Spontaneous Recovery From Hyperglycemia by Regeneration of Pancreatic -Cells in Kir6.2G132S Transgenic Mice
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糖尿病学杂志 2006年第7期
1 Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
2 Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto, Japan
ELISA, enzyme-linked immunosorbent assay; KATP channel, ATP-sensitive K+ channel; DBA, Dolichos biflorus agglutinin; PCNA, proliferating cell nuclear antigen; PDX1, pancreatic duodenal homeobox 1; PGP9.5, protein gene product 9.5; PP, pancreatic polypeptide; TUNEL, transferase-mediated dUTP nick-end labeling; OGTT, oral glucose tolerance test
ABSTRACT
The ATP-sensitive K+ channel (KATP channel) in pancreatic -cells is a critical regulator in insulin secretion. We previously reported that transgenic mice expressing a dominant-negative form (Kir6.2G132S) of Kir6.2, a subunit of the KATP channel, specifically in -cells develop severe hyperglycemia in adults (8 weeks of age). In this study, we conducted a long-term investigation of the phenotype of these transgenic mice. Surprisingly, hyperglycemia was spontaneously improved with concomitant improvement of pancreatic insulin content in the transgenic mice at >25 weeks of age. Insulin-positive cells and pancreatic duodenal homeobox 1 (PDX1)-positive cells both were clearly increased in the older compared with the younger transgenic mice. Interestingly, cells labeled with the lectin Dolichos biflorus agglutinin (DBA), a potential indicator of uncommitted pancreatic epithelial/ductal cells, were detected in the islets of the transgenic mice but not in those of wild-type mice. In addition, a subset of the DBA-labeled cells was positive for PDX1, insulin, glucagon, somatostatin, or pancreatic polypeptide. Moreover, some of the DBA-labeled cells were also positive for a proliferating cell marker. These results show that the Kir6.2G132S transgenic mouse is a useful model for studying -cell regeneration and that DBA-labeled cells participate in the process.
Pancreatic -cells play a central role in controlling blood glucose levels by secreting insulin. Insulin is secreted in a highly regulated manner involving complex intracellular signaling in response to various stimuli (1eC3). Disruption of the system causes disorders of glucose homeostasis, such as diabetes and hypoglycemia (4). In addition, -cell mass is also crucial in the regulation of blood glucose levels. Accumulating evidence suggests that loss of -cell mass can result in a shortage of insulin supply for peripheral tissues that utilize blood glucose, leading to diabetes (5,6). Pancreatic -cell mass is maintained throughout the lifetime by replication of preexisting -cells and/or neogenesis from progenitor cells yet to be identified (7,8). Regeneration of pancreatic -cells has been shown in many animal studies, including chemical induction of diabetes (9eC12), partial pancreatectomy (13,14), cellophane wrapping (15,16), duct ligation (17), and transgenic overexpression of cytokines or growth factors (18eC20). Although neonatal rats injected with streptozotocin later show an improvement of hyperglycemia (21eC23), no good animal models have been reported showing spontaneous recovery from the diabetic state in adults.
ATP-sensitive K+ channels (KATP channels) couple the metabolic state of the cell to its membrane potential and regulate various cellular functions (24,25). In pancreatic -cells, closure of the KATP channels causes depolarization of the plasma membrane and opening of the voltage-dependent Ca2+ channels, allowing the Ca2+ influx that triggers insulin secretion (1eC3). Studies of various KATP channel genetically engineered mice have demonstrated that the -cell KATP channel is critical in both glucose-induced and sulfonylurea-induced insulin secretion (26eC28). We previously generated transgenic mice expressing a dominant-negative form of Kir6.2 (Kir6.2G132S, a substitution of glycine with serine at position 132), the pore-forming subunit of the KATP channel, specifically in the -cells (26). These mice exhibited hypoglycemia with unregulated insulin secretion as neonates, but they developed severe hyperglycemia with almost no insulin response to glucose, which is generally associated with decreased -cell population with age, probably because of accelerated apoptosis (26).
In the course of analyzing the phenotype of Kir6.2G132S transgenic mice, we found that the blood glucose levels in the older transgenic mice fed ad libitum had become greatly improved. In addition, the serum insulin levels were increased in the older compared with the younger transgenic mice. These findings suggested spontaneous regeneration of pancreatic -cells in Kir6.2G132S transgenic mice as they become older.
In the current study, we show that both insulin content and serum insulin levels are increased significantly in older compared with younger transgenic mice. We find that pancreatic -cell regeneration occurs in Kir6.2G132S transgenic mice as they age (>25 weeks of age) and that cells labeled with Dolichos biflorus agglutinin (DBA), a lectin that is a potential marker for uncommitted epithelial/ductal cells in the developing pancreas (29), participate in the regeneration process of pancreatic -cells in Kir6.2G132S transgenic mice. The Kir6.2G132S transgenic mouse is thus a useful model for studying spontaneous regeneration of -cells in vivo.
RESEARCH DESIGN AND METHODS
Kir6.2G132S transgenic mice (M45 line) were originally generated in BDF1 mice as reported previously (26). In this study, we backcrossed the original Kir6.2G132S transgenic mice to C57BL/6J Jms Slc. Mice carrying the transgene were selected by genomic PCR. Female mice were used in this study. All animal experiments were approved by the animal research committees of the Chiba University Graduate School of Medicine (Chiba, Japan) and the Kobe University Graduate School of Medicine.
In vivo studies.
Changes in body weight, blood glucose levels, and serum insulin levels were measured. Blood glucose levels were measured by a GluTest blood glucose monitor (Sanwa Chemicals, Tokyo), and serum insulin levels were determined by an enzyme-linked immunosorbent assay (ELISA) kit (Morinaga, Yokohama, Japan). For measurement of insulin content of whole pancreas, excised pancreata were frozen in liquid nitrogen and disrupted with Cryopress (Microtech Nichion, Funabashi, Japan). The pancreas powder was then suspended in cold acid-ethanol, and insulin was extracted overnight at 4°C (30). The supernatants were diluted and subjected to ELISA as described previously (31).
Morphologic analysis and immunohistochemistry.
Pancreata were fixed with 10% buffered formalin, embedded in paraffin, and cut into sections (4 e蘭 thickness). After being deparaffinized and dehydrated, tissue sections were stained with hematoxylin and eosin. For immunohistochemistry, mice were perfused with phosphate-buffered paraformaldehyde (4%) to fix the tissues. Pancreata removed from the mice were fixed further with the same fixative overnight at 4°C and then dehydrated overnight with 30% sucrose/PBS at 4°C. Cryostat sections (10 e蘭 thick) were prepared from the fixed tissues and subjected to immunostaining.
Primary antibodies used were guinea pig anti-insulin (dilution 1:3,000; Mitsui Pharmaceuticals, Tokyo), rabbit anti-insulin (dilution 1:200; SantaCruz Biotechnology, Santa Cruz, CA), mouse anti-glucagon (dilution 1:3,000; Sigma-Aldrich, Tokyo), rabbit anti-somatostatin (dilution 1:500; Affiniti Research Products, Devon, U.K.), rabbit anti-pancreatic polypeptide (PP; dilution 1:500; Chemicon International, Temecula, CA), rabbit anti-amylase (dilution 1:400; Sigma-Aldrich), biotinylated DBA lectin (dilution 1:500; Vector Labs, Burlingame, CA), fluorescein-conjugated DBA lectin (dilution 1:100; Vector Labs), rabbit antieCprotein gene product 9.5 (PGP9.5; dilution 1:200; Chemicon International), and rabbit antieCproliferating cell nuclear antigen (PCNA; dilution 1:500, Oncogene Research Products, San Diego, CA). AntieCpancreatic duodenal homeobox 1 (PDX1) antiserum (dilution 1:5,000) was raised by immunizing guinea pigs with a 16-mer peptide (SPQPSSIAPLRPQEPR) corresponding to amino acid residues 269eC284 of mouse PDX1.
Secondary antibodies used were Alexa Fluor 488eCconjugated donkey anti-rabbit IgG (dilution 1:500), Alexa Fluor 488eCconjugated donkey anti-mouse IgG (dilution 1:500), Alexa Fluor 488eCconjugated streptavidin (dilution 1:1,000; Molecular Probes, Eugene, OR), Cy3-conjugated donkey anti-guinea pig IgG (dilution 1:500), Cy3-conjugated donkey anti-rabbit IgG (dilution 1:500), and Cy3-conjugated streptavidin (dilution 1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA). When staining PGP9.5, biotinylated goat anti-rabbit IgG (Nichirei, Tokyo) and Cy3- or Alexa Fluor 488eCconjugated streptavidin were used as secondary and tertiary antibodies, respectively. Nuclear staining was performed, using 4'-6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories, Kumamoto, Japan). Stained sections were visualized, using a fluorescent microscope (Olympus, Tokyo) equipped with a charged-coupled device camera (Hamamatsu Photonics, Hamamatsu, Japan).
Quantitative evaluation of islets containing DBA-labeled cells was performed on stained sections. At least 100 islets were examined per mouse, and three mice were analyzed at each individual age. An islet containing at least one DBA-labeled cell was counted as a DBA-positive islet. Results are expressed as the percent of DBA-positive islets.
Detection of apoptosis.
Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, as described previously (26). Light green was used as counterstain.
Quantitative evaluation of -cell area.
Insulin-stained sections were analyzed, using Scion Image software (version beta 4.0.2; Scion, Frederick, MD). A total of 20 random fields from five sections per mouse were examined to determine the area of insulin-positive cells, and three mice were analyzed at each age.
Oral glucose tolerance test.
An oral glucose tolerance test (OGTT) was performed on young (4eC6 weeks of age) and old mice (40eC60 weeks of age) as described previously (32). The mice were fasted for 16eC18 h before OGTT. Blood glucose levels were measured in whole blood with a GluTest blood glucose monitor (Sanwa Chemicals). Serum insulin levels were determined by an ultrahigh-sensitivity rat insulin ELISA kit (Morinaga) according to the manufacturer’s instructions.
RT-PCR analysis.
To confirm expression of Kir6.2G132S transgene, total RNA was isolated from five islets with an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. After treatment with DNase I (Qiagen), first-strand cDNA was synthesized with random primer, using ReverTra Ace (Toyobo, Osaka, Japan). Aliquots of the cDNA were used for PCR amplification with primer sets that were designed from the sequences of the transgene vector of pINSKir6.2G132S (26): sense primer 5'-GCTGCATCAGAAGAGGCCATC-3' corresponds to human preproinsulin gene, and antisense primer 5'-GCCCTGCTCTCGAATGTTCT-3' corresponds to mouse Kir6.2 gene. The expected size of PCR products is 289 bp. PCR (35 cycles) was conducted by a thermal cycler. Each cycle included denature (94°C, 15 s), annealing (60°C, 30 s), and extension (72°C, 1 min). Expression of -actin was used as an internal control.
Statistical analysis.
Results are the means ± SE. Data were analyzed by one-way ANOVA followed by Tukey-Kramer or Dunnett’s multiple comparison tests with StatView software (version 5.0.1; SAS Institute, Cary, NC). P values <0.05 were considered significant.
RESULTS
Blood glucose and serum insulin levels of Kir6.2G132S transgenic mice fed ad libitum.
As reported previously (26), the blood glucose levels at 4eC6 weeks of age in Kir6.2G132S transgenic mice fed ad libitum were significantly higher than in wild-type mice (22.4 ± 0.7 and 8.1 ± 0.3 mmol/l for transgenic and wild-type mice, respectively). In the current study, we found that the blood glucose levels gradually and significantly decreased with age in transgenic compared with wild-type mice, and there was no longer a significant difference between them at 50 weeks of age (10.3 ± 0.8 and 9.1 ± 0.8 mol/l, respectively) (Fig. 1A). On the other hand, the serum insulin levels at 4eC6 weeks of age in Kir6.2G132S transgenic mice fed ad libitum were considerably lower than in wild-type mice (17.4 ± 7.5 and 127.4 ± 24.4 pmol/l, respectively), but they increased in the transgenic mice with age. At 50 weeks, there was no significant difference in serum insulin levels between Kir6.2G132S transgenic and wild-type mice (143 ± 14.9 and 177.1 ± 24.1 pmol/l, respectively) (Fig. 1B).
Glucose tolerance and insulin secretion of Kir6.2G132S transgenic mice.
We then evaluated glucose tolerance and insulin secretion in Kir6.2G132S transgenic mice by OGTT performed by stomach gavage. We previously showed that the Kir6.2G132S mutation severely impaired glucose-induced insulin secretion because of defective KATP channel function and that Kir6.2G132S transgenic mice at 8 weeks of age exhibited severe glucose intolerance (26). In this study, although the fasting blood glucose levels of both younger and older Kir6.2G132S transgenic mice were similar to those of age-matched wild-type mice, both younger and older transgenic mice exhibited glucose tolerance impaired to a similar degree (Fig. 2A). In addition, no insulin response to glucose loading was detected, indicating that KATP channel function remains defective in older Kir6.2G132S transgenic mice (Fig. 2B). This was also confirmed by the finding that the Kir6.2G132S transgene is expressed in the older transgenic mice (Fig. 2C). These findings show that the improved serum insulin levels in the older transgenic mice do not result from normalization of KATP channel function in the pancreatic -cells.
Pancreatic insulin content of Kir6.2G132S transgenic mice.
We then investigated the association of the improved serum insulin levels in Kir6.2G132S transgenic mice with increased insulin content. At 4 weeks of age, the pancreatic insulin content of Kir6.2G132S transgenic mice was significantly lower than that of wild-type mice (60.6 ± 19.3 and 1,225.0 ± 68.4 pmol/pancreas for transgenic and wild-type mice, respectively). In accordance with the improved serum insulin levels, the pancreatic insulin content gradually increased with age. At 50 weeks of age, Kir6.2G132S transgenic mice exhibited pancreatic insulin content comparable to that of wild-type mice (2,054.7 ± 108.8 and 2,233.1 ± 73.1 pmol/pancreas, respectively) (Fig. 3). These findings strongly suggest that the increased serum insulin levels in Kir6.2G132S transgenic mice result from increased pancreatic insulin content.
Morphologic changes in the pancreas of Kir6.2G132S transgenic mice.
Because the restoration of pancreatic insulin content in older Kir6.2G132S transgenic mice suggested that the number of -cells is increased, we performed morphologic analyses of pancreatic islets. The pancreatic islets of Kir6.2G132S transgenic mice were somewhat transparent in comparison with wild-type islets (Figs. 4A and B). Sections stained with hematoxylin and eosin showed that many pancreatic islets of transgenic mice at 4 weeks of age were irregularly shaped, and the number of cells was less than in wild-type mice, but they were recovered by 25 weeks of age (Figs. 4CeCF). As reported previously (26), apoptotic cell death, detected by the TUNEL method, was accelerated in pancreatic islets of Kir6.2G132S transgenic mice at 4 weeks of age (Fig. 4G). The number of islets in cross sections containing TUNEL-positive cells was 59 of 87 islets examined (68%). The frequency of TUNEL-positive cells in a single islet cross section varied between 17 and 65%. In contrast, TUNEL-positive cells in pancreatic islets examined were rarely found in Kir6.2G132S transgenic mice at 25 weeks of age and in wild-type mice (Figs. 4HeCJ). These data indicate that apoptotic cell death of pancreatic -cells is the primary cause of the reduction in insulin content seen in Kir6.2G132S transgenic mice at younger ages, and they suggest that recovery of -cell mass occurs with age.
We next examined expressions of insulin and PDX1 by immunostaining. At 4 weeks of age, the number of insulin-positive cells was reduced significantly in Kir6.2G132S transgenic mice compared with wild-type mice (Figs. 5A and D), as previously reported (26). The -cells of Kir6.2G132S transgenic mice exhibited only weak immunoreactivity for insulin. In addition, only a few PDX1-positive cells in islets of Kir6.2G132S transgenic mice were detected (Fig. 5E). However, the number of insulin-positive cells gradually increased with age. Insulin immunoreactivity also increased (Figs. 5AeCC). The number of PDX1-positive cells increased as well as the number of insulin-positive cells (Figs. 5EeCG). Kir6.2G132S transgenic pancreas at 25 weeks of age was histologically indistinguishable from wild-type pancreas (Figs. 5D and H). Quantitative image analysis confirmed an increase in the -cell area with age in Kir6.2G132S transgenic mice pancreas (Fig. 6). These findings strongly suggest that pancreatic -cells are regenerated in Kir6.2G132S transgenic mice.
We then investigated other endocrine cells in Kir6.2G132S transgenic pancreas. Glucagon-positive cells were found in the central region of pancreatic islets in the younger Kir6.2G132S transgenic mice (Figs. 5I and J), consistent with our previous observation (26). In addition, both - and PP-cell populations were increased in the younger Kir6.2G132S transgenic mice. The distributions of - and PP-cells in the islets of the young transgenic mice also differed from those of wild-type mice. However, the architecture of the islets of Kir6.2G132S transgenic mice gradually improved with age and became almost normal at 25 weeks (Fig. 5C, G, and K).
Although it has been proposed that pancreatic -cells can be derived from -cells in diabetic rodents, based on the finding that insulin/somatostatin double-positive cells are detected in the regenerating islets (9eC11), we were unable to detect insulin/somatostatin double-positive cells in the islets of Kir6.2G132S transgenic mice at any time point examined (data not shown). In addition, neither insulin/glucagon nor insulin/PP double-positive cells were detected (data not shown).
Appearance of DBA-labeled cells in the islets of Kir6.2G132S transgenic mice.
It has been found that regeneration of pancreatic -cells might originate in progenitor cells located in ductal structures (13,33), and DBA, a lectin specific for N-acetylgalactosamine, has been used for staining pancreatic ductal cells (7,34,35). We attempted to stain ductal cells with DBA to determine whether insulin-positive cells are derived from that source. Although ductal structures in the pancreas of wild-type mice were labeled with DBA, no insulin-positive cells were found in the structures examined. Similarly, in the pancreas of Kir6.2G132S transgenic mice, ductal structures were labeled with DBA, and insulin-positive cells were rarely found (data not shown). DBA-labeled cells were not detected in the islets of wild-type mice. In contrast, they were detected frequently in the islets of Kir6.2G132S transgenic mice (Figs. 7AeCD). The frequency of the islets containing DBA-labeled cells in Kir6.2G132S transgenic mice gradually decreased with age (81.1 ± 2.5 and 41.3 ± 5.9% at 4 and 50 weeks of age, respectively) (Fig. 7F).
We then investigated the association of the appearance of DBA-labeled cells in the islets with the increase in -cell number in Kir6.2G132S transgenic mice. Although we were unable to detect DBA/insulin double-positive cells at 4 weeks of age (Fig. 8A), DBA-labeled cells in the islets began to express insulin at 12 weeks of age (Fig. 8B). At 25 weeks of age, a large number of DBA-labeled cells (50eC60% of total DBA-labeled cells within islets) expressed insulin (Fig. 8C). We also examined the expression of PDX1, a transcription factor essential for pancreatic development, in these DBA-labeled cells. Although the number of PDX1-positive cells was markedly reduced in young Kir6.2G132S transgenic islets (<30% of normal islets), some of the PDX1-positive cells were labeled with DBA (Fig. 8D). At 25 weeks of age, a considerable number of DBA-labeled cells expressed PDX1 (Fig. 8F). These results indicate that DBA-labeled cells participate in the process of regeneration of -cells in Kir6.2G132S transgenic mice.
We next investigated whether endocrine cells other than -cells are derived from DBA-labeled cells in Kir6.2G132S transgenic islets. DBA/somatostatin or DBA/PP double-positive cells were detected in Kir6.2G132S transgenic mice at 12 weeks of age and later (Figs. 8G and H). However, DBA/amylase double-positive cells were not detected. Interestingly, DBA-labeled duct-like structures with luminal amylase were formed in the islets of Kir6.2G132S transgenic mice (Fig. 8I). Moreover, cells double-positive for DBA and PCNA, a proliferating marker, were detected (Figs. 8J and K), demonstrating the proliferating capacity of DBA-labeled cells. These findings indicate that the DBA-labeled cells might possess some of the properties of pancreatic progenitor cells.
Because nestin and PGP9.5 have been reported to be potential progenitor markers during regeneration of pancreatic -cells (36eC38), we examined expressions of these marker proteins in islets from Kir6.2G132S transgenic mouse pancreas. Because nestin-positive cells were rarely detected in both young and old Kir6.2G132S transgenic mice (data not shown), it is unlikely that they participate in -cell regeneration in Kir6.2G132S transgenic mice. Increased expression of PGP9.5 has been detected during in vivo regeneration of endocrine cells (36) and in vitro transdifferentiation of insulin-secreting cells (37). We found an increase in the number of PGP9.5-positive cells in Kir6.2G132S transgenic mice (Figs. 8L and M) compared with wild-type mice. PGP9.5-positive cells expressing insulin were also found (Figs. 8L and M). Although the frequency of PGP9.5/insulin double-positive cells was lower than that of DBA/insulin double-positive cells, these results suggests that PGP9.5-positive cells also might contribute in part to -cell regeneration in Kir6.2G132S transgenic mice.
DISCUSSION
We previously found that adult Kir6.2G132S transgenic female mice (at 8 weeks of age) exhibit hyperglycemia with hypoinsulinemia caused by accelerated apoptosis (26). In the current study, we found that hyperglycemia with hypoinsulinemia in Kir6.2G132S transgenic mice recovers significantly with age. We also show that pancreatic insulin content and the number of -cells both increase as the Kir6.2G132S transgenic mice become older. The presence of the Kir6.2G132S mutation in the older Kir6.2G132S transgenic mice excludes the possibility that loss of the transgene is responsible for the recovery of -cell number in the older Kir6.2G132S transgenic mice. In fact, the insulin response to glucose loading was absent in the older Kir6.2G132S transgenic mice. Hence, the recovery of blood glucose and serum insulin are most likely caused by the restoration of pancreatic insulin content resulting from the increased -cell number in the older Kir6.2G132S transgenic mice. In a preliminary study, we found that serum insulin levels also improve with age and that -cell regeneration occurs in old male Kir6.2G132S transgenic mice, although hyperglycemia remains (K.O., K.M., T.M., and S.S., unpublished observations). It has been shown that insulin sensitivity in ovariectomized rats and mice is decreased (39,40) and that treatment with estradiol restores the decreased insulin sensitivity in the rats (39). Thus, the difference in improved hyperglycemia between male and female Kir6.2G132S transgenic mice may result from effects of sex hormones on insulin sensitivity.
Regeneration of pancreatic -cells can occur through several pathways, including neogenesis from progenitor cells located in ductal structures (13,33) or islets (9,10), self-replication of preexisting -cells (7,8), and transdifferentiation from noneC-cells (17,41). Although neonatal rats injected with streptozotocin later show improvement of hyperglycemia, no good animal model has previously been reported of spontaneous recovery from the diabetic state in adults. Although Kir6.2G132S transgenic mice exhibit hyperglycemia in adults, spontaneous recovery from hyperglycemia occurs with increased age. In addition, most -cell regeneration in this model is found within islets, and there are very few insulin-positive cells in ductal structures or exocrine acinar cells. Moreover, as assessed by the expression of PCNA or Ki67, there are no proliferating cells in ductal structures (Figs. 8J and K). These findings indicate that extraislet progenitor cells are not likely to contribute to the restoration of -cells and pancreatic insulin content in Kir6.2G132S transgenic mice.
Interestingly, DBA-labeled cells, which usually represent ductal cells in normal pancreas (7,34,35), were also detected within pancreatic islets of Kir6.2G132S transgenic mice. DBA-labeled cells were detected at highest frequency in Kir6.2G132S transgenic mice at 4 weeks of age. As the number of DBA-labeled cells decreased, the number of -cells increased in parallel with the increased insulin content. Many DBA-labeled cells expressed PDX1, a transcription factor critical in pancreatic development (42,43) that is found not only in mature -cells but also in endocrine progenitor cells in regenerating pancreas (10,11,14,20,44). DBA-labeled cells in the islets of Kir6.2G132S transgenic mice have proliferation potential, as assessed by double-staining with PCNA. Furthermore, DBA-labeled cells expressing insulin, somatostatin, or PP were also detected. These findings indicate that the DBA-labeled cells participate in the process of pancreatic islet cell regeneration in Kir6.2G132S transgenic mice, suggesting that the cells have some properties of progenitor cells, including plasticity in differentiation capacity and proliferation activity. Recently, it has been shown that DBA can be used as a specific marker of the embryonic pancreatic epithelium, which gives rise to endocrine and exocrine cells of developing pancreas (29). It is thus not unlikely that these DBA-labeled cells in adult Kir6.2G132S transgenic islets represent endocrine progenitor cells.
We found that the number of PGP9.5-positive cells increased in Kir6.2G132S transgenic mice islets. PGP9.5 is known as a panendocrine marker and has been reported recently to be a marker of endocrine progenitors (36eC38). Thus, PGP9.5-positive cells may also contribute in part to -cell regeneration in Kir6.2G132S transgenic mice.
We also found that functional ducts containing amylase immunoreactivity in their lumen are formed within the Kir6.2G132S transgenic islets. It has been reported that many ductal structures are formed in the pancreatic islets of mice expressing fibroblast growth factor 10 under control of the PDX1 promoter (45). Because fibroblast growth factor 10 may play an important role in maintaining the proliferating capacity of progenitor cells in embryonic pancreas (46,47), this finding could well indicate the activation of progenitor cells within the islets.
Recent studies have shown that self-replication of preexisting -cells constitutes the primary contribution in the maintenance of -cell mass in adult mice (7,8). However, the frequency of proliferating -cells was similar in Kir6.2G132S transgenic and wild-type mice (data not shown), so it is unlikely that self-replication of -cells is responsible for restoration of -cell number in this case.
In conclusion, our data demonstrate that regeneration of -cells occurs in the pancreatic islets of Kir6.2G132S transgenic mice and that DBA-labeled cells participate in the process. Although the mechanism of regeneration is not known at present, the Kir6.2G132S transgenic mouse should be a useful model for studying spontaneous regeneration of pancreatic -cells in vivo.
ACKNOWLEDGMENTS
This work was supported by a grant-in-aid for specially promoted research and a grant for the 21st Century Center of Excellence program from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from Takeda Chemical Industry.
Part of the study was conducted at the Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, to which K.O., K.M., T.M., and S.S. were formerly affiliated.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Seino S: ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 61:337eC362, 1999
Newgard CB, McGarry JD: Metabolic coupling factors in pancreatic -cell signal transduction. Annu Rev Biochem 64:689eC719, 1995
Rorsman P, Renstrom E: Insulin granule dynamics in pancreatic beta cells. Diabetologia 46:1029eC1045, 2003
DeFronzo RA: Lilly lecture 1987: The triumvirate: beta-cell, muscle, liver: a collusion responsible for NIDDM. Diabetes 37:667eC687, 1988
Kloppel G, Lohr M, Habich K, Oberholzer M, Heitz PU: Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Pathol Res 4:110eC125, 1985
Weir GC, Bonner-Weir S, Leahy JL: Islet mass and function in diabetes and transplantation. Diabetes 39:401eC405, 1990
Dor Y, Brown J, Martinez OI, Melton DA: Adult pancreatic -cells are formed by self-duplication rather than stem-cell differentiation. Nature 429:41eC46, 2004
Georgia S, Bhushan A: cell replication is the primary mechanism for maintaining postnatal cell mass. J Clin Invest 114:963eC968, 2004
Fernandes A, King LC, Guz Y, Stein R, Wright CV, Teitelman G: Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets. Endocrinology 138:1750eC1762, 1997
Guz Y, Nasir I, Teitelman G: Regeneration of pancreatic cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology 142:4956eC4968, 2001
Li L, Yi Z, Seno M, Kojima I: Activin A and betacellulin: effect on regeneration of pancreatic -cells in neonatal streptozotocin-treated rats. Diabetes 53:608eC615, 2004
Waguri M, Yamamoto K, Miyagawa JI, Tochino Y, Yamamori K, Kajimoto Y, Nakajima H, Watada H, Yoshiuchi I, Itoh N, Imagawa A, Namba M, Kuwajima M, Yamasaki Y, Hanafusa T, Matsuzawa Y: Demonstration of two different processes of -cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan. Diabetes 46:1281eC1290, 1997
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. Diabetes 42:1715eC1720, 1993
Sharma A, Zangen DH, Reitz P, Taneja M, Lissauer ME, Miller CP, Weir GC, Habener JF, Bonner-Weir S: The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 48:507eC513, 1999
Rosenberg L, Brown RA, Duguid WP: A new approach to the induction of duct epithelial hyperplasia and nesidioblastosis by cellophane wrapping of the hamster pancreas. J Surg Res 35:63eC72, 1983
Rafaeloff R, Pittenger GL, Barlow SW, Qin XF, Yan B, Rosenberg L, Duguid WP, Vinik AI: Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J Clin Invest 99:2100eC2109, 1997
Wang RN, Kloppel G, Bouwens L: Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 38:1405eC1411, 1995
Gu D, Sarvetnick N: Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. Development 118:33eC46, 1993
Bockman DE, Merlino G: Cytological changes in the pancreas of transgenic mice overexpressing transforming growth factor . Gastroenterology 103:1883eC1892, 1992
Song SY, Gannon M, Washington MK, Scoggins CR, Meszoely IM, Goldenring JR, Marino CR, Sandgren EP, Coffey RJ Jr, Wright CV, Leach SD: Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor . Gastroenterology 117:1416eC1426, 1999
Cantenys D, Portha B, Dutrillaux MC, Hollande E, Roze C, Picon L: Histogenesis of the endocrine pancreas in newborn rats after destruction by streptozotocin: an immunocytochemical study. Virchows Arch B Cell Pathol Incl Mol Pathol 35:109eC122, 1981
Dutrillaux MC, Portha B, Roze C, Hollande E: Ultrastructural study of pancreatic B cell regeneration in newborn rats after destruction by streptozotocin. Virchows Arch B Cell Pathol Incl Mol Pathol 39:173eC185, 1982
Wang RN, Bouwens L, Kloppel G: Beta-cell proliferation in normal and streptozotocin-treated newborn rats: site, dynamics and capacity. Diabetologia 37:1088eC1096, 1994
Ashcroft FM: Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Neurosci 11:97eC118, 1988
Miki T, Seino S: Roles of KATP channels as metabolic sensors in acute metabolic changes. J Mol Cell Cardiol 38:917eC925, 2005
Miki T, Tashiro F, Iwanaga T, Nagashima K, Yoshitomi H, Aihara H, Nitta Y, Gonoi T, Inagaki N, Miyazaki J, Seino S: Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel. Proc Natl Acad Sci U S A 94:11969eC11973, 1997
Miki T, Nagashima K, Tashiro F, Kotake K, Yoshitomi H, Tamamoto A, Gonoi T, Iwanaga T, Miyazaki J, Seino S: Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice. Proc Natl Acad Sci U S A 95:10402eC10406, 1998
Seghers V, Nakazaki M, DeMayo F, Aguilar-Bryan L, Bryan J: Sur1 knockout mice: a model for KATP channel-independent regulation of insulin secretion. J Biol Chem 275:9270eC9277, 2000
Kobayashi H, Spilde TL, Li Z, Marosky JK, Bhatia AM, Hembree MJ, Prasadan K, Preuett BL, Gittes GK: Lectin as a marker for staining and purification of embryonic pancreatic epithelium. Biochem Biophys Res Commun 293:691eC697, 2002
Filipponi P, Marcelli M, Nicoletti I, Pacifici R, Santeusanio F, Brunetti P: Suppressive effect of long term sulfonylurea treatment on A, B, and D cells of normal rat pancreas. Endocrinology 113:1972eC1979, 1983
Minami K, Yano H, Miki T, Nagashima K, Wang CZ, Tanaka H, Miyazaki JI, Seino S: Insulin secretion and differential gene expression in glucose-responsive and -unresponsive MIN6 sublines. Am J Physiol Endocrinol Metab 279:E773eCE781, 2000
Minami K, Morita M, Saraya A, Yano H, Terauchi Y, Miki T, Kuriyama T, Kadowaki T, Seino S: ATP-sensitive K+ channel-mediated glucose uptake is independent of IRS-1/phosphatidylinositol 3-kinase signaling. Am J Physiol Endocrinol Metab 285:E1289eCE1296, 2003
Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, O’Neil JJ: In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 97:7999eC8004, 2000
Watanabe M, Muramatsu T, Shirane H, Ugai K: Discrete distribution of binding sites for Dolichos biflorus agglutinin (DBA) and for peanut agglutinin (PNA) in mouse organ tissues. J Histochem Cytochem 29:779eC780, 1981
Jensen JN, Cameron E, Garay MV, Starkey TW, Gianani R, Jensen J: Recapitulation of elements of embryonic development in adult mouse pancreatic regeneration. Gastroenterology 128:728eC741, 2005
Yokoyama-Hayashi K, Takahashi T, Kakita A, Yamashina S: Expression of PGP9.5 in ductal cells of the rat pancreas during development and regeneration: can it be a marker for pancreatic progenitor cells Endocr J 49:61eC74, 2002
Minami K, Okuno M, Miyawaki K, Okumachi A, Ishizaki K, Oyama K, Kawaguchi M, Ishizuka N, Iwanaga T, Seino S: Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells. Proc Natl Acad Sci U S A 102:15116eC15121, 2005
Yamada S, Terada K, Ueno Y, Sugiyama T, Seno M, Kojima I: Differentiation of adult hepatic stem-like cells into pancreatic endocrine cells. Cell Transplant 14:647eC653, 2005
Kumagai S, Holmang A, Bjorntorp P: The effects of oestrogen and progesterone on insulin sensitivity in female rats. Acta Physiol Scand 149:91eC97, 1993
Puah JA, Bailey CJ: Effect of ovarian hormones on glucose metabolism in mouse soleus muscle. Endocrinology 117:1336eC1340, 1985
Bouwens L: Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 43:332eC336, 1998
Jonsson J, Carlsson L, Edlund T, Edlund H: Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371:606eC609, 1994
Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BL, Wright CV: PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983eC995, 1996
Holland AM, Gonez LJ, Naselli G, Macdonald RJ, Harrison LC: Conditional expression demonstrates the role of the homeodomain transcription factor Pdx1 in maintenance and regeneration of -cells in the adult pancreas. Diabetes 54:2586eC2595, 2005
Yamaoka T, Yoshino K, Yamada T, Yano M, Matsui T, Yamaguchi T, Moritani M, Hata J, Noji S, Itakura M: Transgenic expression of FGF8 and FGF10 induces transdifferentiation of pancreatic islet cells into hepatocytes and exocrine cells. Biochem Biophys Res Commun 292:138eC143, 2002
Bhushan A, Itoh N, Kato S, Thiery JP, Czernichow P, Bellusci S, Scharfmann R: Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128:5109eC5117, 2001
Norgaard GA, Jensen JN, Jensen J: FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development. Dev Biol 264:323eC338, 2003(Kazunobu Oyama, Kohtaro M)
2 Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto, Japan
ELISA, enzyme-linked immunosorbent assay; KATP channel, ATP-sensitive K+ channel; DBA, Dolichos biflorus agglutinin; PCNA, proliferating cell nuclear antigen; PDX1, pancreatic duodenal homeobox 1; PGP9.5, protein gene product 9.5; PP, pancreatic polypeptide; TUNEL, transferase-mediated dUTP nick-end labeling; OGTT, oral glucose tolerance test
ABSTRACT
The ATP-sensitive K+ channel (KATP channel) in pancreatic -cells is a critical regulator in insulin secretion. We previously reported that transgenic mice expressing a dominant-negative form (Kir6.2G132S) of Kir6.2, a subunit of the KATP channel, specifically in -cells develop severe hyperglycemia in adults (8 weeks of age). In this study, we conducted a long-term investigation of the phenotype of these transgenic mice. Surprisingly, hyperglycemia was spontaneously improved with concomitant improvement of pancreatic insulin content in the transgenic mice at >25 weeks of age. Insulin-positive cells and pancreatic duodenal homeobox 1 (PDX1)-positive cells both were clearly increased in the older compared with the younger transgenic mice. Interestingly, cells labeled with the lectin Dolichos biflorus agglutinin (DBA), a potential indicator of uncommitted pancreatic epithelial/ductal cells, were detected in the islets of the transgenic mice but not in those of wild-type mice. In addition, a subset of the DBA-labeled cells was positive for PDX1, insulin, glucagon, somatostatin, or pancreatic polypeptide. Moreover, some of the DBA-labeled cells were also positive for a proliferating cell marker. These results show that the Kir6.2G132S transgenic mouse is a useful model for studying -cell regeneration and that DBA-labeled cells participate in the process.
Pancreatic -cells play a central role in controlling blood glucose levels by secreting insulin. Insulin is secreted in a highly regulated manner involving complex intracellular signaling in response to various stimuli (1eC3). Disruption of the system causes disorders of glucose homeostasis, such as diabetes and hypoglycemia (4). In addition, -cell mass is also crucial in the regulation of blood glucose levels. Accumulating evidence suggests that loss of -cell mass can result in a shortage of insulin supply for peripheral tissues that utilize blood glucose, leading to diabetes (5,6). Pancreatic -cell mass is maintained throughout the lifetime by replication of preexisting -cells and/or neogenesis from progenitor cells yet to be identified (7,8). Regeneration of pancreatic -cells has been shown in many animal studies, including chemical induction of diabetes (9eC12), partial pancreatectomy (13,14), cellophane wrapping (15,16), duct ligation (17), and transgenic overexpression of cytokines or growth factors (18eC20). Although neonatal rats injected with streptozotocin later show an improvement of hyperglycemia (21eC23), no good animal models have been reported showing spontaneous recovery from the diabetic state in adults.
ATP-sensitive K+ channels (KATP channels) couple the metabolic state of the cell to its membrane potential and regulate various cellular functions (24,25). In pancreatic -cells, closure of the KATP channels causes depolarization of the plasma membrane and opening of the voltage-dependent Ca2+ channels, allowing the Ca2+ influx that triggers insulin secretion (1eC3). Studies of various KATP channel genetically engineered mice have demonstrated that the -cell KATP channel is critical in both glucose-induced and sulfonylurea-induced insulin secretion (26eC28). We previously generated transgenic mice expressing a dominant-negative form of Kir6.2 (Kir6.2G132S, a substitution of glycine with serine at position 132), the pore-forming subunit of the KATP channel, specifically in the -cells (26). These mice exhibited hypoglycemia with unregulated insulin secretion as neonates, but they developed severe hyperglycemia with almost no insulin response to glucose, which is generally associated with decreased -cell population with age, probably because of accelerated apoptosis (26).
In the course of analyzing the phenotype of Kir6.2G132S transgenic mice, we found that the blood glucose levels in the older transgenic mice fed ad libitum had become greatly improved. In addition, the serum insulin levels were increased in the older compared with the younger transgenic mice. These findings suggested spontaneous regeneration of pancreatic -cells in Kir6.2G132S transgenic mice as they become older.
In the current study, we show that both insulin content and serum insulin levels are increased significantly in older compared with younger transgenic mice. We find that pancreatic -cell regeneration occurs in Kir6.2G132S transgenic mice as they age (>25 weeks of age) and that cells labeled with Dolichos biflorus agglutinin (DBA), a lectin that is a potential marker for uncommitted epithelial/ductal cells in the developing pancreas (29), participate in the regeneration process of pancreatic -cells in Kir6.2G132S transgenic mice. The Kir6.2G132S transgenic mouse is thus a useful model for studying spontaneous regeneration of -cells in vivo.
RESEARCH DESIGN AND METHODS
Kir6.2G132S transgenic mice (M45 line) were originally generated in BDF1 mice as reported previously (26). In this study, we backcrossed the original Kir6.2G132S transgenic mice to C57BL/6J Jms Slc. Mice carrying the transgene were selected by genomic PCR. Female mice were used in this study. All animal experiments were approved by the animal research committees of the Chiba University Graduate School of Medicine (Chiba, Japan) and the Kobe University Graduate School of Medicine.
In vivo studies.
Changes in body weight, blood glucose levels, and serum insulin levels were measured. Blood glucose levels were measured by a GluTest blood glucose monitor (Sanwa Chemicals, Tokyo), and serum insulin levels were determined by an enzyme-linked immunosorbent assay (ELISA) kit (Morinaga, Yokohama, Japan). For measurement of insulin content of whole pancreas, excised pancreata were frozen in liquid nitrogen and disrupted with Cryopress (Microtech Nichion, Funabashi, Japan). The pancreas powder was then suspended in cold acid-ethanol, and insulin was extracted overnight at 4°C (30). The supernatants were diluted and subjected to ELISA as described previously (31).
Morphologic analysis and immunohistochemistry.
Pancreata were fixed with 10% buffered formalin, embedded in paraffin, and cut into sections (4 e蘭 thickness). After being deparaffinized and dehydrated, tissue sections were stained with hematoxylin and eosin. For immunohistochemistry, mice were perfused with phosphate-buffered paraformaldehyde (4%) to fix the tissues. Pancreata removed from the mice were fixed further with the same fixative overnight at 4°C and then dehydrated overnight with 30% sucrose/PBS at 4°C. Cryostat sections (10 e蘭 thick) were prepared from the fixed tissues and subjected to immunostaining.
Primary antibodies used were guinea pig anti-insulin (dilution 1:3,000; Mitsui Pharmaceuticals, Tokyo), rabbit anti-insulin (dilution 1:200; SantaCruz Biotechnology, Santa Cruz, CA), mouse anti-glucagon (dilution 1:3,000; Sigma-Aldrich, Tokyo), rabbit anti-somatostatin (dilution 1:500; Affiniti Research Products, Devon, U.K.), rabbit anti-pancreatic polypeptide (PP; dilution 1:500; Chemicon International, Temecula, CA), rabbit anti-amylase (dilution 1:400; Sigma-Aldrich), biotinylated DBA lectin (dilution 1:500; Vector Labs, Burlingame, CA), fluorescein-conjugated DBA lectin (dilution 1:100; Vector Labs), rabbit antieCprotein gene product 9.5 (PGP9.5; dilution 1:200; Chemicon International), and rabbit antieCproliferating cell nuclear antigen (PCNA; dilution 1:500, Oncogene Research Products, San Diego, CA). AntieCpancreatic duodenal homeobox 1 (PDX1) antiserum (dilution 1:5,000) was raised by immunizing guinea pigs with a 16-mer peptide (SPQPSSIAPLRPQEPR) corresponding to amino acid residues 269eC284 of mouse PDX1.
Secondary antibodies used were Alexa Fluor 488eCconjugated donkey anti-rabbit IgG (dilution 1:500), Alexa Fluor 488eCconjugated donkey anti-mouse IgG (dilution 1:500), Alexa Fluor 488eCconjugated streptavidin (dilution 1:1,000; Molecular Probes, Eugene, OR), Cy3-conjugated donkey anti-guinea pig IgG (dilution 1:500), Cy3-conjugated donkey anti-rabbit IgG (dilution 1:500), and Cy3-conjugated streptavidin (dilution 1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA). When staining PGP9.5, biotinylated goat anti-rabbit IgG (Nichirei, Tokyo) and Cy3- or Alexa Fluor 488eCconjugated streptavidin were used as secondary and tertiary antibodies, respectively. Nuclear staining was performed, using 4'-6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories, Kumamoto, Japan). Stained sections were visualized, using a fluorescent microscope (Olympus, Tokyo) equipped with a charged-coupled device camera (Hamamatsu Photonics, Hamamatsu, Japan).
Quantitative evaluation of islets containing DBA-labeled cells was performed on stained sections. At least 100 islets were examined per mouse, and three mice were analyzed at each individual age. An islet containing at least one DBA-labeled cell was counted as a DBA-positive islet. Results are expressed as the percent of DBA-positive islets.
Detection of apoptosis.
Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, as described previously (26). Light green was used as counterstain.
Quantitative evaluation of -cell area.
Insulin-stained sections were analyzed, using Scion Image software (version beta 4.0.2; Scion, Frederick, MD). A total of 20 random fields from five sections per mouse were examined to determine the area of insulin-positive cells, and three mice were analyzed at each age.
Oral glucose tolerance test.
An oral glucose tolerance test (OGTT) was performed on young (4eC6 weeks of age) and old mice (40eC60 weeks of age) as described previously (32). The mice were fasted for 16eC18 h before OGTT. Blood glucose levels were measured in whole blood with a GluTest blood glucose monitor (Sanwa Chemicals). Serum insulin levels were determined by an ultrahigh-sensitivity rat insulin ELISA kit (Morinaga) according to the manufacturer’s instructions.
RT-PCR analysis.
To confirm expression of Kir6.2G132S transgene, total RNA was isolated from five islets with an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. After treatment with DNase I (Qiagen), first-strand cDNA was synthesized with random primer, using ReverTra Ace (Toyobo, Osaka, Japan). Aliquots of the cDNA were used for PCR amplification with primer sets that were designed from the sequences of the transgene vector of pINSKir6.2G132S (26): sense primer 5'-GCTGCATCAGAAGAGGCCATC-3' corresponds to human preproinsulin gene, and antisense primer 5'-GCCCTGCTCTCGAATGTTCT-3' corresponds to mouse Kir6.2 gene. The expected size of PCR products is 289 bp. PCR (35 cycles) was conducted by a thermal cycler. Each cycle included denature (94°C, 15 s), annealing (60°C, 30 s), and extension (72°C, 1 min). Expression of -actin was used as an internal control.
Statistical analysis.
Results are the means ± SE. Data were analyzed by one-way ANOVA followed by Tukey-Kramer or Dunnett’s multiple comparison tests with StatView software (version 5.0.1; SAS Institute, Cary, NC). P values <0.05 were considered significant.
RESULTS
Blood glucose and serum insulin levels of Kir6.2G132S transgenic mice fed ad libitum.
As reported previously (26), the blood glucose levels at 4eC6 weeks of age in Kir6.2G132S transgenic mice fed ad libitum were significantly higher than in wild-type mice (22.4 ± 0.7 and 8.1 ± 0.3 mmol/l for transgenic and wild-type mice, respectively). In the current study, we found that the blood glucose levels gradually and significantly decreased with age in transgenic compared with wild-type mice, and there was no longer a significant difference between them at 50 weeks of age (10.3 ± 0.8 and 9.1 ± 0.8 mol/l, respectively) (Fig. 1A). On the other hand, the serum insulin levels at 4eC6 weeks of age in Kir6.2G132S transgenic mice fed ad libitum were considerably lower than in wild-type mice (17.4 ± 7.5 and 127.4 ± 24.4 pmol/l, respectively), but they increased in the transgenic mice with age. At 50 weeks, there was no significant difference in serum insulin levels between Kir6.2G132S transgenic and wild-type mice (143 ± 14.9 and 177.1 ± 24.1 pmol/l, respectively) (Fig. 1B).
Glucose tolerance and insulin secretion of Kir6.2G132S transgenic mice.
We then evaluated glucose tolerance and insulin secretion in Kir6.2G132S transgenic mice by OGTT performed by stomach gavage. We previously showed that the Kir6.2G132S mutation severely impaired glucose-induced insulin secretion because of defective KATP channel function and that Kir6.2G132S transgenic mice at 8 weeks of age exhibited severe glucose intolerance (26). In this study, although the fasting blood glucose levels of both younger and older Kir6.2G132S transgenic mice were similar to those of age-matched wild-type mice, both younger and older transgenic mice exhibited glucose tolerance impaired to a similar degree (Fig. 2A). In addition, no insulin response to glucose loading was detected, indicating that KATP channel function remains defective in older Kir6.2G132S transgenic mice (Fig. 2B). This was also confirmed by the finding that the Kir6.2G132S transgene is expressed in the older transgenic mice (Fig. 2C). These findings show that the improved serum insulin levels in the older transgenic mice do not result from normalization of KATP channel function in the pancreatic -cells.
Pancreatic insulin content of Kir6.2G132S transgenic mice.
We then investigated the association of the improved serum insulin levels in Kir6.2G132S transgenic mice with increased insulin content. At 4 weeks of age, the pancreatic insulin content of Kir6.2G132S transgenic mice was significantly lower than that of wild-type mice (60.6 ± 19.3 and 1,225.0 ± 68.4 pmol/pancreas for transgenic and wild-type mice, respectively). In accordance with the improved serum insulin levels, the pancreatic insulin content gradually increased with age. At 50 weeks of age, Kir6.2G132S transgenic mice exhibited pancreatic insulin content comparable to that of wild-type mice (2,054.7 ± 108.8 and 2,233.1 ± 73.1 pmol/pancreas, respectively) (Fig. 3). These findings strongly suggest that the increased serum insulin levels in Kir6.2G132S transgenic mice result from increased pancreatic insulin content.
Morphologic changes in the pancreas of Kir6.2G132S transgenic mice.
Because the restoration of pancreatic insulin content in older Kir6.2G132S transgenic mice suggested that the number of -cells is increased, we performed morphologic analyses of pancreatic islets. The pancreatic islets of Kir6.2G132S transgenic mice were somewhat transparent in comparison with wild-type islets (Figs. 4A and B). Sections stained with hematoxylin and eosin showed that many pancreatic islets of transgenic mice at 4 weeks of age were irregularly shaped, and the number of cells was less than in wild-type mice, but they were recovered by 25 weeks of age (Figs. 4CeCF). As reported previously (26), apoptotic cell death, detected by the TUNEL method, was accelerated in pancreatic islets of Kir6.2G132S transgenic mice at 4 weeks of age (Fig. 4G). The number of islets in cross sections containing TUNEL-positive cells was 59 of 87 islets examined (68%). The frequency of TUNEL-positive cells in a single islet cross section varied between 17 and 65%. In contrast, TUNEL-positive cells in pancreatic islets examined were rarely found in Kir6.2G132S transgenic mice at 25 weeks of age and in wild-type mice (Figs. 4HeCJ). These data indicate that apoptotic cell death of pancreatic -cells is the primary cause of the reduction in insulin content seen in Kir6.2G132S transgenic mice at younger ages, and they suggest that recovery of -cell mass occurs with age.
We next examined expressions of insulin and PDX1 by immunostaining. At 4 weeks of age, the number of insulin-positive cells was reduced significantly in Kir6.2G132S transgenic mice compared with wild-type mice (Figs. 5A and D), as previously reported (26). The -cells of Kir6.2G132S transgenic mice exhibited only weak immunoreactivity for insulin. In addition, only a few PDX1-positive cells in islets of Kir6.2G132S transgenic mice were detected (Fig. 5E). However, the number of insulin-positive cells gradually increased with age. Insulin immunoreactivity also increased (Figs. 5AeCC). The number of PDX1-positive cells increased as well as the number of insulin-positive cells (Figs. 5EeCG). Kir6.2G132S transgenic pancreas at 25 weeks of age was histologically indistinguishable from wild-type pancreas (Figs. 5D and H). Quantitative image analysis confirmed an increase in the -cell area with age in Kir6.2G132S transgenic mice pancreas (Fig. 6). These findings strongly suggest that pancreatic -cells are regenerated in Kir6.2G132S transgenic mice.
We then investigated other endocrine cells in Kir6.2G132S transgenic pancreas. Glucagon-positive cells were found in the central region of pancreatic islets in the younger Kir6.2G132S transgenic mice (Figs. 5I and J), consistent with our previous observation (26). In addition, both - and PP-cell populations were increased in the younger Kir6.2G132S transgenic mice. The distributions of - and PP-cells in the islets of the young transgenic mice also differed from those of wild-type mice. However, the architecture of the islets of Kir6.2G132S transgenic mice gradually improved with age and became almost normal at 25 weeks (Fig. 5C, G, and K).
Although it has been proposed that pancreatic -cells can be derived from -cells in diabetic rodents, based on the finding that insulin/somatostatin double-positive cells are detected in the regenerating islets (9eC11), we were unable to detect insulin/somatostatin double-positive cells in the islets of Kir6.2G132S transgenic mice at any time point examined (data not shown). In addition, neither insulin/glucagon nor insulin/PP double-positive cells were detected (data not shown).
Appearance of DBA-labeled cells in the islets of Kir6.2G132S transgenic mice.
It has been found that regeneration of pancreatic -cells might originate in progenitor cells located in ductal structures (13,33), and DBA, a lectin specific for N-acetylgalactosamine, has been used for staining pancreatic ductal cells (7,34,35). We attempted to stain ductal cells with DBA to determine whether insulin-positive cells are derived from that source. Although ductal structures in the pancreas of wild-type mice were labeled with DBA, no insulin-positive cells were found in the structures examined. Similarly, in the pancreas of Kir6.2G132S transgenic mice, ductal structures were labeled with DBA, and insulin-positive cells were rarely found (data not shown). DBA-labeled cells were not detected in the islets of wild-type mice. In contrast, they were detected frequently in the islets of Kir6.2G132S transgenic mice (Figs. 7AeCD). The frequency of the islets containing DBA-labeled cells in Kir6.2G132S transgenic mice gradually decreased with age (81.1 ± 2.5 and 41.3 ± 5.9% at 4 and 50 weeks of age, respectively) (Fig. 7F).
We then investigated the association of the appearance of DBA-labeled cells in the islets with the increase in -cell number in Kir6.2G132S transgenic mice. Although we were unable to detect DBA/insulin double-positive cells at 4 weeks of age (Fig. 8A), DBA-labeled cells in the islets began to express insulin at 12 weeks of age (Fig. 8B). At 25 weeks of age, a large number of DBA-labeled cells (50eC60% of total DBA-labeled cells within islets) expressed insulin (Fig. 8C). We also examined the expression of PDX1, a transcription factor essential for pancreatic development, in these DBA-labeled cells. Although the number of PDX1-positive cells was markedly reduced in young Kir6.2G132S transgenic islets (<30% of normal islets), some of the PDX1-positive cells were labeled with DBA (Fig. 8D). At 25 weeks of age, a considerable number of DBA-labeled cells expressed PDX1 (Fig. 8F). These results indicate that DBA-labeled cells participate in the process of regeneration of -cells in Kir6.2G132S transgenic mice.
We next investigated whether endocrine cells other than -cells are derived from DBA-labeled cells in Kir6.2G132S transgenic islets. DBA/somatostatin or DBA/PP double-positive cells were detected in Kir6.2G132S transgenic mice at 12 weeks of age and later (Figs. 8G and H). However, DBA/amylase double-positive cells were not detected. Interestingly, DBA-labeled duct-like structures with luminal amylase were formed in the islets of Kir6.2G132S transgenic mice (Fig. 8I). Moreover, cells double-positive for DBA and PCNA, a proliferating marker, were detected (Figs. 8J and K), demonstrating the proliferating capacity of DBA-labeled cells. These findings indicate that the DBA-labeled cells might possess some of the properties of pancreatic progenitor cells.
Because nestin and PGP9.5 have been reported to be potential progenitor markers during regeneration of pancreatic -cells (36eC38), we examined expressions of these marker proteins in islets from Kir6.2G132S transgenic mouse pancreas. Because nestin-positive cells were rarely detected in both young and old Kir6.2G132S transgenic mice (data not shown), it is unlikely that they participate in -cell regeneration in Kir6.2G132S transgenic mice. Increased expression of PGP9.5 has been detected during in vivo regeneration of endocrine cells (36) and in vitro transdifferentiation of insulin-secreting cells (37). We found an increase in the number of PGP9.5-positive cells in Kir6.2G132S transgenic mice (Figs. 8L and M) compared with wild-type mice. PGP9.5-positive cells expressing insulin were also found (Figs. 8L and M). Although the frequency of PGP9.5/insulin double-positive cells was lower than that of DBA/insulin double-positive cells, these results suggests that PGP9.5-positive cells also might contribute in part to -cell regeneration in Kir6.2G132S transgenic mice.
DISCUSSION
We previously found that adult Kir6.2G132S transgenic female mice (at 8 weeks of age) exhibit hyperglycemia with hypoinsulinemia caused by accelerated apoptosis (26). In the current study, we found that hyperglycemia with hypoinsulinemia in Kir6.2G132S transgenic mice recovers significantly with age. We also show that pancreatic insulin content and the number of -cells both increase as the Kir6.2G132S transgenic mice become older. The presence of the Kir6.2G132S mutation in the older Kir6.2G132S transgenic mice excludes the possibility that loss of the transgene is responsible for the recovery of -cell number in the older Kir6.2G132S transgenic mice. In fact, the insulin response to glucose loading was absent in the older Kir6.2G132S transgenic mice. Hence, the recovery of blood glucose and serum insulin are most likely caused by the restoration of pancreatic insulin content resulting from the increased -cell number in the older Kir6.2G132S transgenic mice. In a preliminary study, we found that serum insulin levels also improve with age and that -cell regeneration occurs in old male Kir6.2G132S transgenic mice, although hyperglycemia remains (K.O., K.M., T.M., and S.S., unpublished observations). It has been shown that insulin sensitivity in ovariectomized rats and mice is decreased (39,40) and that treatment with estradiol restores the decreased insulin sensitivity in the rats (39). Thus, the difference in improved hyperglycemia between male and female Kir6.2G132S transgenic mice may result from effects of sex hormones on insulin sensitivity.
Regeneration of pancreatic -cells can occur through several pathways, including neogenesis from progenitor cells located in ductal structures (13,33) or islets (9,10), self-replication of preexisting -cells (7,8), and transdifferentiation from noneC-cells (17,41). Although neonatal rats injected with streptozotocin later show improvement of hyperglycemia, no good animal model has previously been reported of spontaneous recovery from the diabetic state in adults. Although Kir6.2G132S transgenic mice exhibit hyperglycemia in adults, spontaneous recovery from hyperglycemia occurs with increased age. In addition, most -cell regeneration in this model is found within islets, and there are very few insulin-positive cells in ductal structures or exocrine acinar cells. Moreover, as assessed by the expression of PCNA or Ki67, there are no proliferating cells in ductal structures (Figs. 8J and K). These findings indicate that extraislet progenitor cells are not likely to contribute to the restoration of -cells and pancreatic insulin content in Kir6.2G132S transgenic mice.
Interestingly, DBA-labeled cells, which usually represent ductal cells in normal pancreas (7,34,35), were also detected within pancreatic islets of Kir6.2G132S transgenic mice. DBA-labeled cells were detected at highest frequency in Kir6.2G132S transgenic mice at 4 weeks of age. As the number of DBA-labeled cells decreased, the number of -cells increased in parallel with the increased insulin content. Many DBA-labeled cells expressed PDX1, a transcription factor critical in pancreatic development (42,43) that is found not only in mature -cells but also in endocrine progenitor cells in regenerating pancreas (10,11,14,20,44). DBA-labeled cells in the islets of Kir6.2G132S transgenic mice have proliferation potential, as assessed by double-staining with PCNA. Furthermore, DBA-labeled cells expressing insulin, somatostatin, or PP were also detected. These findings indicate that the DBA-labeled cells participate in the process of pancreatic islet cell regeneration in Kir6.2G132S transgenic mice, suggesting that the cells have some properties of progenitor cells, including plasticity in differentiation capacity and proliferation activity. Recently, it has been shown that DBA can be used as a specific marker of the embryonic pancreatic epithelium, which gives rise to endocrine and exocrine cells of developing pancreas (29). It is thus not unlikely that these DBA-labeled cells in adult Kir6.2G132S transgenic islets represent endocrine progenitor cells.
We found that the number of PGP9.5-positive cells increased in Kir6.2G132S transgenic mice islets. PGP9.5 is known as a panendocrine marker and has been reported recently to be a marker of endocrine progenitors (36eC38). Thus, PGP9.5-positive cells may also contribute in part to -cell regeneration in Kir6.2G132S transgenic mice.
We also found that functional ducts containing amylase immunoreactivity in their lumen are formed within the Kir6.2G132S transgenic islets. It has been reported that many ductal structures are formed in the pancreatic islets of mice expressing fibroblast growth factor 10 under control of the PDX1 promoter (45). Because fibroblast growth factor 10 may play an important role in maintaining the proliferating capacity of progenitor cells in embryonic pancreas (46,47), this finding could well indicate the activation of progenitor cells within the islets.
Recent studies have shown that self-replication of preexisting -cells constitutes the primary contribution in the maintenance of -cell mass in adult mice (7,8). However, the frequency of proliferating -cells was similar in Kir6.2G132S transgenic and wild-type mice (data not shown), so it is unlikely that self-replication of -cells is responsible for restoration of -cell number in this case.
In conclusion, our data demonstrate that regeneration of -cells occurs in the pancreatic islets of Kir6.2G132S transgenic mice and that DBA-labeled cells participate in the process. Although the mechanism of regeneration is not known at present, the Kir6.2G132S transgenic mouse should be a useful model for studying spontaneous regeneration of pancreatic -cells in vivo.
ACKNOWLEDGMENTS
This work was supported by a grant-in-aid for specially promoted research and a grant for the 21st Century Center of Excellence program from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from Takeda Chemical Industry.
Part of the study was conducted at the Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, to which K.O., K.M., T.M., and S.S. were formerly affiliated.
FOOTNOTES
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REFERENCES
Seino S: ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 61:337eC362, 1999
Newgard CB, McGarry JD: Metabolic coupling factors in pancreatic -cell signal transduction. Annu Rev Biochem 64:689eC719, 1995
Rorsman P, Renstrom E: Insulin granule dynamics in pancreatic beta cells. Diabetologia 46:1029eC1045, 2003
DeFronzo RA: Lilly lecture 1987: The triumvirate: beta-cell, muscle, liver: a collusion responsible for NIDDM. Diabetes 37:667eC687, 1988
Kloppel G, Lohr M, Habich K, Oberholzer M, Heitz PU: Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Pathol Res 4:110eC125, 1985
Weir GC, Bonner-Weir S, Leahy JL: Islet mass and function in diabetes and transplantation. Diabetes 39:401eC405, 1990
Dor Y, Brown J, Martinez OI, Melton DA: Adult pancreatic -cells are formed by self-duplication rather than stem-cell differentiation. Nature 429:41eC46, 2004
Georgia S, Bhushan A: cell replication is the primary mechanism for maintaining postnatal cell mass. J Clin Invest 114:963eC968, 2004
Fernandes A, King LC, Guz Y, Stein R, Wright CV, Teitelman G: Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets. Endocrinology 138:1750eC1762, 1997
Guz Y, Nasir I, Teitelman G: Regeneration of pancreatic cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology 142:4956eC4968, 2001
Li L, Yi Z, Seno M, Kojima I: Activin A and betacellulin: effect on regeneration of pancreatic -cells in neonatal streptozotocin-treated rats. Diabetes 53:608eC615, 2004
Waguri M, Yamamoto K, Miyagawa JI, Tochino Y, Yamamori K, Kajimoto Y, Nakajima H, Watada H, Yoshiuchi I, Itoh N, Imagawa A, Namba M, Kuwajima M, Yamasaki Y, Hanafusa T, Matsuzawa Y: Demonstration of two different processes of -cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan. Diabetes 46:1281eC1290, 1997
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. Diabetes 42:1715eC1720, 1993
Sharma A, Zangen DH, Reitz P, Taneja M, Lissauer ME, Miller CP, Weir GC, Habener JF, Bonner-Weir S: The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 48:507eC513, 1999
Rosenberg L, Brown RA, Duguid WP: A new approach to the induction of duct epithelial hyperplasia and nesidioblastosis by cellophane wrapping of the hamster pancreas. J Surg Res 35:63eC72, 1983
Rafaeloff R, Pittenger GL, Barlow SW, Qin XF, Yan B, Rosenberg L, Duguid WP, Vinik AI: Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J Clin Invest 99:2100eC2109, 1997
Wang RN, Kloppel G, Bouwens L: Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 38:1405eC1411, 1995
Gu D, Sarvetnick N: Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. Development 118:33eC46, 1993
Bockman DE, Merlino G: Cytological changes in the pancreas of transgenic mice overexpressing transforming growth factor . Gastroenterology 103:1883eC1892, 1992
Song SY, Gannon M, Washington MK, Scoggins CR, Meszoely IM, Goldenring JR, Marino CR, Sandgren EP, Coffey RJ Jr, Wright CV, Leach SD: Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor . Gastroenterology 117:1416eC1426, 1999
Cantenys D, Portha B, Dutrillaux MC, Hollande E, Roze C, Picon L: Histogenesis of the endocrine pancreas in newborn rats after destruction by streptozotocin: an immunocytochemical study. Virchows Arch B Cell Pathol Incl Mol Pathol 35:109eC122, 1981
Dutrillaux MC, Portha B, Roze C, Hollande E: Ultrastructural study of pancreatic B cell regeneration in newborn rats after destruction by streptozotocin. Virchows Arch B Cell Pathol Incl Mol Pathol 39:173eC185, 1982
Wang RN, Bouwens L, Kloppel G: Beta-cell proliferation in normal and streptozotocin-treated newborn rats: site, dynamics and capacity. Diabetologia 37:1088eC1096, 1994
Ashcroft FM: Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Neurosci 11:97eC118, 1988
Miki T, Seino S: Roles of KATP channels as metabolic sensors in acute metabolic changes. J Mol Cell Cardiol 38:917eC925, 2005
Miki T, Tashiro F, Iwanaga T, Nagashima K, Yoshitomi H, Aihara H, Nitta Y, Gonoi T, Inagaki N, Miyazaki J, Seino S: Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel. Proc Natl Acad Sci U S A 94:11969eC11973, 1997
Miki T, Nagashima K, Tashiro F, Kotake K, Yoshitomi H, Tamamoto A, Gonoi T, Iwanaga T, Miyazaki J, Seino S: Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice. Proc Natl Acad Sci U S A 95:10402eC10406, 1998
Seghers V, Nakazaki M, DeMayo F, Aguilar-Bryan L, Bryan J: Sur1 knockout mice: a model for KATP channel-independent regulation of insulin secretion. J Biol Chem 275:9270eC9277, 2000
Kobayashi H, Spilde TL, Li Z, Marosky JK, Bhatia AM, Hembree MJ, Prasadan K, Preuett BL, Gittes GK: Lectin as a marker for staining and purification of embryonic pancreatic epithelium. Biochem Biophys Res Commun 293:691eC697, 2002
Filipponi P, Marcelli M, Nicoletti I, Pacifici R, Santeusanio F, Brunetti P: Suppressive effect of long term sulfonylurea treatment on A, B, and D cells of normal rat pancreas. Endocrinology 113:1972eC1979, 1983
Minami K, Yano H, Miki T, Nagashima K, Wang CZ, Tanaka H, Miyazaki JI, Seino S: Insulin secretion and differential gene expression in glucose-responsive and -unresponsive MIN6 sublines. Am J Physiol Endocrinol Metab 279:E773eCE781, 2000
Minami K, Morita M, Saraya A, Yano H, Terauchi Y, Miki T, Kuriyama T, Kadowaki T, Seino S: ATP-sensitive K+ channel-mediated glucose uptake is independent of IRS-1/phosphatidylinositol 3-kinase signaling. Am J Physiol Endocrinol Metab 285:E1289eCE1296, 2003
Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, O’Neil JJ: In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 97:7999eC8004, 2000
Watanabe M, Muramatsu T, Shirane H, Ugai K: Discrete distribution of binding sites for Dolichos biflorus agglutinin (DBA) and for peanut agglutinin (PNA) in mouse organ tissues. J Histochem Cytochem 29:779eC780, 1981
Jensen JN, Cameron E, Garay MV, Starkey TW, Gianani R, Jensen J: Recapitulation of elements of embryonic development in adult mouse pancreatic regeneration. Gastroenterology 128:728eC741, 2005
Yokoyama-Hayashi K, Takahashi T, Kakita A, Yamashina S: Expression of PGP9.5 in ductal cells of the rat pancreas during development and regeneration: can it be a marker for pancreatic progenitor cells Endocr J 49:61eC74, 2002
Minami K, Okuno M, Miyawaki K, Okumachi A, Ishizaki K, Oyama K, Kawaguchi M, Ishizuka N, Iwanaga T, Seino S: Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells. Proc Natl Acad Sci U S A 102:15116eC15121, 2005
Yamada S, Terada K, Ueno Y, Sugiyama T, Seno M, Kojima I: Differentiation of adult hepatic stem-like cells into pancreatic endocrine cells. Cell Transplant 14:647eC653, 2005
Kumagai S, Holmang A, Bjorntorp P: The effects of oestrogen and progesterone on insulin sensitivity in female rats. Acta Physiol Scand 149:91eC97, 1993
Puah JA, Bailey CJ: Effect of ovarian hormones on glucose metabolism in mouse soleus muscle. Endocrinology 117:1336eC1340, 1985
Bouwens L: Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 43:332eC336, 1998
Jonsson J, Carlsson L, Edlund T, Edlund H: Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371:606eC609, 1994
Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BL, Wright CV: PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983eC995, 1996
Holland AM, Gonez LJ, Naselli G, Macdonald RJ, Harrison LC: Conditional expression demonstrates the role of the homeodomain transcription factor Pdx1 in maintenance and regeneration of -cells in the adult pancreas. Diabetes 54:2586eC2595, 2005
Yamaoka T, Yoshino K, Yamada T, Yano M, Matsui T, Yamaguchi T, Moritani M, Hata J, Noji S, Itakura M: Transgenic expression of FGF8 and FGF10 induces transdifferentiation of pancreatic islet cells into hepatocytes and exocrine cells. Biochem Biophys Res Commun 292:138eC143, 2002
Bhushan A, Itoh N, Kato S, Thiery JP, Czernichow P, Bellusci S, Scharfmann R: Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128:5109eC5117, 2001
Norgaard GA, Jensen JN, Jensen J: FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development. Dev Biol 264:323eC338, 2003(Kazunobu Oyama, Kohtaro M)