might have potential therapeutic value for type 1 and type 2
diabetes, diseases that are characterized by insufficient beta cell
mass (Butler et al., 2007). Although transplantation of cadaveric
human islets can normalize blood glucose levels in type 1 diabetes
patients, the scarcity of donors limits this therapy (Shapiro et al.,
2006). Expanding donor islets in vitro or in vivo by activating beta
1
Department of Developmental Biology and Cancer Research and Molecular Biology,
The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah
Medical School, Jerusalem 91120, Israel. 2Department of Systems Biology, Harvard
Medical School, Boston, MA 02115, USA. 3Cavendish Laboratory, Department of
Physics, J. J. Thomson Avenue, University of Cambridge, Cambridge CB3 0HE, UK.
4
Department of Stem Cell and Regenerative Biology, Howard Hughes Medical
Institute, Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue,
Cambridge, MA 02138, USA. 5Department of Metabolic and Vascular Diseases,
Hoffmann-La Roche, Nutley, NJ 07110, USA.
*Present address: Developmental Biology Program, Sloan-Kettering Institute, 1275
York Avenue, New York, NY 10021, USA.
†
Author for correspondence ([email protected])
Accepted 23 July 2010
cell replication would have a significant impact on the utility of
clinical islet transplantation. A second therapeutic solution involves
the use of external stimuli to induce beta cell regeneration in vivo.
To succeed, both approaches require significant beta cell
expansion, probably necessitating numerous divisions of each
individual beta cell. However, it is unclear whether a replicating
beta cell has the capacity to divide again and what might control
this decision.
Several studies have shown that all beta cells have a similar
replicative potential, with no sub-population of replicationprivileged cells (Brennand et al., 2007; Teta et al., 2007). This
suggests that replicated beta cells are no more likely to divide
again than undivided beta cells. Further analysis suggested that,
post-division, beta cells become dramatically less likely to reenter the cell cycle, as they enter a prolonged ‘refractory period’,
estimated in months, during which they cannot divide again
(Teta et al., 2007). This idea was supported by a recent study
suggesting that a single beta cell undergoes only two or three
replications over the course of its life time (Desgraz and Herrera,
2009).
The limited replicative capability of a single beta cell contrasts
with reports that indicate a dramatic increase in beta cell mass from
birth to maturity (Dor et al., 2004; Finegood et al., 1995).
Additionally, beta cell mass is known to undergo a several-fold
expansion during regeneration and in response to physiological
demand (Cano et al., 2008; Kulkarni et al., 2004; Nir et al., 2007).
To understand the mechanisms of beta cell expansion under
conditions of both normal and compensatory beta cell division, it
is crucial to characterize the proliferative capacity of a single beta
cell, and to investigate the nature of the beta cell replication
refractory period (Teta et al., 2007).
Here we describe a novel and broadly applicable pulse-chase
assay designed to study the post-replication dynamics of cells in
vivo. Using this assay we can identify and study cells that have
replicated, exited mitosis and returned to cycle (‘re-entered cells’).
We report that dividing beta cells in adult mice quickly re-enter the
DEVELOPMENT
KEY WORDS: Beta cells, Regeneration, Diabetes, Mouse
pool of replication-competent cells and are able to divide again a
few days after mitosis. Clonal analysis of beta cells independently
supports this finding. We show that organismal aging lengthens the
time needed for replicated beta cells to return to the cell cycle; by
contrast, beta cell injury and glucose metabolism shorten the postmitosis quiescence period of beta cells and increase the likelihood
of individual beta cells to divide again.
MATERIALS AND METHODS
Mice
Animal care and experiments were approved by the Institutional Animal
Care and Use Committee of the Hebrew University. We used either ICR
mice from Harlan Israel or Insulin-rtTA; TET-DTA mice (Nir et al., 2007)
(bDTA) that were given doxycycline for 7 days in the drinking water [200
g/ml doxycycline in 2% (w/v) sucrose] at 4 weeks of age.
BrdU labeling was performed at 4-5 or 6 weeks of age in ICR or bDTA
mice, respectively. In the experiement on older mice, 3-month-old ICR
mice were used. BrdU (Sigma-Aldrich) was dissolved in PBS and injected
intraperitoneally (i.p.) (1 mg/g body weight) three times a day every 3
hours for 2 days. Mice were sacrificed 16 hours, 2 days, 5 days, 1 week
and 2 weeks after the last injection. In experiments with a single pulse,
BrdU was injected once and mice were sacrificed after 4, 28 and 48 hours.
Glucokinase activator (GKA) (Grimsby et al., 2003) was diluted in
saline containing 20% DMSO and 1% Tween 80 and injected i.p. at 0.04
mg/g body weight.
Immunostaining
Pancreas was fixed with 4% buffered formaldehyde for 4 hours. Paraffin
sections (5 m) were rehydrated and antigen retrieval was performed using
a Biocare pressure cooker and citrate buffer. The following primary
antibodies were used: guinea pig anti-insulin (1:500, DAKO), rabbit antiKi67 (1:200, Neomarkers), mouse anti-BrdU (1:300, Amersham/GE
Healthcare), rabbit anti-cyclin D2 (1:400, Santa Cruz) and rabbit antipHH3 (1:100, Cell Signaling). For DNA counterstaining on acinar tissue,
Sytox (1:500, Invitrogen) was used. Secondary antibodies were from
Jackson ImmunoResearch: anti-guinea pig Cy2 (1:200), anti-mouse Cy3
(1:500), anti-rabbit Cy5 (1:500). Immunofluorescence images were
captured using a Nikon C1 confocal microscope. Four mice were used for
each data point. Approximately 30 islets or 3000 beta cells were counted
per 1-month-old animal and 50 islets or 8000-10,000 cells in 3-month-old
animals using ImageJ.
Statistics
Statistical analyses were performed using a two-tailed Student’s t-test
(P<0.05). Results are reported with a mean standard deviation.
Modeling
Predictions for the likelihood of cell cycle re-entry (Fig. 1B,C) were generated
by simulation of an expanding cell population, implemented in MATLAB
(MathWorks). The predictions were further confirmed by theoretical analysis
(see Fig. S6 in the supplementary material). In the simulation, each cell is
assigned a life time between birth and mitosis that is drawn at random from
a cell cycle distribution with a refractory period that is either short (days) or
long (months) compared with the average cell division rate (see Fig. S6 in the
supplementary material). To mimic the action of BrdU labeling, all cell
divisions occurring within a short period of time during the simulation are
‘labeled’. The likelihood of cell cycle re-entry is then calculated as the ratio
of the division rates (i.e. the number of cell divisions per cell per unit time) of
the labeled and total cell populations. The simulations assume no cell loss and
a steady decline in the average cell division rate.
RESULTS
An assay for studying cell cycle re-entry of
recently replicated cells
To identify previously divided cells that have returned to the cell
cycle, we developed a pulse-chase protocol based on the thymidine
analog BrdU. One-month-old mice were injected with BrdU (six
Development 137 (19)
injections over 48 hours – the pulse period) to label replicating
cells, and sacrificed at one of several subsequent time points (the
chase period). Beta cells that replicated during the pulse period
should stain for BrdU, whereas beta cells that were dividing at the
time of sacrifice should stain for the general cell cycle marker Ki67
(Gerdes et al., 1991; Key et al., 1993). The fraction of recently
replicated beta cells that re-entered the cell cycle can be determined
by staining beta cells for BrdU and Ki67 at the chase time periods
and calculating the percentage of BrdU+ Ki67+ cells among the
BrdU-labeled population (Fig. 1A). By comparing the likelihood
of replication among the general beta cell population and among
pulse-labeled beta cells, this novel pulse-chase assay can
distinguish between models of beta cell replication dynamics.
Moreover, it can provide an accurate estimate of the duration of the
post-replication refractory period of beta cells and, therefore, of the
time necessary for a replicated cell to re-enter the pool of
replication-competent cells. We generated a simple model (Fig. 1B)
to demonstrate the expected behavior of beta cells if there were no
refractory period, if there were a short refractory period, or if there
were a prolonged refractory period, as has been suggested (see
Introduction). To distinguish between these possibilities, we can
compare the fraction of all replicating beta cells (percentage Ki67+
beta cells; here termed the normal cell division rate) with the
fraction of currently replicating beta cells among the population of
BrdU+ beta cells (percentage BrdU+ Ki67+ among BrdU+ beta
cells; here termed the replicated cell division rate). If, at a given
time after pulse, the replicated cell division rate is less than the
normal cell division rate, this means that replicated beta cells
exhibit a replication refractory period. The first chase time when
the two are equal represents the point at which the likelihood of a
replicated beta cell to re-enter the cell cycle equals the likelihood
of a non-divided cell to divide, and therefore represents the end of
the refractory period (Fig. 1C).
We performed control experiments to establish the validity of
our assay. We first assessed whether Ki67 is indeed expressed and
then downregulated post-mitosis in BrdU-labeled beta cells. Four
hours after a single injection of BrdU, all BrdU+ cells were Ki67+
(Fig. 1D,F). Twenty-eight hours after the injection, 15% of BrdU+
cells expressed Ki67 and 48 hours after the injection only 1% costained for BrdU and Ki67. This demonstrates that Ki67 is
downregulated in BrdU-labeled cells post-mitosis. Therefore, a
subsequent increase in the percentage of co-stained cells must
represent cells that have re-entered the cell cycle. Furthermore, to
quantify exactly when BrdU-labeled beta cells exited the cell cycle,
we co-stained for BrdU+ and phospho-histone H3 (pHH3), a
replication marker that is immediately downregulated post-mitosis
(see Fig. S1A,B in the supplementary material). Three hours after
a single injection of BrdU, nearly all BrdU+ beta cells co-stained
for pHH3, whereas at 12 hours post-injection only 40% of BrdU+
beta cells co-stained for pHH3. By 24 hours post-injection, almost
no BrdU+ cells co-stained for pHH3, indicating that by this time all
BrdU+ cells have exited the cell cycle.
In addition, we verified that beta cells incorporating BrdU
undergo productive mitosis. We counted the percentage of BrdU+
beta cells present at 4, 28 and 48 hours after a single pulse. If
most BrdU-labeled cells divide, the percentage of BrdU+ beta
cells should roughly double after mitosis, when one cell becomes
two. Indeed, at 4 hours we found 2.5% BrdU+ beta cells, whereas
by 48 hours we detected 4.2% BrdU+ beta cells (Fig. 1E),
suggesting that most BrdU-labeled beta cells undergo mitosis.
Furthermore, we quantified the percentage of BrdU+ cells found
in doublets 4 hours and 48 hours after a BrdU pulse (see Fig. S2
DEVELOPMENT
3206 RESEARCH ARTICLE
Cell cycle re-entry of beta cells
RESEARCH ARTICLE 3207
in the supplementary material). We performed our analysis by
confocal microscopy on paraffin sections stained for insulin and
BrdU. Theoretically, we expected to observe about one-third of
BrdU+ cells in doublets, reflecting cell divisions that fall within
the confocal optical plane, but not above or below the plane. By
48 hours post-injection, 40% of BrdU+ cells were found in
doublets (see Fig. S2 in the supplementary material). These data
further confirm that most BrdU-labeled beta cells undergo
productive mitosis.
Replicated beta cells regain replicative potential
after a short period of quiescence
After performing the 2-day pulse protocol, we analyzed the
percentage of BrdU+ beta cells at the chase time periods to confirm
the labeling of a large group of replicated beta cells. Two days after
the last BrdU injection, over 11% of the beta cell population were
BrdU+, representing an increase in BrdU labeling over day 0 and
indicating that although almost all beta cells have exited the cell
cycle by day 0, there is still a small amount of residual mitosis (Fig.
2A). At subsequent time points, the percentage of BrdU+ cells
slightly declined, which we attribute both to the expansion of the
total beta cell population and to the BrdU dilution effect caused by
cells that returned to cycle.
To establish the percentage of ‘re-entered cells’ at each time
point and the beta cell replication refractory period, we counted the
percentage of Ki67+ cells among the BrdU+ beta cell population
(replicated cell division rate) and compared it with the percentage
of total beta cells expressing Ki67 (normal cell division rate) (Fig.
2B,D). Two days after the last BrdU injection, 0.5% of BrdUlabeled beta cells expressed Ki67, in contrast to the total population
in which 3.5% of beta cells expressed Ki67, representing a
significant difference and supporting the idea of a refractory period
of beta cells. By day 5, the percentage of the BrdU+ population
expressing Ki67 increased to 2.5%, while the total beta cell
population expressed Ki67 in 5% of cells, still yielding a significant
difference between the two groups. However, by day 7, and again
on days 12 and 16, there was no significant difference between the
replicated cell division rate and the normal cell division rate. When
the replicating cell division rate was compared with the normal cell
division rate, on day 2 replicated beta cells were only 15% as likely
DEVELOPMENT
Fig. 1. An assay for measuring the beta cell postreplication quiescence period. (A)Outline of the pulsechase 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.
3208 RESEARCH ARTICLE
Development 137 (19)
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.
only 2 days replicated acinar cells are equally as likely to replicate
as the normal acinar cell population (see Fig. S4 in the
supplementary material). These results differ dramatically from the
re-entry rate of the beta cell, which is only 15% 2 days after
mitosis, showing that replicated acinar cells more rapidly regain
their potential to divide.
Clonal analysis confirms a short quiescence period
To further examine the ability of beta cells to re-enter the cell cycle,
we analyzed the size of pulse-labeled clones derived from single
beta cells at 1-2 months post-labeling (Fig. 3A). If the duration of
the refractory period extends beyond the clone age of 1-2 months,
then pulse-labeled cells should give rise only to one-cell and twocell clones. By contrast, a shorter refractory period would allow
successive divisions, leading to larger clones (Fig. 3B).
For clonal analysis, we analyzed in detail the clone fate data
originally presented by Brennand et al. (Brennand et al., 2007). This
system indelibly labels individual beta cells with both GFP and RFP
at extremely low efficiencies (0.1-0.5%) using a tamoxifendependent Cre-lox system. Mice were labeled at 4 to 8 weeks of age.
At 4 days post-labeling, 90% of the islets containing labeled cells had
only one labeled cell, and the remaining 10% contained two labeled
cells. Therefore, given the low labeling efficiency, single cell-derived
clones were defined as clusters of labeled cells within a single islet.
DEVELOPMENT
to replicate, yet by day 7 they were 70% as likely to replicate, and
by day 12 they were equally as likely to replicate (Fig. 2C). Indeed,
although there was a significant increase in the likelihood of cell
cycle re-entry from days 2 to 5 and 5 to 7, there was no statistically
significant increase in the likelihood from 7 days onward. These
results indicate that within ~1 week after mitosis, a beta cell regains
its normal potential to divide. These data are consistent with a short
refractory period (Fig. 1B,C), after which previously divided beta
cells re-enter the pool of beta cells that can respond to mitogenic
stimuli.
To confirm these findings using an additional marker of cell
cycle entry, we compared the replicated cell division rate with the
normal cell division rate using pHH3 instead of Ki67. At day 7
post-BrdU, 0.75% of BrdU+ beta cells were pHH3+, whereas 0.8%
of the total beta cell population was pHH3+ (see Fig. S3 in the
supplementary material). This suggests that at day 7, BrdU+ beta
cells were 90% as likely to replicate as normal beta cells. These
results again demonstrate that divided beta cells regain their normal
replication rate after ~1 week.
To compare the beta cell re-entry behavior with that of a
different cell population, we performed the pulse-chase assay
analysis on pancreatic acinar cells. Two days after the last BrdU
injection, the normal cell division rate and the replicated cell
division rate were nearly identical at 1.5%, demonstrating that after
Fig. 3. Clonal analysis using RipCreER-MADM mice supports
multiple divisions of the same beta cell and a short postreplication 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 longterm 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.
Following a 1- to 2-month chase period, clones were sampled and
their size scored from single random sections (Fig. 3C) (Brennand et
al., 2007). The results show that only 16 of 45 clones (35%)
contained 1-2 cells following a 1-month chase period, and only 3 of
40 clones (8%) contained 1-2 cells after 2 months. Therefore, it
appears that the majority of cells are capable of dividing at least
twice within a period of 1 month. Remarkably, the clonal analysis
also revealed the presence of larger clones, indicating that cells may
divide repeatedly within the chase period. At 1 month post-labeling,
5 out of 45 clones (11%) contained at least 10 cells, and at 2 months
post-labeling 6 out of 40 clones (15%) contained at least 16 cells.
The largest clones at 1 month contained 26 cells, suggesting that a
single cell might occasionally divide four or five times within this
time period, or approximately once per week. Taken together, the
clone fate data provide independent support for the notion that
replicated beta cells rapidly regain their ability to re-enter the cell
cycle. Furthermore, these results dramatically demonstrate that beta
cells in adult mice are capable of multiple divisions.
The post-replication quiescence period is
prolonged with age
The rate of beta cell replication drops dramatically with age in
rodents and humans (Chen et al., 2009; Dhawan et al., 2009;
Finegood et al., 1995; Krishnamurthy et al., 2006; Meier et al.,
RESEARCH ARTICLE 3209
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.
2008; Rankin and Kushner, 2009; Teta et al., 2005; Tschen et al.,
2009; Wong et al., 2009). However, it is not known how age affects
the likelihood of an individual beta cell to divide again after it had
completed mitosis. To examine the role of aging in the regulation
of cell cycle re-entry of beta cells, we performed our pulse-chase
assay on older mice. Given the necessity of labeling a significant
population of replicating beta cells with BrdU, we chose 3-monthold mice, which still maintain a considerable rate of beta cell
replication. Mice were injected with BrdU according to our
standard protocol (Fig. 1A) and sacrificed at 5, 7, 12 and 16 days.
At 5 days, 5% of all beta cells were BrdU+ (Fig. 4A). Interestingly,
the percentage of BrdU-labeled cells did not significantly decline
during the remainder of the chase period, supporting the assertion
that the decline in BrdU+ cells found in the 1-month chase period
resulted from a significant beta cell expansion while the BrdU+
cells were in the refractory period.
DEVELOPMENT
Cell cycle re-entry of beta cells
3210 RESEARCH ARTICLE
Development 137 (19)
We then quantified the replicated cell division rate (the
percentage of Ki67+ cells among the BrdU+ population) and the
normal cell division rate (the percentage of Ki67+ among the total
beta cell population), at each of the chase time periods (Fig. 4B).
Whereas in 1-month-old mice at 7 days of chase there was no
significant difference between the two rates, in 3-month-old mice
the replicated cells showed significantly lower cell division
throughout the entire chase period. Furthermore, 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, 40% as likely to replicate on
day 12, and 30% as likely to replicate on day 16, as compared with
the general population (Fig. 5C). Thus, although beta cells in older
mice can, in principle, re-enter the cell cycle rapidly after cell
division, they are less likely to do so than beta cells in younger
mice.
Regenerating and glucose-stimulated beta cells
have a shortened quiescence period
We next investigated whether the duration of the beta cell postreplication quiescence period is under physiological control, and
specifically whether it could be shortened in response to specific
stimuli. First, using a transgenic mouse model for conditional
ablation of beta cells (Nir et al., 2007), we examined the beta cell
quiescence period in regenerating islets. Treatment of 4-week-old
Insulin-rtTA; TET-DTA mice with doxycycline for 1 week caused
hyperglycemia (blood glucose of 550 mg/dl on average, compared
with 140 mg/dl on average in control littermates) and ablation of
the majority of beta cells. Mice were then injected with BrdU as
described above (Fig. 1A) and sacrificed 2 and 5 days after the last
injection. Surprisingly, after just 2 days the replicated cell division
rate (the percentage of Ki67+ cells among the BrdU+ population)
equaled the normal cell division rate (the percentage of Ki67+ cells
among the total beta cell population), each proliferating at 6% (Fig.
5A,C). This phenotype continued on day 5, when both groups
again proliferated at 6%. When the replicating cell division rate
was compared with the normal cell division rate, on day 2 and
again on day 5 the replicated cells were equally as likely as normal
cells to enter the cell cycle (Fig. 5B). These results indicate that in
contrast to the normal adult beta cell, which requires 7 days to
regain its normal proliferative capacity, regenerating beta cells
almost immediately recover from replication, essentially
eliminating the quiescence period.
We hypothesized that the shortened beta cell post-replication
quiescence period might be a result of increased glucose signaling
due to hyperglycemia in Insulin-rtTA; TET-DTA mice. To
determine whether higher glucose levels can shorten the beta cell
post-replication quiescence period, we treated mice with a
pharmacological activator of glucokinase, glucokinase activator
(GKA), shown previously to reduce the S0.5 for glucose and
increase the Vmax of glucokinase in beta cells (Grimsby et al.,
2003). This drug causes mild systemic hypoglycemia (due to
DEVELOPMENT
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 InsulinrtTA; 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.
Cell cycle re-entry of beta cells
RESEARCH ARTICLE 3211
increased insulin secretion), but within the beta cell it increases
glycolysis and mimics the effect of systemic hyperglycemia. After
six injections of BrdU over 2 days, mice were injected with
vehicle or GKA and sacrificed 24 hours later. As expected, GKA
treatment lowered the average blood glucose levels from 115 to
45 mg/dl (Fig. 5D). Mice receiving the vehicle demonstrated a
0.25% replicated cell division rate and a 3.5% normal cell division
rate (comparable to values for wild-type mice not receiving
vehicle). By contrast, mice receiving GKA exhibited a 2.8%
replicated cell division rate and a 4% normal cell division rate
(Fig. 5E). When compared as a ratio, vehicle-treated replicated
beta cells were 5% as likely to divide as normal beta cells,
whereas replicated beta cells of mice receiving GKA were 70% as
likely to divide again compared with the normal beta cell
population (Fig. 5F). Similar results were obtained when mice
were sacrificed 2 days after GKA administration, when the
glucose-lowering effect had vanished (see Fig. S5 in the
supplementary material).
These results show that an increased rate of glucose metabolism
shortens the post-replication quiescence period of beta cells.
Furthermore, they suggest that the dramatic shortening of the
quiescence period in regenerating beta cells in the Insulin-rtTA;
TET-DTA model is a result of hyperglycemia.
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.
If cyclin D2 is indeed a key factor in the control of beta cell
cycle re-entry, our models with a shortened beta cell quiescence
period should show a faster post-mitotic return of normal cyclin D2
levels. By contrast, beta cells in older mice (having a lengthened
refractory period) should exhibit a slower return of cyclin D2. To
investigate whether hyperglycemia induces faster upregulation of
cyclin D2, we compared vehicle- and GKA-treated mice on day
zero (16 hours after the last BrdU injection). Control mice
expressed cyclin D2 in just 28% of BrdU+ cells, in contrast to the
quiescent beta cell population in which 80% of the cells expressed
cyclin D2 (Fig. 6C). By contrast, on day zero, 52% of the BrdU+
beta cells in regenerating Insulin-rtTA; TET-DTA mice were cyclin
D2+. Similarly, in mice injected with GKA 24 hours before
sacrifice 58% of BrdU+ cells expressed cyclin D2. In both InsulinrtTA; TET-DTA and GKA models, the fraction of BrdU+ cells that
expressed cyclin D2 was significantly higher than in untreated
mice. To examine whether beta cells in older mice upregulated
cyclin D2 at a slower rate after mitosis, we compared the
DEVELOPMENT
Cyclin D2 is downregulated during beta cell
replication and slowly returns to basal levels by a
glucose-sensitive mechanism
We considered possible molecular mechanisms that could account
for the regulation of the quiescence period. Cyclin D2 is essential
for beta cell proliferation (Georgia and Bhushan, 2004; Kushner,
2006; Kushner et al., 2005). Although the dynamics of cyclin D2
expression during cell division have not been studied before, cyclin
D1 is known to be downregulated in vitro during the S/G2 phases
of the cell cycle in other cell types (Baldin et al., 1993; Lukas et
al., 1994). We hypothesized that such a phenomenon could occur
in replicating beta cells in vivo, and that low levels of cyclin D2
might persist after mitosis. This in turn could account for the
quiescence period by preventing beta cells from returning to the
cell cycle until cyclin D2 is re-expressed.
To examine cyclin D2 expression during the cell cycle of beta
cells in vivo, we injected 1-month-old ICR mice with BrdU and
sacrificed after 2 hours. Beta cells that incorporated BrdU co-stained
for cyclin D2 25% of the time, as compared with the quiescent beta
cell population which stained positive 80% of the time, indicating
that cyclin D2 is downregulated during the S phase of replicating
beta cells in vivo (Fig. 6A,B). To further investigate the regulation
of cyclin D2 during the cell cycle and post-mitosis, we injected mice
with BrdU and sacrificed at later time points up to 350 hours postBrdU (Fig. 6B). Strikingly, replicating beta cells remained largely
cyclin D2-negative even after mitosis had ended, and very slowly
started to re-express the gene. At 7 and 23 hours post-labeling, only
~20% of BrdU-positive beta cells expressed cyclin D2; 30 hours
after labeling 40% of BrdU-positive cells expressed cyclin D2, and
at 150 hours post-labeling 70% of BrdU-positive cells co-stained for
cyclin D2. As beta cells need cyclin D2 to replicate, these results
suggest that the decrease in beta cell cyclin D2 during cell division
and immediately after mitosis prevents cells from re-entering the cell
cycle. This assertion is strengthened by the correlation between the
time necessary for the replicated beta cell population to regain its
normal ability to replicate (~7 days) and the time necessary for the
replicated beta cells to express levels of cyclin D2 found in quiescent
cells (150 hours, or 6 days).
percentage of cyclin D2+ BrdU+ cells in 1- and 3-month-old beta
cells on day 5 (Fig. 6D). At both ages, beta cells expressed cyclin
D2 in 70% of Brdu+ cells, suggesting that the return of cyclin D2
levels post-mitosis occurs at a similar rate in each.
These data suggest that metabolic demand might control the
replication refractory period of beta cells via the rate of cyclin D2
return post-mitosis. Further work will be required to test this idea.
However, aging is likely to affect the replication refractory period
via other mechanisms.
DISCUSSION
We conclude that adult beta cells are capable of multiple sequential
divisions and, post-mitosis, quickly regain their potential to
replicate. Using our pulse-chase assay and clonal analysis, we show
that post-mitotic beta cells re-enter the pool of replicationcompetent cells after a short quiescence period lasting ~1 week.
This recovery period is lengthened in older beta cells, but can be
shortened by physiological conditions that increase the rate of
glucose metabolism, possibly by upregulating cyclin D2.
Previous reports have demonstrated that replication is the
primary method for beta cell expansion and regeneration
(Brennand et al., 2007; Dor et al., 2004; Georgia and Bhushan,
2004; Meier et al., 2008; Nir et al., 2007; Teta et al., 2007), but the
replicative potential of a single beta cell has remained unclear. The
large clusters of labeled beta cells in our pulse-chase clonal analysis
demonstrate that single beta cells are capable of numerous
divisions. Furthermore, in BrdU pulse-chase experiments, the
appearance of post-mitotic cells that have re-entered the cell cycle
demonstrates the ability of a beta cell to replicate multiple times.
These findings are consistent with previous studies showing
dramatic beta cell mass expansion from birth to maturity,
necessitating multiple divisions of individual beta cells (Finegood
et al., 1995). Very recently, a report employing clonal analysis of
Ngn3+ endocrine progenitor cells suggested that beta cells replicate
only once or twice throughout the life time of a mouse (Desgraz
and Herrera, 2009). This estimation differs considerably from our
findings, and is difficult to reconcile with the significant expansion
and turnover of beta cells during postnatal life. The reason for the
differences between the two studies remains to be determined.
Our study shows that after a short period of quiescence lasting
~7 days, replicated beta cells from 1-month-old mice are equally
as likely to divide again when compared with the normal beta cell
population. Although we do not find any significant difference in
the likelihood of re-entry after day 7, we cannot rule out the
possibility of a slight increase in the likelihood beyond day 7.
Additionally, there might be a small population of cells with a
longer refractory period that is difficult to detect with our assay.
Indeed, clonal analysis showed multiple very small clones that
might be representative of cells that remain in an extended period
of quiescence. However, the presence of small clones is equally
consistent with a normal distribution of cells dividing at random.
Furthermore, even though there is a possibility that some cells have
entered a long refractory period, they would represent a very small
proportion of the beta cell population given that the overall
likelihood of a beta cell to re-enter the cell cycle returns to normal
after ~1 week.
Recently, using a novel assay of serial thymidine analog
labeling, Teta et al. demonstrated that all beta cells maintain equal
proliferative potential and suggested that, post-replication, beta
cells enter a long refractory period of several months (Teta et al.,
2007). Our results are consistent with their study, as we have
demonstrated a short refractory period in 1-month-old mice,
Development 137 (19)
whereas their studies were performed on 3-month-old mice.
Indeed, at 3 months of age we also see an extended refractory
period. However, we believe that our method, which directly
measures the time to cell cycle re-entry and relies on two very
different epitopes (BrdU and Ki67, in contrast to CldU and IdU in
Teta et al.), is a more straightforward assay for quantifying the
duration of the refractory period, as the continuous labeling assay
might lead to an overestimate of the true period length (see Fig. S6
in the supplementary material).
The lengthened quiescence period in older mice and the
shortened quiescence period in response to beta cell ablation and
GKA stimulation provide a novel insight into the mechanisms of
beta cell proliferation. Previous studies have demonstrated that
ablation can increase the overall rate of beta cell division (Cano et
al., 2008; Nir et al., 2007), whereas aging decreases the rate of beta
cell replication (Finegood et al., 1995; Krishnamurthy et al., 2006;
Meier et al., 2008; Rankin and Kushner, 2009; Tschen et al., 2009).
Our work suggests that beyond the direct replicative effect that
glucose, regeneration and aging have on beta cells, these factors
might also regulate the pool of cells that can replicate by
controlling the recovery time of quiescent post-mitotic cells. More
generally, our results raise the idea that the duration of a postmitosis refractory period could be a control point in tissue
dynamics. For example, it would be interesting to examine whether
tumor cells differ from their cell of origin in their post-replication
quiescence period. If such differences exist, the underlying
molecular mechanisms might provide an interesting target for
intervention.
Notably, we found that beta cell regeneration in the Insulin-rtTA;
TET-DTA model, and simulation of glycolysis with GKA,
considerably shorten the quiescence period, potentially by
upregulating the expression of cyclin D2 from the low levels
present during and after replication. Previous studies have
demonstrated that cyclin D2 is important for beta cell replication,
explaining why recently replicated beta cells lacking this protein
might be unable to re-enter the cell cycle. Interestingly, cyclin D2
is not necessary for acinar cell replication, suggesting a possible
explanation for why acinar cells do not maintain a detectable
refractory period. Several studies have demonstrated that the
activation of pathways that upregulate cyclin D2 increases beta cell
proliferation. Most notable among these pathways is high glucose,
which has been shown to increase beta cell cyclin D2 levels
(Alonso et al., 2007), supporting our assertion that both beta cell
ablation and GKA directly increase cyclin D2 and allow beta cells
to rapidly re-enter the proliferative pool. The molecular mechanism
by which glucose regulates the levels of cyclin D2 in beta cells is
currently under investigation. More experiments are necessary to
understand why cyclin D2 is downregulated during S phase and
what regulatory factors cause its slow return after mitosis.
Interestingly, we did not observe a difference in the post-mitotic
regulation of cyclin D2 in young and older mice. Previous work
has demonstrated that as beta cells age, they express higher levels
of cell cycle inhibitors such as INK4A/p16 (Cdkn2a)
(Krishnamurthy et al., 2006). We speculate that the higher level of
CDK inhibitors is involved in the delay of cell cycle re-entry with
age. Thus, the post-replication quiescence period of beta cells
might be regulated by modulators of CDK activity: cyclin D2 in
the context of glucose stimulation and CDK inhibitors in the
context of aging. Indeed, it is likely that several levels of cell cycle
regulation are involved in the re-entry of beta cells, as cyclin D2
returns at a faster rate than the termination of the refractory period.
Although the absence of cyclin D2 could prevent replicated beta
DEVELOPMENT
3212 RESEARCH ARTICLE
cells from returning to the pool of dividing cells, it does not
necessarily control the process alone. Further work will be
necessary to determine the role of CDK inhibitors and other cell
cycle proteins in the regulation of the refractory period. We also
acknowledge that the current work provides only a correlation
between cyclin D2 levels and the duration of the refractory period.
Demonstration of a causal role for cyclin D2 in the refractory
period will require direct manipulation of the protein. Our
BrdU/Ki67 pulse-chase assay provides a new tool for identifying
cells returning to the cell cycle and for establishing the likelihood
of cell cycle re-entry. Further investigation will be necessary to
examine re-entry in other adult tissues and in beta cells under
different physiological stimuli, such as pregnancy or a high-fat diet.
Finally, these results might have several clinical implications.
First, the capacity of beta cells to undergo multiple divisions in
vivo further supports the theoretical feasibility of regenerative
therapy as a treatment for diabetes. Recent reports have
demonstrated the existence of beta cell proliferation in normal and
diabetic patients, suggesting the opportunity for further beta cell
expansion (Kassem et al., 2000; Meier et al., 2008; Meier et al.,
2006). Second, although islet transplantation is an effective
treatment for type 1 diabetes, the scarcity of donor islets
significantly limits its therapeutic scope. Our results suggest that
existing donor islets might be induced into significant proliferation
in vitro, thereby increasing the number of available donor islets.
Lastly, our ability to regulate the post-mitotic quiescent period
using a pharmacological activator suggests interesting new
directions for the expansion of beta cell mass in vivo.
Acknowledgements
We thank Doug Melton and Ittai Ben-Porath for critical reading of the
manuscript and Michal Maatouf, Avigail Dreazen and Judith Magenheim for
assistance. This work was supported by grants from the Beta Cell Biology
Consortium (NIH), Juvenile Diabetes Research Foundation, Israel Science
Foundation, the EU FP6, FP7 BetaCellTherapy consortium and the Helmsley
Foundation (to Y.D.). A.M.K. is supported by an EPSRC LSI fellowship
(F043325/1. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.054304/-/DC1
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DEVELOPMENT
Cell cycle re-entry of beta cells