Very Slow Turnover of -Cells in Aged Adult Mice
http://www.100md.com
糖尿病学杂志 2005年第9期
1 Division of Endocrinology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
2 Division of Endocrinology, Children’s Hospital, Boston, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Although many signaling pathways have been shown to promote -cell growth, surprisingly little is known about the normal life cycle of preexisting -cells or the signaling pathways required for -cell survival. Adult -cells have been speculated to have a finite life span, with ongoing adult -cell replication throughout life to replace lost cells. However, little solid evidence supports this idea. To more accurately measure adult -cell turnover, we performed continuous long-term labeling of proliferating cells with the DNA precursor analog 5-bromo-2-deoxyuridine (BrdU) in 1-year-old mice. We show that -cells of aged adult mice have extremely low rates of replication, with minimal evidence of turnover. Although some pancreatic components acquired BrdU label in a linear fashion, only 1 in 1,400 adult -cells were found to undergo replication per day. We conclude that adult -cells are very long lived.
Inadequate islet mass is a central finding in both type 1 and type 2 diabetes, resulting in an absolute or relative insulin deficiency and subsequent metabolic complications (1,2eC3). Over the past decade, much has been learned about the specific signaling pathways that direct embryonic islet development (4) and postnatal islet growth (5). To compensate for increased metabolic demands, adult islet mass increases dramatically over the first year of life in rodents (6) and may represent growth within preexisting islets because islet density does not typically increase as mice age (7,8). It has been known for some time that adult -cell growth is dependent on cyclin-dependent kinase-4 (9). Recently, we (10) and others (11) showed that cyclins D2 and D1 are the likely partners with cyclin-dependent kinase-4 to promote G1 cell cycle progression in adult -cells. Remarkably, new evidence suggests that much of adult -cell growth may occur by replication of insulin-positive cells and not by -cell neogenesis (12). However, it is possible that -cell neogenesis may exist under some circumstances (13,14). Surprisingly little is known about the factors that promote adult -cell survival, although some amount of -cell apoptosis occurs in normal islets (15) as well as in pathological states (16). Although the absolute -cell death rate is unknown, -cell death appears to be a rare event. Supporting this notion, -cell area increases severalfold throughout adulthood, despite an apparent decline in daily proliferation rate (6,17).
Despite the lack of direct evidence, it has been largely assumed that -cells have a finite life span, with dying cells replenished by new -cells on an ongoing basis (12,14,17). One method of estimating cell life span is to measure the growth of total cell populations and cellular proliferation rate. In this model, the cell turnover rate is reflected by the proportion of cellular proliferation that does not contribute to cell population growth. In the simplest example of a stable population of cells with no net growth, cellular proliferation must be explained by ongoing loss and reflect the true cellular turnover rate. Because of inherent inaccuracies in measuring both cell growth and cellular proliferation rates, this technique is particularly well suited to organs or cell populations that are fairly stable or change very slowly. However, even in easily modeled cell populations, estimates of cell life span by this method are nonetheless indirect and do not readily account for nonproliferative neogenesis (i.e., cellular trans-differentiation).
Using -cell proliferation data from young rats, Finegood et al. (17) showed that -cell proliferation rates decline from 20% per day in pups to 10% per day in adolescence and then to 2% in early adulthood. Based on limited data in aged animals, the authors assumed that -cell proliferation rates stabilize at 2% throughout adulthood, and they estimated -cell life span to be 1eC3 months. Similarly, Montanya et al. (6) found that intraislet proliferation rates declined to 0.8% per day in 1-year-old rats and was stable thereafter (0.2% 5-bromo-2-deoxyuridine [BrdU]-positive cells after a 6-h BrdU label). However, the authors estimated -cell proliferation by immunostaining with a cocktail of antisera specific to noneC-cell hormones and BrdU, which also counts proliferating noneChormone-containing islet cells. We hypothesized that -cell replication rates might continue to decline into adulthood, making estimates of -cell life span in young animals not applicable to the life span of mature -cells. However, little reliable data exists on -cell proliferation in aged mice to address this question.
To estimate adult -cell turnover, we performed continuous long-term BrdU labeling in aged adult mice, a technique previously used to measure the life span of lymphocytes (18) and ganglionic glia (19). Unlike the standard 6-h BrdU label, long-term BrdU labeling allowed us to calculate -cell proliferation in terms of the total cell population, giving us better insight into the rate of -cell turnover and life span. A priori, we predicted at least three possible models of BrdU labeling of -cells using continuous BrdU (Fig. 1). If -cells rapidly turn over, as predicted by Finegood et al. (17), near complete -cell BrdU labeling would occur if the period of label exceeded the typical -cell life span. In contrast, if -cells exhibit slow turn over, limited accrual of BrdU label would occur in -cells. Finally, heterogeneity within islets might result in nonlinear BrdU labeling: a limited population of rapidly dividing but short-lived -cells could coexist with slowly proliferating long-lived -cells. Surprisingly, our results show that adult mouse -cells acquire BrdU in a very slow manner and appear to be very long lived, with 1 in 1,400 mature -cells turning over per day.
RESEARCH DESIGN AND METHODS
Male wild-type c57BL/6 x 129Sv mixed genetic background mice were derived from a colony of cyclin D1+/eC::D2+/eC mice generously provided by Peter Sicinski, genotyped as previously described (20,21). Mice were maintained at a barrier animal facility in the Harvard School of Public Health and fed mouse diet 5020 9F (9% fat calculated by weight, 21.6% fat calculated by kilocalories; PMI Nutrition International, Richmond, IN). Random-fed glucose measurements were performed using a Glucometer Elite (Bayer). Male and female wild-type BALB/c mice (purchased from Taconic) were housed at the animal facility at the Children’s Hospital of Philadelphia and fed a similar diet (mouse diet 5015; PMI Nutrition International).
Statistics.
All results are reported as the means ± SE for equivalent groups. Results were compared with independent t tests (unpaired and two tailed) reported as P values.
Immunohistochemistry.
Pancreas samples were dissected from fed mice and fixed with 4% paraformaldehyde/PBS solution overnight. Then, 5-e longitudinal sections of paraffin blocks were rehydrated with xylene, followed by decreasing concentrations of ethanol, microwaved in 0.01 mol/l sodium citrate (pH 6.0) for 20 min, and permeabilized with 1% Triton X-100 in PBS before primary antisera incubation. Primary antisera were guinea pig anti-insulin (Zymed Laboratories Inc., South San Francisco, CA), rabbit anti-glucagon (Zymed), rabbit anti-somatostatin (Zymed), rabbit anti-pancreatic polypeptide (Zymed), and rat anti-BrdU (BU1/75; Accurate Chemical, Westbury, NY). Secondary antibodies were highly cross-adsorbed and labeled with Cy2 or Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclear staining was performed with 4',6 diamino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR).
Long-term BrdU proliferation analysis.
Mice were labeled continuously with BrdU by substituting drinking water bottles with bottles that contained 1 mg/ml BrdU. To ensure that the BrdU was bioactive, water bottles were completely wrapped with aluminum foil (to prevent light exposure), and the BrdU water solution was changed weekly with freshly prepared solution. Paraformaldehyde-fixed paraffin sections prepared from killed animals were stained with DAPI/insulin/BrdU, and images were acquired from all visible multicellular islets at 20x magnification. BrdU-positive -cell ratios were calculated as the means ± SE of BrdU-positive -cells over total -cells per section, two sections per animal. Individual animal results each represent an average of 46 islets counted per animal (range 40eC71) comprising an average of 12,613 -cells (range 3,445eC34,171). Counted -cells were estimated by measuring the cross-sectional -cell area of each acquired islet using the lasso tool and dividing by mean -cell size determined from multiple islets. Acinar cell proliferation analysis was performed by randomly acquiring five DAPI/insulin/BrdU images (20x magnification) of acinar cells from each section. Acinar cells were counted for total BrdU- and total DAPI-positive nuclei. Individual animal results of pancreatic acinar tissue each represent 10 fields counted per animal comprising an average of 1,455 cells (range 813eC3,802). Preadipocyte proliferation analysis was performed in a similar manner by acquiring five DAPI/insulin/BrdU images (20x magnification) of peripancreatic adipose tissue from each animal. Individual animal results of peripancreatic adipose tissue each represent an average of 10 fields counted per animal (range 5eC20) comprising an average of 801 cells (range 325-1592). Perigonadal fat pads were fixed and processed as per pancreata and stained with antisera against BrdU. Five DAPI/BrdU images were acquired at 20x magnification and counted for BrdU and DAPI adipocytes. Individual animal results of perigonadal fat pads each represent an average of 11 fields counted per animal (range 7eC15) comprising an average of 1,036 cells (range 424eC1,604).
Islet morphometry.
-Cell area was measured by acquiring at least 10 adjacent nonoverlapping images from each insulin-stained section, three sections per animal, using an Axioskop 2 plus mot with a 10x objective and a 0.63x converter (Carl Zeiss MicroImaging, Thornwood, NY) and captured with a Hamamatsu Orca ER digital camera. Images were analyzed for area with Open Lab 4.0 software, using the lasso tool to select individual islets and using individually optimized settings to maximize region-of-interest accuracy. Results of -cell quantification were expressed as the percentage of the total surveyed area containing cells positive for insulin. Results represent the average from 14 total animals (11 mice up to 12 months of age and 3 at 14 months of age), each with an average of 102 fields counted per animal (range 57eC148).
Short-term BrdU islet proliferation analysis.
Short-term BrdU labeling -cell proliferation studies in aged mice were performed by injecting five different 9- to 12-month-old mice, also from the same genetic cohort of c57BL/6 x 129Sv background, with BrdU (100 e/g body wt; Roche) 6 h before they were killed. Triple-label DAPI/insulin/BrdU immunohistochemistry images were acquired from rehydrated paraformaldehyde-fixed, paraffin-embedded sections. All visible islets per pancreatic section (five sections per animal) were analyzed to determine total BrdU/insulin copositive cells and total islet count per section. Mean -cell size was determined by counting all insulin/DAPI copositive nuclei in at least five islets per animal. The -cell replication rate was then estimated by determining the mean islet size by measuring the cross-sectional area of all islets in the section. BrdU-positive -cell ratios were then calculated as the means ± SE of BrdU-positive -cells. Results represent the average of five total animals, each with an average of 439 islets counted per animal (range 256eC636) comprising an average of 27,302 cells per animal (range 6,415eC57,395).
Short-term BrdU labeling -cell proliferation studies in young mice were performed by injecting 5-day-old pups with BrdU 6 h before they were killed, as described above. The largest islets in each pancreatic section (two sections per animal) were analyzed to determine total BrdU-insulin copositive cells and the total islet count per section. Results represent the average of seven total animals, each with an average of 11 islets counted per animal (range 6eC17) comprising an average of 733 cells per animal (range 343eC1,215).
Islet apoptosis analysis.
Apoptosis analysis was performed on pancreas sections from 9- to 12-month-old male mice by the transferase-mediated dUTP nick-end labeling (TUNEL) method using Cy2-labeled reagents (Roche) on rehydrated and trypsin-predigested pancreas sections. Sections were subsequently stained for insulin and then stained with appropriate Cy3 secondary antisera, as above. All visible islets per pancreatic section (five sections per animal) were analyzed for the total number of DAPI/TUNEL/insulin copositive cells and for the total number of islets per section, and they were analyzed as per proliferation analysis. Results represent the average of 14 total animals, each with an average of 597 islets counted per animal (range 244eC814) comprising an average of 138,868 cells per animal (range 23,738eC365,232).
Low-dose streptozotocin treatment.
Mice were treated with five daily injections of low-dose (30 mg/kg) streptozotocin in 0.1 mol/l sodium citrate buffer (pH 4.5) or control sodium citrate buffer. Mice were given BrdU in drinking water continuously until they were killed 15 days after the end of streptozotocin treatment. Pancreata were processed and DAPI/insulin/BrdU images acquired as for other long-term BrdU-exposed mice. Results were averaged from individual animals (three per treatment), each of which represent an average of 45 islets counted per animal (range 28eC69) comprising an average of 8,686 -cells (range 3,859eC14,176).
RESULTS
Long-term BrdU exposure is well tolerated in adult mice.
To estimate adult -cell turnover, we analyzed -cell replication rates in adult islets by continuously treating 1-year-old mice with BrdU in drinking water for various lengths of time (2, 4, 8, and 16 weeks) immediately followed by killing. Mice tolerated long-term continuous BrdU exposure well, although one mouse died 2 weeks into BrdU labeling from unknown causes. Long-term BrdU exposure had little effect on body weight (data not shown). Similarly, long-term BrdU appeared to have little effect on glucose homeostasis; although blood glucose was slightly decreased in BrdU-treated mice over time, the trend was nonsignificant (r2 = 0.101). There was also no relationship between BrdU incorporation rates and glucose levels measured before the mice were killed (r2 = 0.006) (data not shown).
At the time they were killed, mice were grossly normal, with normal-appearing organs. Total pancreatic -cell area was very high (5.4 ± 1.1% total pancreas area). -Cell area was widely variable, with individual animal results ranging from 1.2 to 14.4% total pancreas area, which presumably reflects animal-to-animal differences in peripheral insulin resistance and subsequent insulin production within this aged mixed genetic background (c57BL/6 x 129Sv) cohort. Notably, no association was observed between the length of BrdU exposure and -cell area or between age at time of death and -cell area (data not shown).
Long-term BrdU labels rapidly proliferating cells in a predictable manner.
Pancreatic replication rates were measured by performing DAPI/insulin/BrdU triple staining of pancreatic sections. Most cells within peripancreatic lymph nodes were BrdU positive by 4 weeks, illustrating that long-term BrdU exposure efficiently labels rapidly turning over cells in vivo (Fig. 2). Similarly, peripancreatic fat depots were examined for evidence of proliferation in resident preadipocytes, which proliferate in vivo (22). Pancreatic preadipocytes proliferated slower than lymphoid cells, but after 16 weeks of BrdU labeling, many cells displayed evidence of past proliferation (Figs. 2 and 5A). Overall, pancreatic preadipocytes displayed evidence of past proliferation at a rate of 4.76 ± 0.49% per day, with individual animal results ranging from 2.55 ± 0.18 to 8.21 ± 0.97% per day. To verify that pancreatic fat depots were representative of other fat depots, we also examined proliferation in suprapubic fat pads from mice after 8 weeks of BrdU exposure (n = 3). Preadipocytes from perigonadal fat pads proliferated at a rate of 6.21 ± 0.38% per day, in agreement with our results with pancreatic fat depots.
Modest proliferation of pancreatic ductal and acinar cells.
Within the pancreas are rare insulin-positive pancreatic ductal cells, which have been speculated to serve as a reservoir for -cell progenitors in the adult pancreas (14,23). Notably, pancreatic ductal cells had infrequent evidence of past proliferation across all groups, although BrdU-positive ductal cells were more common in pancreata from mice labeled with BrdU for longer periods (Fig. 2). Similarly, insulin-positive pancreatic ductal cells appeared to be largely postmitotic in the mice examined, with extremely few insulin-positive ductal cells displaying BrdU immunoreactivity (Fig. 2, far right panels). Thus, at least in the basal state in aged adult mice, insulin-positive ductal cells do not appear to be a rapidly dividing precursor population.
Similar to ductal cells, pancreatic exocrine cells acquired BrdU label in a slower manner, with 25% of acinar cells showing evidence of past proliferation by 16 weeks (Figs. 2 and 5A). Acinar cell proliferation was 0.194 ± 0.039% per day, with individual animal results ranging from 0.079 ± 0.020 to 8.21 ± 0.97% per day. Thus, our results correspond to 1 in 513 acinar cells proliferating per day, and they suggest that most acinar cells are in a mostly quiescent state.
Minimal -cell proliferation in aged adult mice.
In contrast to other cells contained within pancreatic preparations, insulin/BrdU copositive cells were fairly rare and comprised only a small portion of total -cells, even in mice exposed to BrdU for very long periods (Figs. 3 and 5A). -Cell proliferation rates averaged 0.0701 ± 0.0227% per day, far lower than expected given previous data of 3% daily -cell proliferation in rats (12). However, considerable animal-to-animal heterogeneity was observed, and -cell proliferation rates ranged from 0.0085 ± 0.0020 to 0.3113 ± 0.0433% per day, a 36-fold difference. Strikingly, several animals had almost no evidence of past -cell proliferation, with as few as 1 in 11,764 -cells turning over per day in one animal. Other animals had much more -cell proliferation, with as many as 1 in 321 -cells turning over per day. Because of the large animal-to-animal variation in -cell proliferation rates, there was little correlation between the period of label and the percent of BrdU-positive -cells (r2 = 0.298) (Fig. 5A). In contrast, higher-proliferating tissues showed less animal-to-animal variation and better correlation in between the period of label and proliferation (peripancreatic adipocytes, r2 = 0.786; pancreatic acinar cells, r2 = 0.528) (Fig. 5A).
In an attempt to moderate the effect of genetic variation, 8-month-old male mice from a secondary and more homogeneous background, BALB/c, were continuously labeled with BrdU for 1 week. -Cell proliferation rates were slightly higher, with an average of 0.679 ± 0.06% per day (n = 8). Although these mice also showed significant animal-to-animal variation (from 0.456 ± 0.06 to 0.978 ± 0.06% per day) (Fig. 5C), proliferation rates were still <1%. We also labeled 8-month-old female BALB/c mice for 1 week, and they had lower rates of proliferation (0.242 ± 0.064% per day) and showed greater variation, with some females labeling from as few as 1 in 2,165 cells per day to as many as 1 in 176 cells (n = 8). Overall, our data imply that adult -cell proliferation is a very rare event, with 1 in 1,400 cells replicating within a 24-h period for 1-year-old mice.
Minimal proliferation of glucagon-, somatostatin-, or pancreatic polypeptideeCcontaining cells in aged adult mice.
To examine whether our findings of low adult -cell proliferation applied to other hormone-containing islet cells, we immunostained pancreata with antibodies against glucagon, somatostatin, or pancreatic polypeptide as well as BrdU. Proliferating glucagon-, somatostatin-, or pancreatic polypeptideeCcontaining cells were very rare in all groups, even after 16 weeks of continuous BrdU labeling (Fig. 4). Thus, our findings of infrequent proliferation in glucagon-, somatostatin-, and pancreatic polypeptideeCcontaining cells is consistent with our findings in -cells, suggesting that adult islet endocrine cells are largely quiescent.
No evidence for BrdU breakdown during labeling.
Problems with long-term BrdU labeling could provide a possible alternate explanation for our findings of low adult -cells and replication -cells and other endocrine-containing islet cells. If BrdU-containing water solution rapidly degraded at room temperature in drinking water bottles, only partial BrdU labeling would result. As a precaution against this possibility, drinking water bottles were wrapped with aluminum foil, and the BrdU water solution was changed weekly. To evaluate for potential BrdU breakdown, we performed additional BrdU labeling in two young male mice (12 weeks of age) using recycled BrdU drinking water obtained from mouse long-term labeling experiments after 1 week of use. We found that this recycled water in young mice experiment resulted in high rates of -cell proliferation: 4.8 ± 0.9 and 15.8 ± 2.0% per day, respectively (Fig. 6A). Hence, BrdU-containing water solution was still highly active after 1 week in our experiments. We conclude that the low rates of BrdU incorporation in adult -cells cannot be explained by BrdU breakdown. Furthermore, our results suggest -cell proliferation declines as mice age, confirming previous reports (12).
Minimal -cell proliferation in aged adult mice measured with short-term BrdU.
Our results with long-term BrdU labeling suggest that far less -cell turnover occurs than previously appreciated. However, a possible alternate explanation for our findings is that very long-term continuous exposure to BrdU somehow impairs -cell proliferation in a nonlinear manner, resulting in progressively lower rates of -cell proliferation after prolonged BrdU exposure. Long-term BrdU exposure is generally well tolerated in mice, and to our knowledge has not been reported to impair -cell proliferation. Nonetheless, to rule out the possibility that long-term BrdU impaired proliferation, we performed extensive -cell proliferation analysis of pancreata after a short pulse of BrdU (6 h) in an identical cohort of mice of similar age from both backgrounds as well as 5-day-old newborn mice from the c57BL/6 x 129Sv mixed background. We found minimal evidence of proliferation in pancreata from 1-year-old c57BL/6 x 129Sv mice: 0.010 ± 0.003% of -cells were BrdU positive after a 6-h label, corresponding to a daily -cell proliferation rate of 0.039 ± 0.01% (n = 5). In comparison, 8-month-old mice from the BALB/c background showed slightly elevated proliferation: 0.107 ± 0.032% over 6 h, corresponding to a daily proliferation rate of 0.426 ± 0.129% (n = 8). These values are very similar to the overall daily -cell proliferation rates we obtained with long-term BrdU labeling (0.0701 ± 0.0227% at 1 year and 0.678 ± 0.060% at 8 months), and this suggests that long-term BrdU labeling accurately measures -cell replication. In comparison to our studies in aged adult mice, 5-day-old wild-type mice had a very high rate of -cell proliferation: 6.7 ± 1.0% of -cells were BrdU positive after a 6-h label, corresponding to a daily -cell proliferation rate of 26.8 ± 3.8% (n = 7).
Streptozotocin-inducible -cell replication detected in aged adult mice.
Because -cell proliferation in our aged cohort of mice was so much lower than expected, we performed low-dose streptozotocin administration to further test our ability to accurately detect -cell replication in aged mice and to examine whether aged adult -cells had undergone permanent mitotic exit. Low-dose streptozotocin has been widely used to induce -cell proliferation in rodents, and it is associated with hypoglycemia and hyperglycemia followed by recovery when used in small repeated doses (24). We treated a cohort of 1-year-old mice with low-dose streptozotocin (30 mg/kg) for 7 days followed by 14 days of recovery, while being continuously labeled with BrdU in drinking water. As expected, low-dose streptozotocineCtreated mice had a 10-fold induction of BrdU/insulin-positive -cells compared with islets of control treated mice (from 0.08% per day to 0.93% per day), although the response to streptozotocin was variable (Fig. 6B, Table 1). Islets frequently had evidence of infiltrating T-cells (a known consequence of low-dose streptozotocin treatment) that were consistently BrdU positive. Thus, long-term BrdU infusion is capable of detecting high rates of -cell proliferation induced by provocative stimuli, supporting the observation that the true static rate of -cell proliferation in aged adult mice is very low. Furthermore, we conclude that aged adult -cells may be capable of entering mitosis under some circumstances.
Minimal -cell death detected in aged adult mice.
To further study the low rates of -cell turnover observed in our model, we performed extensive TUNEL analysis of pancreata from our BrdU-labeled studies. Similar to our results with BrdU, TUNEL-positive -cells were extremely rare (mean TUNEL-positive content 0.0011 ± 0.0003%). Thus, our results of very rare -cell proliferation and similarly rare TUNEL-positive -cell death suggest that adult -cell turnover rates are extremely low.
No correlation between islet size and -cell replication in aged adult mice.
It has been speculated that new -cells largely grow within small islets, replicating from insulin-positive ductal cells or solitary pancreatic -cells to expand into larger islets (14,23). In such a model, smaller islets should be highly proliferative, with greater -cell BrdU incorporation rates than larger islets. However, we found no such inverse correlation between islet size and BrdU incorporation rate in islets examined after any length of BrdU infusion or after low-dose streptozotocin administration (Fig. 7). Thus, our results are consistent with an intraislet model of -cell expansion, with insulin-positive cells proliferating in islets of all sizes at roughly equivalent rates.
DISCUSSION
It has been known for some time that -cell replication declines as rodents age, but it was widely assumed that proliferation rates eventually stabilized at a rate of 1eC3% per day (6,17,25). Using long-term BrdU labeling, we now show that -cells proliferate very slowly in 1-year-old mice, with 1 in 1,400 cells replicating per day. In our cohort, we confirm previous findings of an age-dependent decline in -cell replication, but we show that -cell replication eventually approaches 0 (Fig. 5B). Thus, the low -cell replication rates observed in our study of aged mice challenge preexisting notions of adult -cell turnover that implied that ongoing replication of -cells or -cell precursors was necessary for maintenance of adult -cell number (12,17). Given that mice on a similar genetic background live for 24eC30 months (26), it seems highly likely that most -cells present in 1-year-old mice would still be alive for the duration of a mouse’s natural life span. Furthermore, our findings may be widely applicable to human islets: -cell turnover is infrequent in human pancreata (2).
Because -cells replicate very slowly, we find that long-term BrdU labeling is a very useful tool in estimating adult -cell turnover. The analysis involved in short-term labeling is much more laborious, in that a greater number of sections must be analyzed per animal because so few cells are actually dividing within a 6-h time span (an average of 27,302 -cells were analyzed per animal for the 6-h BrdU label versus an average of 12,613 -cells per animal in the long term). By measuring long-term growth over the total population of insulin-secreting cells, we not only minimized the amount of labor involved, but we were able to measure more proliferation events, minimizing the standard error. Long-term labeling also allowed correlation between islet size and the -cell proliferation rate: instead of smaller islets becoming larger ones, our results show islets of any size have the potential to expand. One potential limitation of long-term BrdU labeling is with a heterogeneous cell population: if a cell divides twice while exposed to BrdU, long-term BrdU labeling would underrepresent the true -cell replication rate (Fig. 1). Notably, we find that long- and short-term BrdU label give equivalent results. Therefore, if special populations of frequently proliferating -cells exist in islets, they likely represent a small percentage of total -cells.
In comparison to previously published studies where -cell turnover was observed in 1eC3% of adult -cells in rats (6,17), we found that -cell turnover is a very rare event in aged adult mice. Why are our results so much lower than those previously observed Animal age seems to be the most important factor: we performed our studies in aged mice to better differentiate -cell turnover from -cell expansion, which occurs through the first year of life. Although animal species could also explain the different results, this seems unlikely. Important technical differences probably also contribute to our findings of lower rates of -cell proliferation. Several previous studies examined proliferation using BrdU antisera combined with a noneC-cell antisera cocktail against glucagon, somatostatin, and pancreatic polypeptide, followed by immunoperoxidase detection with secondary antisera (6,17,27), a technique that identifies BrdU-positive cells within islets. Notably, this technique cannot distinguish proliferating insulin-positive cells from hormone-negative cells. To address these limitations, we directly detected proliferating -cells using antisera against BrdU and insulin in combination with fluorescent secondary antisera. We found that many proliferating cells within aged adult islets are actually hormone negative (for insulin, glucagon, somatostatin, or pancreatic polypeptide); several such examples can be seen in Fig. 3 (red arrows). Thus, estimates of -cell replication that use measurement of intraislet replication could be biased by inclusion of these hormone-negative cells.
Adult -cells grow in response to increased metabolic demands, allowing the pancreas to adapt and produce sufficient insulin (28). In our aged cohort, we were able to stimulate -cell replication in aged adult mice using the -cell toxin streptozotocin in low doses. This data would imply that aged adult -cells have not undergone terminal mitotic exit. However, it is important to note that the response to streptozotocin was quite variable. Given that the cohort was small (three animals per group), it remains possible that variation within this cohort might under- or overrepresent the -cell proliferative response to this provocative stimulus in aged animals. Nonetheless, given the low rate of basal -cell proliferation, it seems possible that aged -cells might not always be able to enter cell cycle from such a profoundly quiescent state. Thus, in states of increased metabolic demand, inadequate adult -cell adaptation and growth could result in insufficient -cell mass, impaired -cell function, and type 2 diabetes.
In summary, we show that -cell replication is a rare event in aged adult mice, with minimal evidence of -cell turnover. We conclude that aged murine -cells are in a mostly quiescent G0 state. Although it has been assumed that signaling pathways that promote -cell growth are also active in -cell remodeling and adaptation, this has not been formally proven in most cases except for the insulin/IGF signaling molecule insulin receptor substrate 2 (5,13,29). Therefore, it is possible that some factors shown to regulate -cell growth in adolescence might not be required for adult -cell survival. Similarly, other -cell mitogenic signals might only be involved in adult -cell adaptation, allowing -cells to emerge from the quiescent G0 state to enter the G1 state and divide. Moreover, human type 2 diabetes could conceivably occur from either of two scenarios: inadequate -cell growth or insufficient -cell adaptation. If true, drugs that promote -cell adaptation might be especially effective diabetes treatments, even in the face of severe insulin resistance. We anticipate that by characterizing normal -cell turnover, future studies will expand on this concept to characterize the signaling pathways that regulate adult -cell life span, turnover, and adaptation. Such studies will hopefully give rise to novel therapies for patients with type 1 and type 2 diabetes.
FOOTNOTES
BrdU, 5-bromo-2-deoxyuridine; DAPI, 4',6 diamino-2-phenylindole; TUNEL, transferase-mediated dUTP nick-end labeling.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Foulis AK, Liddle CN, Farquharson MA, Richmond JA, Weir RS: The histopathology of the pancreas in type 1 (insulin-dependent) diabetes mellitus: a 25 year review of death in patients under 20 years of age in the United Kingdom. Diabetologia29 :267 eC274,1986
Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes52 :102 eC110,2003
Yoon KH, Ko SH, Cho JH, Lee JM, Ahn YB, Song KH, Yoo SJ, Kang MI, Cha BY, Lee KW, Son HY, Kang SK, Kim HS, Lee IK, Bonner-Weir S: Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrinol Metab88 :2300 eC2308,2003
Jensen J: Gene regulatory factors in pancreatic development. Dev Dyn229 :176 eC200,2004
Dickson LM, Rhodes CJ: Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt Am J Physiol Endocrinol Metab287 :E192 eCE198,2004
Montanya E, Nacher V, Biarnes M, Soler J: Linear correlation between beta-cell mass and body weight throughout the lifespan in Lewis rats: role of beta-cell hyperplasia and hypertrophy. Diabetes49 :1341 eC1346,2000
Kushner JA, Ye J, Schubert M, Burks DJ, Dow MA, Flint CL, Dutta S, Wright CV, Montminy MR, White MF: Pdx1 restores beta cell function in Irs2 knockout mice. J Clin Invest109 :1193 eC1201,2002
Bock T, Pakkenberg B, Buschard K: Increased islet volume but unchanged islet number in ob/ob mice. Diabetes52 :1716 eC1722,2003
Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, Barbacid M: Loss of cdk4 expression causes insulin-deficient diabetes and cdk4 activation results in -islet cell hyperplasia. Nat Genet22 :44 eC52,1999
Kushner JA, Ciemerych MA, Sicinska E, Wartschow LM, Teta M, Long SY, Sicinski P, White MF: Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth. Mol Cell Biol25 :3752 eC3762,2005
Georgia S, Bhushan A: Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass. J Clin Invest114 :963 eC968,2004
Dor Y, Brown J, Martinez OI, Melton DA: Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature429 :41 eC46,2004
Lin X, Taguchi A, Park S, Kushner JA, Li F, Li Y, White MF: Dysregulation of insulin receptor substrate 2 in beta cells and brain causes obesity and diabetes. J Clin Invest114 :908 eC916,2004
Bonner-Weir S, Toschi E, Inada A, Reitz P, Fonseca SY, Aye T, Sharma A: The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatr Diabetes5 (Suppl. 2) :16 eC22,2004
Pick A, Clark J, Kubstrup C, Levisetti M, Pugh W, Bonner-Weir S, Polonsky KS: Role of apoptosis in failure of -cell mass compensation for insulin resistance and -cell defects in the male Zucker diabetic fatty rat. Diabetes47 :358 eC364,1998
Mathis D, Vence L, Benoist C: Beta-cell death during progression to diabetes. Nature414 :792 eC798,2001
Finegood DT, Scaglia L, Bonner Weir S: Dynamics of beta-cell mass in the growing rat pancreas: estimation with a simple mathematical model. Diabetes44 :249 eC256,1995
Bonhoeffer S, Mohri H, Ho D, Perelson AS: Quantification of cell turnover kinetics using 5-bromo-2'-deoxyuridine. J Immunol164 :5049 eC5054,2000
Elson K, Ribeiro RM, Perelson AS, Simmons A, Speck P: The life span of ganglionic glia in murine sensory ganglia estimated by uptake of bromodeoxyuridine. Exp Neurol186 :99 eC103,2004
Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA: Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell82 :621 eC630,1995
Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner H, Park MY, Robker RL, Richards JS, McGinnis LK, Biggers JD, Eppig JJ, Bronson RT, Elledge SJ, Weinberg RA: Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature384 :470 eC474,1996
Hausman DB, DiGirolamo M, Bartness TJ, Hausman GJ, Martin RJ: The biology of white adipocyte proliferation. Obes Rev2 :239 eC254,2001
Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE: A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development. Diabetes42 :1715 eC1720,1993
Like AA, Rossini AA: Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus. Science193 :415 eC417,1976
Swenne I: Effects of aging on the regenerative capacity of the pancreatic B-cell of the rat. Diabetes32 :14 eC19,1983
Bluher M, Kahn BB, Kahn CR: Extended longevity in mice lacking the insulin receptor in adipose tissue. Science299 :572 eC574,2003
Montana E, Bonner Weir S, Weir GC: Transplanted beta cell response to increased metabolic demand: changes in beta cell replication and mass. J Clin Invest93 :1577 eC1582,1994
Bonner-Weir S, Deery D, Leahy JL, Weir GC: Compensatory growth of pancreatic -cells in adult rats after short-term glucose infusion. Diabetes38 :49 eC53,1989
Kubota N, Terauchi Y, Tobe K, Yano W, Suzuki R, Ueki K, Takamoto I, Satoh H, Maki T, Kubota T, Moroi M, Okada-Iwabu M, Ezaki O, Nagai R, Ueta Y, Kadowaki T, Noda T: Insulin receptor substrate 2 plays a crucial role in beta cells and the hypothalamus. J Clin Invest114 :917 eC927,2004(Monica Teta, Simon Y. Lon)
2 Division of Endocrinology, Children’s Hospital, Boston, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Although many signaling pathways have been shown to promote -cell growth, surprisingly little is known about the normal life cycle of preexisting -cells or the signaling pathways required for -cell survival. Adult -cells have been speculated to have a finite life span, with ongoing adult -cell replication throughout life to replace lost cells. However, little solid evidence supports this idea. To more accurately measure adult -cell turnover, we performed continuous long-term labeling of proliferating cells with the DNA precursor analog 5-bromo-2-deoxyuridine (BrdU) in 1-year-old mice. We show that -cells of aged adult mice have extremely low rates of replication, with minimal evidence of turnover. Although some pancreatic components acquired BrdU label in a linear fashion, only 1 in 1,400 adult -cells were found to undergo replication per day. We conclude that adult -cells are very long lived.
Inadequate islet mass is a central finding in both type 1 and type 2 diabetes, resulting in an absolute or relative insulin deficiency and subsequent metabolic complications (1,2eC3). Over the past decade, much has been learned about the specific signaling pathways that direct embryonic islet development (4) and postnatal islet growth (5). To compensate for increased metabolic demands, adult islet mass increases dramatically over the first year of life in rodents (6) and may represent growth within preexisting islets because islet density does not typically increase as mice age (7,8). It has been known for some time that adult -cell growth is dependent on cyclin-dependent kinase-4 (9). Recently, we (10) and others (11) showed that cyclins D2 and D1 are the likely partners with cyclin-dependent kinase-4 to promote G1 cell cycle progression in adult -cells. Remarkably, new evidence suggests that much of adult -cell growth may occur by replication of insulin-positive cells and not by -cell neogenesis (12). However, it is possible that -cell neogenesis may exist under some circumstances (13,14). Surprisingly little is known about the factors that promote adult -cell survival, although some amount of -cell apoptosis occurs in normal islets (15) as well as in pathological states (16). Although the absolute -cell death rate is unknown, -cell death appears to be a rare event. Supporting this notion, -cell area increases severalfold throughout adulthood, despite an apparent decline in daily proliferation rate (6,17).
Despite the lack of direct evidence, it has been largely assumed that -cells have a finite life span, with dying cells replenished by new -cells on an ongoing basis (12,14,17). One method of estimating cell life span is to measure the growth of total cell populations and cellular proliferation rate. In this model, the cell turnover rate is reflected by the proportion of cellular proliferation that does not contribute to cell population growth. In the simplest example of a stable population of cells with no net growth, cellular proliferation must be explained by ongoing loss and reflect the true cellular turnover rate. Because of inherent inaccuracies in measuring both cell growth and cellular proliferation rates, this technique is particularly well suited to organs or cell populations that are fairly stable or change very slowly. However, even in easily modeled cell populations, estimates of cell life span by this method are nonetheless indirect and do not readily account for nonproliferative neogenesis (i.e., cellular trans-differentiation).
Using -cell proliferation data from young rats, Finegood et al. (17) showed that -cell proliferation rates decline from 20% per day in pups to 10% per day in adolescence and then to 2% in early adulthood. Based on limited data in aged animals, the authors assumed that -cell proliferation rates stabilize at 2% throughout adulthood, and they estimated -cell life span to be 1eC3 months. Similarly, Montanya et al. (6) found that intraislet proliferation rates declined to 0.8% per day in 1-year-old rats and was stable thereafter (0.2% 5-bromo-2-deoxyuridine [BrdU]-positive cells after a 6-h BrdU label). However, the authors estimated -cell proliferation by immunostaining with a cocktail of antisera specific to noneC-cell hormones and BrdU, which also counts proliferating noneChormone-containing islet cells. We hypothesized that -cell replication rates might continue to decline into adulthood, making estimates of -cell life span in young animals not applicable to the life span of mature -cells. However, little reliable data exists on -cell proliferation in aged mice to address this question.
To estimate adult -cell turnover, we performed continuous long-term BrdU labeling in aged adult mice, a technique previously used to measure the life span of lymphocytes (18) and ganglionic glia (19). Unlike the standard 6-h BrdU label, long-term BrdU labeling allowed us to calculate -cell proliferation in terms of the total cell population, giving us better insight into the rate of -cell turnover and life span. A priori, we predicted at least three possible models of BrdU labeling of -cells using continuous BrdU (Fig. 1). If -cells rapidly turn over, as predicted by Finegood et al. (17), near complete -cell BrdU labeling would occur if the period of label exceeded the typical -cell life span. In contrast, if -cells exhibit slow turn over, limited accrual of BrdU label would occur in -cells. Finally, heterogeneity within islets might result in nonlinear BrdU labeling: a limited population of rapidly dividing but short-lived -cells could coexist with slowly proliferating long-lived -cells. Surprisingly, our results show that adult mouse -cells acquire BrdU in a very slow manner and appear to be very long lived, with 1 in 1,400 mature -cells turning over per day.
RESEARCH DESIGN AND METHODS
Male wild-type c57BL/6 x 129Sv mixed genetic background mice were derived from a colony of cyclin D1+/eC::D2+/eC mice generously provided by Peter Sicinski, genotyped as previously described (20,21). Mice were maintained at a barrier animal facility in the Harvard School of Public Health and fed mouse diet 5020 9F (9% fat calculated by weight, 21.6% fat calculated by kilocalories; PMI Nutrition International, Richmond, IN). Random-fed glucose measurements were performed using a Glucometer Elite (Bayer). Male and female wild-type BALB/c mice (purchased from Taconic) were housed at the animal facility at the Children’s Hospital of Philadelphia and fed a similar diet (mouse diet 5015; PMI Nutrition International).
Statistics.
All results are reported as the means ± SE for equivalent groups. Results were compared with independent t tests (unpaired and two tailed) reported as P values.
Immunohistochemistry.
Pancreas samples were dissected from fed mice and fixed with 4% paraformaldehyde/PBS solution overnight. Then, 5-e longitudinal sections of paraffin blocks were rehydrated with xylene, followed by decreasing concentrations of ethanol, microwaved in 0.01 mol/l sodium citrate (pH 6.0) for 20 min, and permeabilized with 1% Triton X-100 in PBS before primary antisera incubation. Primary antisera were guinea pig anti-insulin (Zymed Laboratories Inc., South San Francisco, CA), rabbit anti-glucagon (Zymed), rabbit anti-somatostatin (Zymed), rabbit anti-pancreatic polypeptide (Zymed), and rat anti-BrdU (BU1/75; Accurate Chemical, Westbury, NY). Secondary antibodies were highly cross-adsorbed and labeled with Cy2 or Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclear staining was performed with 4',6 diamino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR).
Long-term BrdU proliferation analysis.
Mice were labeled continuously with BrdU by substituting drinking water bottles with bottles that contained 1 mg/ml BrdU. To ensure that the BrdU was bioactive, water bottles were completely wrapped with aluminum foil (to prevent light exposure), and the BrdU water solution was changed weekly with freshly prepared solution. Paraformaldehyde-fixed paraffin sections prepared from killed animals were stained with DAPI/insulin/BrdU, and images were acquired from all visible multicellular islets at 20x magnification. BrdU-positive -cell ratios were calculated as the means ± SE of BrdU-positive -cells over total -cells per section, two sections per animal. Individual animal results each represent an average of 46 islets counted per animal (range 40eC71) comprising an average of 12,613 -cells (range 3,445eC34,171). Counted -cells were estimated by measuring the cross-sectional -cell area of each acquired islet using the lasso tool and dividing by mean -cell size determined from multiple islets. Acinar cell proliferation analysis was performed by randomly acquiring five DAPI/insulin/BrdU images (20x magnification) of acinar cells from each section. Acinar cells were counted for total BrdU- and total DAPI-positive nuclei. Individual animal results of pancreatic acinar tissue each represent 10 fields counted per animal comprising an average of 1,455 cells (range 813eC3,802). Preadipocyte proliferation analysis was performed in a similar manner by acquiring five DAPI/insulin/BrdU images (20x magnification) of peripancreatic adipose tissue from each animal. Individual animal results of peripancreatic adipose tissue each represent an average of 10 fields counted per animal (range 5eC20) comprising an average of 801 cells (range 325-1592). Perigonadal fat pads were fixed and processed as per pancreata and stained with antisera against BrdU. Five DAPI/BrdU images were acquired at 20x magnification and counted for BrdU and DAPI adipocytes. Individual animal results of perigonadal fat pads each represent an average of 11 fields counted per animal (range 7eC15) comprising an average of 1,036 cells (range 424eC1,604).
Islet morphometry.
-Cell area was measured by acquiring at least 10 adjacent nonoverlapping images from each insulin-stained section, three sections per animal, using an Axioskop 2 plus mot with a 10x objective and a 0.63x converter (Carl Zeiss MicroImaging, Thornwood, NY) and captured with a Hamamatsu Orca ER digital camera. Images were analyzed for area with Open Lab 4.0 software, using the lasso tool to select individual islets and using individually optimized settings to maximize region-of-interest accuracy. Results of -cell quantification were expressed as the percentage of the total surveyed area containing cells positive for insulin. Results represent the average from 14 total animals (11 mice up to 12 months of age and 3 at 14 months of age), each with an average of 102 fields counted per animal (range 57eC148).
Short-term BrdU islet proliferation analysis.
Short-term BrdU labeling -cell proliferation studies in aged mice were performed by injecting five different 9- to 12-month-old mice, also from the same genetic cohort of c57BL/6 x 129Sv background, with BrdU (100 e/g body wt; Roche) 6 h before they were killed. Triple-label DAPI/insulin/BrdU immunohistochemistry images were acquired from rehydrated paraformaldehyde-fixed, paraffin-embedded sections. All visible islets per pancreatic section (five sections per animal) were analyzed to determine total BrdU/insulin copositive cells and total islet count per section. Mean -cell size was determined by counting all insulin/DAPI copositive nuclei in at least five islets per animal. The -cell replication rate was then estimated by determining the mean islet size by measuring the cross-sectional area of all islets in the section. BrdU-positive -cell ratios were then calculated as the means ± SE of BrdU-positive -cells. Results represent the average of five total animals, each with an average of 439 islets counted per animal (range 256eC636) comprising an average of 27,302 cells per animal (range 6,415eC57,395).
Short-term BrdU labeling -cell proliferation studies in young mice were performed by injecting 5-day-old pups with BrdU 6 h before they were killed, as described above. The largest islets in each pancreatic section (two sections per animal) were analyzed to determine total BrdU-insulin copositive cells and the total islet count per section. Results represent the average of seven total animals, each with an average of 11 islets counted per animal (range 6eC17) comprising an average of 733 cells per animal (range 343eC1,215).
Islet apoptosis analysis.
Apoptosis analysis was performed on pancreas sections from 9- to 12-month-old male mice by the transferase-mediated dUTP nick-end labeling (TUNEL) method using Cy2-labeled reagents (Roche) on rehydrated and trypsin-predigested pancreas sections. Sections were subsequently stained for insulin and then stained with appropriate Cy3 secondary antisera, as above. All visible islets per pancreatic section (five sections per animal) were analyzed for the total number of DAPI/TUNEL/insulin copositive cells and for the total number of islets per section, and they were analyzed as per proliferation analysis. Results represent the average of 14 total animals, each with an average of 597 islets counted per animal (range 244eC814) comprising an average of 138,868 cells per animal (range 23,738eC365,232).
Low-dose streptozotocin treatment.
Mice were treated with five daily injections of low-dose (30 mg/kg) streptozotocin in 0.1 mol/l sodium citrate buffer (pH 4.5) or control sodium citrate buffer. Mice were given BrdU in drinking water continuously until they were killed 15 days after the end of streptozotocin treatment. Pancreata were processed and DAPI/insulin/BrdU images acquired as for other long-term BrdU-exposed mice. Results were averaged from individual animals (three per treatment), each of which represent an average of 45 islets counted per animal (range 28eC69) comprising an average of 8,686 -cells (range 3,859eC14,176).
RESULTS
Long-term BrdU exposure is well tolerated in adult mice.
To estimate adult -cell turnover, we analyzed -cell replication rates in adult islets by continuously treating 1-year-old mice with BrdU in drinking water for various lengths of time (2, 4, 8, and 16 weeks) immediately followed by killing. Mice tolerated long-term continuous BrdU exposure well, although one mouse died 2 weeks into BrdU labeling from unknown causes. Long-term BrdU exposure had little effect on body weight (data not shown). Similarly, long-term BrdU appeared to have little effect on glucose homeostasis; although blood glucose was slightly decreased in BrdU-treated mice over time, the trend was nonsignificant (r2 = 0.101). There was also no relationship between BrdU incorporation rates and glucose levels measured before the mice were killed (r2 = 0.006) (data not shown).
At the time they were killed, mice were grossly normal, with normal-appearing organs. Total pancreatic -cell area was very high (5.4 ± 1.1% total pancreas area). -Cell area was widely variable, with individual animal results ranging from 1.2 to 14.4% total pancreas area, which presumably reflects animal-to-animal differences in peripheral insulin resistance and subsequent insulin production within this aged mixed genetic background (c57BL/6 x 129Sv) cohort. Notably, no association was observed between the length of BrdU exposure and -cell area or between age at time of death and -cell area (data not shown).
Long-term BrdU labels rapidly proliferating cells in a predictable manner.
Pancreatic replication rates were measured by performing DAPI/insulin/BrdU triple staining of pancreatic sections. Most cells within peripancreatic lymph nodes were BrdU positive by 4 weeks, illustrating that long-term BrdU exposure efficiently labels rapidly turning over cells in vivo (Fig. 2). Similarly, peripancreatic fat depots were examined for evidence of proliferation in resident preadipocytes, which proliferate in vivo (22). Pancreatic preadipocytes proliferated slower than lymphoid cells, but after 16 weeks of BrdU labeling, many cells displayed evidence of past proliferation (Figs. 2 and 5A). Overall, pancreatic preadipocytes displayed evidence of past proliferation at a rate of 4.76 ± 0.49% per day, with individual animal results ranging from 2.55 ± 0.18 to 8.21 ± 0.97% per day. To verify that pancreatic fat depots were representative of other fat depots, we also examined proliferation in suprapubic fat pads from mice after 8 weeks of BrdU exposure (n = 3). Preadipocytes from perigonadal fat pads proliferated at a rate of 6.21 ± 0.38% per day, in agreement with our results with pancreatic fat depots.
Modest proliferation of pancreatic ductal and acinar cells.
Within the pancreas are rare insulin-positive pancreatic ductal cells, which have been speculated to serve as a reservoir for -cell progenitors in the adult pancreas (14,23). Notably, pancreatic ductal cells had infrequent evidence of past proliferation across all groups, although BrdU-positive ductal cells were more common in pancreata from mice labeled with BrdU for longer periods (Fig. 2). Similarly, insulin-positive pancreatic ductal cells appeared to be largely postmitotic in the mice examined, with extremely few insulin-positive ductal cells displaying BrdU immunoreactivity (Fig. 2, far right panels). Thus, at least in the basal state in aged adult mice, insulin-positive ductal cells do not appear to be a rapidly dividing precursor population.
Similar to ductal cells, pancreatic exocrine cells acquired BrdU label in a slower manner, with 25% of acinar cells showing evidence of past proliferation by 16 weeks (Figs. 2 and 5A). Acinar cell proliferation was 0.194 ± 0.039% per day, with individual animal results ranging from 0.079 ± 0.020 to 8.21 ± 0.97% per day. Thus, our results correspond to 1 in 513 acinar cells proliferating per day, and they suggest that most acinar cells are in a mostly quiescent state.
Minimal -cell proliferation in aged adult mice.
In contrast to other cells contained within pancreatic preparations, insulin/BrdU copositive cells were fairly rare and comprised only a small portion of total -cells, even in mice exposed to BrdU for very long periods (Figs. 3 and 5A). -Cell proliferation rates averaged 0.0701 ± 0.0227% per day, far lower than expected given previous data of 3% daily -cell proliferation in rats (12). However, considerable animal-to-animal heterogeneity was observed, and -cell proliferation rates ranged from 0.0085 ± 0.0020 to 0.3113 ± 0.0433% per day, a 36-fold difference. Strikingly, several animals had almost no evidence of past -cell proliferation, with as few as 1 in 11,764 -cells turning over per day in one animal. Other animals had much more -cell proliferation, with as many as 1 in 321 -cells turning over per day. Because of the large animal-to-animal variation in -cell proliferation rates, there was little correlation between the period of label and the percent of BrdU-positive -cells (r2 = 0.298) (Fig. 5A). In contrast, higher-proliferating tissues showed less animal-to-animal variation and better correlation in between the period of label and proliferation (peripancreatic adipocytes, r2 = 0.786; pancreatic acinar cells, r2 = 0.528) (Fig. 5A).
In an attempt to moderate the effect of genetic variation, 8-month-old male mice from a secondary and more homogeneous background, BALB/c, were continuously labeled with BrdU for 1 week. -Cell proliferation rates were slightly higher, with an average of 0.679 ± 0.06% per day (n = 8). Although these mice also showed significant animal-to-animal variation (from 0.456 ± 0.06 to 0.978 ± 0.06% per day) (Fig. 5C), proliferation rates were still <1%. We also labeled 8-month-old female BALB/c mice for 1 week, and they had lower rates of proliferation (0.242 ± 0.064% per day) and showed greater variation, with some females labeling from as few as 1 in 2,165 cells per day to as many as 1 in 176 cells (n = 8). Overall, our data imply that adult -cell proliferation is a very rare event, with 1 in 1,400 cells replicating within a 24-h period for 1-year-old mice.
Minimal proliferation of glucagon-, somatostatin-, or pancreatic polypeptideeCcontaining cells in aged adult mice.
To examine whether our findings of low adult -cell proliferation applied to other hormone-containing islet cells, we immunostained pancreata with antibodies against glucagon, somatostatin, or pancreatic polypeptide as well as BrdU. Proliferating glucagon-, somatostatin-, or pancreatic polypeptideeCcontaining cells were very rare in all groups, even after 16 weeks of continuous BrdU labeling (Fig. 4). Thus, our findings of infrequent proliferation in glucagon-, somatostatin-, and pancreatic polypeptideeCcontaining cells is consistent with our findings in -cells, suggesting that adult islet endocrine cells are largely quiescent.
No evidence for BrdU breakdown during labeling.
Problems with long-term BrdU labeling could provide a possible alternate explanation for our findings of low adult -cells and replication -cells and other endocrine-containing islet cells. If BrdU-containing water solution rapidly degraded at room temperature in drinking water bottles, only partial BrdU labeling would result. As a precaution against this possibility, drinking water bottles were wrapped with aluminum foil, and the BrdU water solution was changed weekly. To evaluate for potential BrdU breakdown, we performed additional BrdU labeling in two young male mice (12 weeks of age) using recycled BrdU drinking water obtained from mouse long-term labeling experiments after 1 week of use. We found that this recycled water in young mice experiment resulted in high rates of -cell proliferation: 4.8 ± 0.9 and 15.8 ± 2.0% per day, respectively (Fig. 6A). Hence, BrdU-containing water solution was still highly active after 1 week in our experiments. We conclude that the low rates of BrdU incorporation in adult -cells cannot be explained by BrdU breakdown. Furthermore, our results suggest -cell proliferation declines as mice age, confirming previous reports (12).
Minimal -cell proliferation in aged adult mice measured with short-term BrdU.
Our results with long-term BrdU labeling suggest that far less -cell turnover occurs than previously appreciated. However, a possible alternate explanation for our findings is that very long-term continuous exposure to BrdU somehow impairs -cell proliferation in a nonlinear manner, resulting in progressively lower rates of -cell proliferation after prolonged BrdU exposure. Long-term BrdU exposure is generally well tolerated in mice, and to our knowledge has not been reported to impair -cell proliferation. Nonetheless, to rule out the possibility that long-term BrdU impaired proliferation, we performed extensive -cell proliferation analysis of pancreata after a short pulse of BrdU (6 h) in an identical cohort of mice of similar age from both backgrounds as well as 5-day-old newborn mice from the c57BL/6 x 129Sv mixed background. We found minimal evidence of proliferation in pancreata from 1-year-old c57BL/6 x 129Sv mice: 0.010 ± 0.003% of -cells were BrdU positive after a 6-h label, corresponding to a daily -cell proliferation rate of 0.039 ± 0.01% (n = 5). In comparison, 8-month-old mice from the BALB/c background showed slightly elevated proliferation: 0.107 ± 0.032% over 6 h, corresponding to a daily proliferation rate of 0.426 ± 0.129% (n = 8). These values are very similar to the overall daily -cell proliferation rates we obtained with long-term BrdU labeling (0.0701 ± 0.0227% at 1 year and 0.678 ± 0.060% at 8 months), and this suggests that long-term BrdU labeling accurately measures -cell replication. In comparison to our studies in aged adult mice, 5-day-old wild-type mice had a very high rate of -cell proliferation: 6.7 ± 1.0% of -cells were BrdU positive after a 6-h label, corresponding to a daily -cell proliferation rate of 26.8 ± 3.8% (n = 7).
Streptozotocin-inducible -cell replication detected in aged adult mice.
Because -cell proliferation in our aged cohort of mice was so much lower than expected, we performed low-dose streptozotocin administration to further test our ability to accurately detect -cell replication in aged mice and to examine whether aged adult -cells had undergone permanent mitotic exit. Low-dose streptozotocin has been widely used to induce -cell proliferation in rodents, and it is associated with hypoglycemia and hyperglycemia followed by recovery when used in small repeated doses (24). We treated a cohort of 1-year-old mice with low-dose streptozotocin (30 mg/kg) for 7 days followed by 14 days of recovery, while being continuously labeled with BrdU in drinking water. As expected, low-dose streptozotocineCtreated mice had a 10-fold induction of BrdU/insulin-positive -cells compared with islets of control treated mice (from 0.08% per day to 0.93% per day), although the response to streptozotocin was variable (Fig. 6B, Table 1). Islets frequently had evidence of infiltrating T-cells (a known consequence of low-dose streptozotocin treatment) that were consistently BrdU positive. Thus, long-term BrdU infusion is capable of detecting high rates of -cell proliferation induced by provocative stimuli, supporting the observation that the true static rate of -cell proliferation in aged adult mice is very low. Furthermore, we conclude that aged adult -cells may be capable of entering mitosis under some circumstances.
Minimal -cell death detected in aged adult mice.
To further study the low rates of -cell turnover observed in our model, we performed extensive TUNEL analysis of pancreata from our BrdU-labeled studies. Similar to our results with BrdU, TUNEL-positive -cells were extremely rare (mean TUNEL-positive content 0.0011 ± 0.0003%). Thus, our results of very rare -cell proliferation and similarly rare TUNEL-positive -cell death suggest that adult -cell turnover rates are extremely low.
No correlation between islet size and -cell replication in aged adult mice.
It has been speculated that new -cells largely grow within small islets, replicating from insulin-positive ductal cells or solitary pancreatic -cells to expand into larger islets (14,23). In such a model, smaller islets should be highly proliferative, with greater -cell BrdU incorporation rates than larger islets. However, we found no such inverse correlation between islet size and BrdU incorporation rate in islets examined after any length of BrdU infusion or after low-dose streptozotocin administration (Fig. 7). Thus, our results are consistent with an intraislet model of -cell expansion, with insulin-positive cells proliferating in islets of all sizes at roughly equivalent rates.
DISCUSSION
It has been known for some time that -cell replication declines as rodents age, but it was widely assumed that proliferation rates eventually stabilized at a rate of 1eC3% per day (6,17,25). Using long-term BrdU labeling, we now show that -cells proliferate very slowly in 1-year-old mice, with 1 in 1,400 cells replicating per day. In our cohort, we confirm previous findings of an age-dependent decline in -cell replication, but we show that -cell replication eventually approaches 0 (Fig. 5B). Thus, the low -cell replication rates observed in our study of aged mice challenge preexisting notions of adult -cell turnover that implied that ongoing replication of -cells or -cell precursors was necessary for maintenance of adult -cell number (12,17). Given that mice on a similar genetic background live for 24eC30 months (26), it seems highly likely that most -cells present in 1-year-old mice would still be alive for the duration of a mouse’s natural life span. Furthermore, our findings may be widely applicable to human islets: -cell turnover is infrequent in human pancreata (2).
Because -cells replicate very slowly, we find that long-term BrdU labeling is a very useful tool in estimating adult -cell turnover. The analysis involved in short-term labeling is much more laborious, in that a greater number of sections must be analyzed per animal because so few cells are actually dividing within a 6-h time span (an average of 27,302 -cells were analyzed per animal for the 6-h BrdU label versus an average of 12,613 -cells per animal in the long term). By measuring long-term growth over the total population of insulin-secreting cells, we not only minimized the amount of labor involved, but we were able to measure more proliferation events, minimizing the standard error. Long-term labeling also allowed correlation between islet size and the -cell proliferation rate: instead of smaller islets becoming larger ones, our results show islets of any size have the potential to expand. One potential limitation of long-term BrdU labeling is with a heterogeneous cell population: if a cell divides twice while exposed to BrdU, long-term BrdU labeling would underrepresent the true -cell replication rate (Fig. 1). Notably, we find that long- and short-term BrdU label give equivalent results. Therefore, if special populations of frequently proliferating -cells exist in islets, they likely represent a small percentage of total -cells.
In comparison to previously published studies where -cell turnover was observed in 1eC3% of adult -cells in rats (6,17), we found that -cell turnover is a very rare event in aged adult mice. Why are our results so much lower than those previously observed Animal age seems to be the most important factor: we performed our studies in aged mice to better differentiate -cell turnover from -cell expansion, which occurs through the first year of life. Although animal species could also explain the different results, this seems unlikely. Important technical differences probably also contribute to our findings of lower rates of -cell proliferation. Several previous studies examined proliferation using BrdU antisera combined with a noneC-cell antisera cocktail against glucagon, somatostatin, and pancreatic polypeptide, followed by immunoperoxidase detection with secondary antisera (6,17,27), a technique that identifies BrdU-positive cells within islets. Notably, this technique cannot distinguish proliferating insulin-positive cells from hormone-negative cells. To address these limitations, we directly detected proliferating -cells using antisera against BrdU and insulin in combination with fluorescent secondary antisera. We found that many proliferating cells within aged adult islets are actually hormone negative (for insulin, glucagon, somatostatin, or pancreatic polypeptide); several such examples can be seen in Fig. 3 (red arrows). Thus, estimates of -cell replication that use measurement of intraislet replication could be biased by inclusion of these hormone-negative cells.
Adult -cells grow in response to increased metabolic demands, allowing the pancreas to adapt and produce sufficient insulin (28). In our aged cohort, we were able to stimulate -cell replication in aged adult mice using the -cell toxin streptozotocin in low doses. This data would imply that aged adult -cells have not undergone terminal mitotic exit. However, it is important to note that the response to streptozotocin was quite variable. Given that the cohort was small (three animals per group), it remains possible that variation within this cohort might under- or overrepresent the -cell proliferative response to this provocative stimulus in aged animals. Nonetheless, given the low rate of basal -cell proliferation, it seems possible that aged -cells might not always be able to enter cell cycle from such a profoundly quiescent state. Thus, in states of increased metabolic demand, inadequate adult -cell adaptation and growth could result in insufficient -cell mass, impaired -cell function, and type 2 diabetes.
In summary, we show that -cell replication is a rare event in aged adult mice, with minimal evidence of -cell turnover. We conclude that aged murine -cells are in a mostly quiescent G0 state. Although it has been assumed that signaling pathways that promote -cell growth are also active in -cell remodeling and adaptation, this has not been formally proven in most cases except for the insulin/IGF signaling molecule insulin receptor substrate 2 (5,13,29). Therefore, it is possible that some factors shown to regulate -cell growth in adolescence might not be required for adult -cell survival. Similarly, other -cell mitogenic signals might only be involved in adult -cell adaptation, allowing -cells to emerge from the quiescent G0 state to enter the G1 state and divide. Moreover, human type 2 diabetes could conceivably occur from either of two scenarios: inadequate -cell growth or insufficient -cell adaptation. If true, drugs that promote -cell adaptation might be especially effective diabetes treatments, even in the face of severe insulin resistance. We anticipate that by characterizing normal -cell turnover, future studies will expand on this concept to characterize the signaling pathways that regulate adult -cell life span, turnover, and adaptation. Such studies will hopefully give rise to novel therapies for patients with type 1 and type 2 diabetes.
FOOTNOTES
BrdU, 5-bromo-2-deoxyuridine; DAPI, 4',6 diamino-2-phenylindole; TUNEL, transferase-mediated dUTP nick-end labeling.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Foulis AK, Liddle CN, Farquharson MA, Richmond JA, Weir RS: The histopathology of the pancreas in type 1 (insulin-dependent) diabetes mellitus: a 25 year review of death in patients under 20 years of age in the United Kingdom. Diabetologia29 :267 eC274,1986
Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes52 :102 eC110,2003
Yoon KH, Ko SH, Cho JH, Lee JM, Ahn YB, Song KH, Yoo SJ, Kang MI, Cha BY, Lee KW, Son HY, Kang SK, Kim HS, Lee IK, Bonner-Weir S: Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrinol Metab88 :2300 eC2308,2003
Jensen J: Gene regulatory factors in pancreatic development. Dev Dyn229 :176 eC200,2004
Dickson LM, Rhodes CJ: Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt Am J Physiol Endocrinol Metab287 :E192 eCE198,2004
Montanya E, Nacher V, Biarnes M, Soler J: Linear correlation between beta-cell mass and body weight throughout the lifespan in Lewis rats: role of beta-cell hyperplasia and hypertrophy. Diabetes49 :1341 eC1346,2000
Kushner JA, Ye J, Schubert M, Burks DJ, Dow MA, Flint CL, Dutta S, Wright CV, Montminy MR, White MF: Pdx1 restores beta cell function in Irs2 knockout mice. J Clin Invest109 :1193 eC1201,2002
Bock T, Pakkenberg B, Buschard K: Increased islet volume but unchanged islet number in ob/ob mice. Diabetes52 :1716 eC1722,2003
Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, Barbacid M: Loss of cdk4 expression causes insulin-deficient diabetes and cdk4 activation results in -islet cell hyperplasia. Nat Genet22 :44 eC52,1999
Kushner JA, Ciemerych MA, Sicinska E, Wartschow LM, Teta M, Long SY, Sicinski P, White MF: Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth. Mol Cell Biol25 :3752 eC3762,2005
Georgia S, Bhushan A: Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass. J Clin Invest114 :963 eC968,2004
Dor Y, Brown J, Martinez OI, Melton DA: Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature429 :41 eC46,2004
Lin X, Taguchi A, Park S, Kushner JA, Li F, Li Y, White MF: Dysregulation of insulin receptor substrate 2 in beta cells and brain causes obesity and diabetes. J Clin Invest114 :908 eC916,2004
Bonner-Weir S, Toschi E, Inada A, Reitz P, Fonseca SY, Aye T, Sharma A: The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatr Diabetes5 (Suppl. 2) :16 eC22,2004
Pick A, Clark J, Kubstrup C, Levisetti M, Pugh W, Bonner-Weir S, Polonsky KS: Role of apoptosis in failure of -cell mass compensation for insulin resistance and -cell defects in the male Zucker diabetic fatty rat. Diabetes47 :358 eC364,1998
Mathis D, Vence L, Benoist C: Beta-cell death during progression to diabetes. Nature414 :792 eC798,2001
Finegood DT, Scaglia L, Bonner Weir S: Dynamics of beta-cell mass in the growing rat pancreas: estimation with a simple mathematical model. Diabetes44 :249 eC256,1995
Bonhoeffer S, Mohri H, Ho D, Perelson AS: Quantification of cell turnover kinetics using 5-bromo-2'-deoxyuridine. J Immunol164 :5049 eC5054,2000
Elson K, Ribeiro RM, Perelson AS, Simmons A, Speck P: The life span of ganglionic glia in murine sensory ganglia estimated by uptake of bromodeoxyuridine. Exp Neurol186 :99 eC103,2004
Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA: Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell82 :621 eC630,1995
Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner H, Park MY, Robker RL, Richards JS, McGinnis LK, Biggers JD, Eppig JJ, Bronson RT, Elledge SJ, Weinberg RA: Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature384 :470 eC474,1996
Hausman DB, DiGirolamo M, Bartness TJ, Hausman GJ, Martin RJ: The biology of white adipocyte proliferation. Obes Rev2 :239 eC254,2001
Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE: A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development. Diabetes42 :1715 eC1720,1993
Like AA, Rossini AA: Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus. Science193 :415 eC417,1976
Swenne I: Effects of aging on the regenerative capacity of the pancreatic B-cell of the rat. Diabetes32 :14 eC19,1983
Bluher M, Kahn BB, Kahn CR: Extended longevity in mice lacking the insulin receptor in adipose tissue. Science299 :572 eC574,2003
Montana E, Bonner Weir S, Weir GC: Transplanted beta cell response to increased metabolic demand: changes in beta cell replication and mass. J Clin Invest93 :1577 eC1582,1994
Bonner-Weir S, Deery D, Leahy JL, Weir GC: Compensatory growth of pancreatic -cells in adult rats after short-term glucose infusion. Diabetes38 :49 eC53,1989
Kubota N, Terauchi Y, Tobe K, Yano W, Suzuki R, Ueki K, Takamoto I, Satoh H, Maki T, Kubota T, Moroi M, Okada-Iwabu M, Ezaki O, Nagai R, Ueta Y, Kadowaki T, Noda T: Insulin receptor substrate 2 plays a crucial role in beta cells and the hypothalamus. J Clin Invest114 :917 eC927,2004(Monica Teta, Simon Y. Lon)