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-Cell Proliferation, and Islet Neogenesis
Department of Anatomy and Cell Biology, State University of New York (M.V., Y.G., M.R., G.T.), Brooklyn, New York 11203; Department of Medical Nutrition, Suzuka University of Medical Science and Technology (M.F.), Suzuka, Mie 510-02, Japan; and Howard Hughes Medical Institute, University of Chicago (G.W., D.S.), Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Gladys Teitelman, Ph.D., Department of Anatomy and Cell Biology, State University of New York, Health Science Center, 450 Clarkson Avenue, Brooklyn, New York 11203. E-mail: gteitelman{at}downstate.com.
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-cell phenotype. Thus, in contrast to controls, -cells of mutant mice had protracted expression of Nkx 6.1 and Pdx-1, but did not express Brn-4. Islets of adult mutant mice also contained cells coexpressing insulin and somatostatin, an immature cell type found only in islets of the wild-type strain during development. In addition to the effects on islet cell differentiation, the absence of PC2 activity resulted in a 3-fold increase in the rate of proliferation of proglucagon cells during the perinatal period. This increase contributed to the development of -cell hyperplasia during postnatal life. Furthermore, the total ß-cell volume was increased 2-fold in adult mutants compared with controls. This increase was due to islet neogenesis, as the number of islets per section was significantly higher in knockout mice compared with wild-type mice, whereas both strains had similar rates of IN cell proliferation. These results indicate that hormones processed by PC2 affected processes that regulate islet cell differentiation and maturation in embryos and adults. |
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-cell apoptosis in adults (4). To further elucidate the role of pancreatic hormones in islet cell development, we examined mice with a deletion in the gene encoding for prohormone convertase-2 (PC2). PC2 is a member of a family of serine proteases that, in conjunction with PC1/3, is responsible for the maturation of a large number of hormone and neuropeptide precursors (5, 6). In pancreatic islets, ß-cells contain both PC2 and PC1/3, which act together to process proinsulin to insulin (7, 8, 9). PC2 is also expressed in glucagon (GLU) cells of the pancreas where it selectively converts proglucagon (proGLU) to GLU (7, 10). In the L cells of the intestine, proGLU is processed by PC1/3, resulting in the formation of glucagon-like peptide-1 (10). Mice lacking PC2 activity due to a disruption of the cognate gene by targeted mutagenesis fail to convert prosomatostatin and proGLU to the active forms of the hormones and have increased levels of intermediate forms of proinsulin in the circulation (7, 8, 11).
Adult PC2 knockout (KO) mice show a dramatic hypertrophy and hyperplasia of the -cells, which we termed -proGLU cells to differentiate them from -cells of controls, and of the 
and pancreatic polypeptide (PP) cells without alteration in the normal location of these cells in the periphery of islets (7). Although PC2 KO mice have multiple hormonal deficiencies (12, 13, 14, 15, 16), the administration of glucagon leads to a reduction in the number of -proGLU cells due to increased apoptosis (4). However, the -proGLU cell hyperplasia found in mutant mice could be due not only to a reduction in the number of cells that die, but also to an increase in the rate of -proGLU cell proliferation, which would suggest that molecules processed by PC2 regulate the cell cycle of -cells. Therefore, our objectives were, first, to determine whether the lack of the mature hormones affected islet cell differentiation and maturation and, second, to ascertain whether the mutation produced a dysregulation in the rate of -proGLU cell proliferation.
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Materials and Methods |
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Adult mice were perfused through the heart with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer and postfixed for several hours in the same fixative. Embryos were fixed overnight by immersion in the same fixative solution. The fixed tissues were infiltrated in 30% sucrose and mounted in embedding matrix (Lipshaw Co., Pittsburgh, PA), and 10-µm cryostat sections were collected onto gelatin-coated slides. The animal procedures used in these studies were approved by the Downstate animal care and use committee.
Source of antibodies
Guinea pig antibodies to bovine insulin and rat C peptide were purchased from Linco Research, Inc. (St. Charles, MO). Rabbit antiserum to human glucagon was purchased from Calbiochem (San Diego, CA). Rabbit antisera to human PP and to somatostatin (SOM) were supplied by Peninsula Laboratories (Belmont, CA). Monoclonal antisera to insulin and glucagon were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Rat antiserum to SOM was purchased from Protos (New York, NY). Rabbit antisera to Pdx-1, Nkx 2.2, Isl-1, Nkx6.1, and Ngn3 were generous gifts from C. V. E. Wright (Vanderbilt University, Nashville, TN), T. Jessel (Columbia University, New York, NY), O. Madsen (Hagedorn Research Institute, Gentofte, Denmark), and M. German (University of California, San Francisco, CA), respectively. Antiserum to Pax 6 was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Ames, IA). Antibodies were used at the following dilutions: guinea pig antiinsulin and antirat C-peptide antisera at 1:400; monoclonal antiserum to insulin at 1:2,000, monoclonal antiserum to glucagon at 1:6,000 and to Pax 6 at 1:1,000; rabbit antiserum to human glucagon at 1:12,000, to human SOM at 1:8,000, and to proinsulin at 1:5,000; rabbit antihuman PP antiserum at 1:10,000; rabbit antimouse PDX-1 antiserum at 1:8,000 and antirat SOM antiserum at 1:2,000; and rabbit antiserum to PC1/3 at 1:5,000 and to Nkx2.2, Isl-1, and Nkx 6.1 at 1:2,000.
Secondary antibodies
Biotinylated goat antirabbit IgG and avidin-labeled peroxidase were purchased from Vector Laboratories, Inc. (Burlingame, CA). Alexa Fluor 488 antimouse, antirat, and antirabbit IgG and Alexa Fluor 594 antiguinea pig, antirabbit, and antimouse IgG were purchased from Molecular Probes (Eugene, OR). For double-labeling using rabbit and guinea pig antisera, the secondary antibody used to visualize the guinea pig antibodies (purchased from Jackson ImmunoResearch Laboratories, West Grove, PA) did not cross-react with rabbit antibodies. All secondary IgGs were used at a 1:200 dilution. After completion of the staining procedure, sections were covered with two or three drops of Prolong Antifade solution (Molecular Probes) and dried at room temperature before examination.
Immunocytochemistry of cryostat sections using peroxidase techniques
These techniques have been previously described (17). In brief, the sections were incubated sequentially in an empirically derived optimal dilution of control serum or primary antibody raised in species X, a biotinylated IgG solution, and a peroxidase-avidin complex for 30 min. After these incubations, the bound peroxidase was visualized with diaminobenzidene, and the sections were dehydrated and mounted with Permount (Fisher Scientific, Pittsburgh, PA).
Confocal microscopy
An LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc., New York, NY) fitted with an Axiovert 100M microscope (Carl Zeiss, Inc.) was used with a x63 1.4NA pan Apochromat objective (Carl Zeiss, Inc.). Excitation on LSM 510 U was performed with a 15-mW argon ion laser running at 75% power emitting at 488 nm, a 1.0-mW helium/neon laser emitting at 543 nm, and a 5.0-mW helium/neon laser emitting at 633 nm. Emissions were collected using a 505- to 530-nm bandpass filter to collect Alexa green emissions and a 560- to 615-nm bandpass filter to collect Alexa red emissions. The settings for all experiments were 3–5% transmittance of the 488-nm band and 20–25% of the 546-nm band. In addition, 0.7-µm vertical steps were used with a vertical optical resolution of less than 1.0 µm.
Determination of cell proliferation in vivo
Mice received a single dose of [3H]thymidine (10 µCi/gm, ip; NEN Life Science Products, Boston, MA; specific activity, 
80 Ci/ mmol), perfused with fixative solution 1 h later, the pancreas was removed, and the sections were processed for immunohistochemistry. After the diaminobenzidene step, sections were dehydrated with ascending alcohols, cleared in xylene, rehydrated, air-dried, and dipped in Ilford L-4 emulsion (Polysciences, Warrington, PA) at 50 C diluted 1:1 with distilled water. The coated slides were air-dried and exposed in light-proof boxes with desiccant for 40 C for 2–3 wk. At the end of the exposure period, the slides were developed, fixed, counterstained with hematoxylin, dehydrated, cleared, and mounted with a coverslip.
Determination of the percentage of islet cells
The relative number of or ß-cells per islet volume or exocrine tissue was determined in sections immunostained for GLU or IN and counterstained with hematoxylin by the point-sampling method (18) using a 300-point ocular grid at a total magnification of x400.The average number of stained cells per islet volume was calculated according to the formula F = h/n, in which h is the number of hits over stained cells, and n is the number of points scored over islets (18). The relative - or ß-cell volume was calculated by dividing the number of points over immunostained cells over the total number of points for that section. At least 10,000 points from three pancreases/age/strain were calculated for each experiment. The ß-cell mass was calculated by multiplying ß-cell relative volume by pancreas weight for individual animals.
Measurement of -cell size and perimeter of islets
Sections were projected on the screen of a video monitor, and the area was measured using the NIH Image program for Macintosh (http://rsb.info.nih.gov./nih-image). The area of -proGlu cells was measured in at least 40 cells from 10 islets/mice/3 mice per strain/age. The same program was used to measure the perimeter of islets in 9 sections/pancreas, 3 pancreata/strain.
Statistical analysis
All values indicate the mean ± SEM. For comparison between two groups, the unpaired t test (two-tailed) was used. P < 0.05 was considered significant.
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). In contrast to controls, the pancreas of e-13 KO embryos did not contain cells expressing IN (Fig. 1B) or the insulin precursor molecule proinsulin (not shown). In mutants, IN+ cells first appeared on e-15 (Fig. 1C), and on e-17, the pancreas of PC2 KO contained a large number of ß-cells (Fig. 1D). These data indicate a delay in the appearance of IN in developing KO pancreas compared with littermate controls. SOM- and PP-containing cells first appeared on e-15 and postnatal d 1 (P-1), respectively, in both control and KO mice (not shown).
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, A–C). Cells coexpressing IN and proGLU were not found in islets on e-17 or P-2, or in adult mutant mice (not shown).
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-cells throughout life (17) and by -cells on e-10, but not in older embryos or adults (19). However, examination of the pancreas of PC2-null mutant mice revealed the presence of a significant number of Pdx-1+/pro-GLU+ cells in islets on e-15 (not shown) and in P-2 KO mice (Fig. 2D
). As expected, these cells were not seen in islets of heterozygote (not shown) or littermate controls (not shown). Pdx-1+/proGLU cells were also observed in all islets of P-15 and 6-month-old KO mice (not shown). In contrast to GLU cells of controls (24), -proGLU cells of embryonic and adult mutant mice did not express the pou homeodomain protein Brn-4 (Fig. 2I). Pancreatic islets of normal and mutant mice also differ in the patter of expression of the homeobox gene Nkx 6.1. In controls, Nkx 6.1 is expressed in almost all epithelial cells of the pancreatic primordia. Starting on e-14, Nkx 6.1 expression becomes restricted to ß-cells (24, 25, 26). However, cells expressing Nkx 6.1 and proGLU were found in islets of PC2 KO mice on e-16 (not shown) and P-2 (Fig. 2E), but not in islets of adults (not shown).
The deletion of PC2 did not affect the expression of Ngn3, which in normal mice is transiently expressed in pancreatic precursor cells during development (reviewed in Ref.26). Similarly, cells expressing Ngn3 were found in the pancreas of PC2 mutant mice on e-15 (Fig. 3A), but not on e-17 (Fig. 3B) or in adults (not shown). Likewise, the pattern of expression of the transcription factors Isl-1 and Nkx2.2 in mutants was similar to that reported in controls (25, 26, 27). Thus, cells expressing Nkx2.2 (Fig. 2F) and Isl-1 (Fig. 2G) were seen in islets of mutant mice throughout life, and both of these factors were expressed by -proGLU cells. Pax 6 was not detected in islets of e-15 and P-2 mutants and littermate controls (not shown), but was expressed by all islet cells of adults in both strains of mice (Fig. 4). -ProGLU cells of mutant mice also showed properties characteristic of mature -cells. In control mice, glucagon cells transiently express PC1/3 during development, but not in adults (28, 29). Similarly, the -proGLU cells of adult PC2 mutant mice lacked PC1/3 expression (Fig. 2J).
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-proGLU cells, islets of PC2 mutant mice were populated by other immature cell types. Thus, pancreatic islets of neonatal (not shown) and adult KO mice contained cells coexpressing IN and SOM (Fig. 5, D–F). In contrast, in controls, these cells were found in embryos (Fig. 5, A–C), but not in islets of postnatal and adult mice (not shown). Islets containing SOM/IN cells in mutant mice were generally of small size (perimenter, 408.43 ± 29.06 µm; n = 10). A second type of islet present in the pancreas of adult KO mice was usually larger and had a central core of ß-cells that expressed IN, but not SOM, while the -cells located in the periphery of the islet contained only SOM (Fig. 5, G–I). Some of these islets had a narrow rim of non-ß-cells (Fig. 4, G–I), whereas in other islets these cells formed a wide mantle (not shown). At no time were cells coexpressing SOM and GLU found in the pancreas of KO mice (not shown).
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-cell proliferation was more than 3-fold higher in e-17.5 KO than in WT littermates (Fig. 6). No significant difference was found in the labeling index of -proGLU cells from P-15 KO (5.8 ± 0.35) and WT (4.5 ± 0.29) littermate controls, whereas at 3 months the replication rate of -proGLU cells in both strains was extremely low (<0.1%). In contrast to -proGLU cells, the replication rate of IN+ cells was similar in e-17 KO and WT animals (Fig. 6) and was decreased in both strains to very low levels in adults. These observations demonstrate that the mitotic stimulus in embryos was not due to a growth-promoting effect of the mutation on all islet cells. The rate of proliferation of - and PP cells was not determined in these studies.
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-proGlu cell proliferation during the perinatal period resulted in the appearance of -cell hyperplasia in the islets of KO mice. This growth was already detected in islets of 2-d-old mice (Fig. 7C), became prominent on postnatal d 15 (Fig. 7D), and was also seen in islets of adults (4). It has been proposed that GLU cell hyperplasia was the result of hypoglycemia present in adults. However, the glucose levels in KO embryos were normal, because heterozygote mothers were euglycemic (7). Similarly, neonatal and early postnatal mutant mice were euglycemic (blood glucose, 80–100 mg/dl; n = 6), indicating that the increased rate of GLU proliferation was not induced by low levels of glucose.
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-proGLU cell size during postnatal development. Thus, although the mean area (±SEM) of individual -proGLU cells was 20% larger in KO (51.03 ± 4.24 µm2) than in WT (41.5 ± 2.05 µm2) mice on P-15, the mean area of -proGlu cells of 3.5-month-old KO mice (98.5 ± 5.03 µm2) was almost 3-fold larger than that in littermate controls (39.01 ± 1.7 µm2) (this study and Ref.7). The sizes of - and PP cells were similar in KO and controls (not shown). Adult PC2 KO mice had a higher rate of exocrine cell proliferation in adult mutants (10.68 ± 0.27; n = 3; 1670 cells scored) than in littermate controls (0.53 ± 0.09; n = 4; 1940 cells scored). No significant differences were found between KO and WT mice in the rate of proliferation of ductal cells (1.3 ± 0.14 vs. 1.1 ± 0.12). The increase in the rate of cell proliferation in the exocrine compartment contributed to an augmentation of pancreas weight [KO, 0.25 ± 0.04 (n = 3); WT, 0.145 ± 0.03 (n = 3)]. Examination of liver, adrenal gland, kidney, and spleen failed to detect a generalized organomegalia, and adult KO and WT animals had similar weights (not shown).
Islet neogenesis in pancreas of KO mice
Next we sought to determine whether the ablation of PC2 activity conferred other immature traits to the pancreas of mutant mice. We examined whether islet neogenesis, a process normally restricted to embryonic and early postnatal stages (30), occurred in the pancreas of adult PC2 KO strain. Three common criteria used to determine whether an increase in islet cell mass is due to islet neogenesis are 1) the presence of islet cells budding from the duct; 2) the presence of an increased number of small islets, which are assumed to be newly formed; and 3) whether there is an augmentation of ß-cell mass that cannot be accounted for by ß-cell proliferation. The pancreas of KO mutant mice contained few islets that appeared to emerge from ducts. However, as indicated in Table 1, the number of arbitrarily defined small (2–10 endocrine cells) and medium to large islets (>10 endocrine cells /islet) in sections of pancreas from KO was 3-fold higher than that in littermate WT mice. Due to the presence of non-ß-cell hyperplasia and -cell hypertrophy, the relative ß-cell volume per islet was lower in KO mice compared with that in controls (Fig. 8A). However, the total ß-cell volume was higher in KO mice than in controls (Fig. 8B). The fact that the number of islets was significantly higher in the mutant than in the WT strain and that the rate of ß-cell proliferation was similar in both strains indicates that the increase in ß-cell volume in PC2 null mice was due to islet cell neogenesis.
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Discussion |
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When the first insulin cells differentiate in mutant mice on e-15 of development, the pancreas contained cells coexpressing the proteins encoded by the IN and GLU genes. The presence GLU+/IN+ cells in the pancreas of normal mouse embryos during the protodifferentiated stage (32), which in mouse embryos extends from e-10 to e-13 of development, is well established (19, 20, 21, 23, 24, 25). The disappearance of the GLU+/IN+ cells coincided with a burst of ß-cell differentiation and the appearance of cell types expressing only IN or GLU (31, 32). The present findings indicate that insulin cells coexpress the GLU gene when they first differentiate regardless of whether they appear at the initial or the more advanced stage of pancreatic morphogenesis. As cell labeling studies indicate that GLU+/IN+ cells do not generate mature ß-cells (33), the role of these bihormonal cells in islet cell development remains unclear.
Previous studies suggest that IN+ and SOM+ cells originate from common precursors and that IN+/SOM+ cells are intermediate cells in that pathway (17, 19, 34, 35). In agreement with this observation, SOM+/IN+ cells are detected in islets of control embryos after midgestation, but are not found after birth, although this period is characterized by the presence of islet neogenesis (30). However, the disruption of the PC2 gene resulted in the presence of SOM+/IN+ cells in islets of postnatal and adult KO mice. Islets containing these cells probably were newly formed, because they lacked a thick mantle of non-ß cells, reflecting the process of neogenesis observed in this strain of mouse. Presumably, these newly formed islets had a normal proportion of ß- to non-ß-cells when they first appeared, and the increase in the non-ß-cell population occurred after the islet developed. It is likely that concomitant with the increase in the size of the islet mantle, the SOM+/IN+ cells matured into cells expressing only IN or SOM. These observations raise the hypothesis that in postnatal WT mice, precursors that generate the ß- and -cells of new islets do not go through an intermediate stage in which they coexpress both hormones. The fact that newly formed islets of postnatal PC2 KO mice contained SOM+/IN+ cells suggests that the precursors generating these cells are similar to the embryonic precursors of controls.
The molecular mechanisms determining the appearance of the different islet cell types during normal development are poorly understood. However, a large number of recently identified transcription factors have been shown to affect pancreatic development (reviewed in Refs. 26 and 35). The abrogation of PC2 activity did not affect the temporal expression or cellular distribution of Ngn3, Nkx2.2, and Isl-1, which appeared to be similar in controls and mutant mice. Pax-6 has been reported to be expressed in normal mice from early development (35). The lack of Pax6 expression in embryos and neonates of both PC2 mutant and littermate controls suggests that this factor was present during pre- and early postnatal development at levels below the sensitivity of the techniques used in the present study. In adults, all islet cells of mutant and control mice expressed Pax 6.
The lack of the convertase, however, perturbed the expression of Nkx 6.1, Pdx-1, and Brn-4. It has been suggested that precursor cells in normal embryos are characterized by a temporal sequence of activation and inactivation of different transcription factors (25, 26, 36). According to this proposition, islet precursors initially express Ngn3, Pdx-1, Nkx2.2, and NKx6.1 (25). Precursors that become -cells initiate the expression of Brn4, inhibit Nkx6.1 and Pdx-1, and activate the GLU gene (25). Moreover, it was recently proposed that a prerequisite for -cell differentiation was the loss of Pdx-1 function and the appearance of Brn4 (37, 38). However, the fact that -ProGLU cells of mutant mice had protracted expression of NKx6.1 and Pdx-1 argue that the expression of those two transcription factors does not affect the initiation of expression of the GLU gene. Moreover, -proGLU cells lacked Brn4 expression, suggesting that the activation of this transcription factor is a late event in -cell differentiation.
A more plausible model of -cell differentiation is one in which there is an initial population of -cell precursors that transiently coexpress the GLU gene, Nkx6.1, and Pdx1, and a late population of progenitors in which Nkx6.1 and Pdx-1 expression is inhibited concomitantly with the activation of Brn-4 and GLU. According to this scenario, the -proGLU cells of PC2 KO mice would be generated only by the early precursor pool throughout life and would maintain the immature phenotype for an extended period of time. Taken together, the persistence of Nkx6.1/proGLU, Pdx-1/ProGLU, and SOM/IN cells in pancreatic islets of PC2 KO mice suggests that the characteristics of the islet precursor cells rapidly evolve with time in normal development, but that this evolution proceeds at a much slower pace when the PC2 gene is mutated. These observations also suggest that the evolution of the precursor population is regulated by factors normally processed by PC2.
The abrogation of PC2 activity also resulted in the appearance of -cell hyperplasia, which developed soon after birth and, as reported by others (4), persisted throughout life. The growth of the relative -cell mass was partly due to a higher rate of -proGLU cell proliferation in KO than in WT mice during prenatal and presumably early postnatal life. The rate of GLU cell proliferation decreased with time after birth, reaching very low levels in 3-month-old controls and KO mice. If the number of cells produced during the perinatal period was similar to the number that die, the increased cell proliferation of -proGLU cells would not result in -cell hyperplasia. However, PC2 KO mice were unable to eliminate the supernumerary -proGLU cells (4). These observations suggest that in KO mice, -cells generated during development and early postnatal stages persist throughout life.
Adult mutant PC2 mice showed an increase in islet neogenesis. In control mice islet neogenesis occurs mainly during the late prenatal and early postnatal stages, and then islet growth slows down to undetectable levels (39). In contrast, the pancreas of postnatal and adult PC2-null mice contained significantly higher numbers of small and medium/large islets per tissue section than littermate controls. In addition, the ß-cell volume in the pancreas of adult KO mice was twice that in the WT strain. This increase was not due to ß-cell proliferation, as the replication rate of IN+ cells in KO and WT strains was very low. Although some islets appeared to bud from the duct, most small islets were scattered between the acinii and were not connected to ducts. These observations support the proposition that the mutation in the PC2 gene induced the formation of new islets during postnatal life and that these islets were generated by unidentified precursors located in the exocrine tissue. The identity of the signals promoting islet neogenesis in this KO strain remains to be determined.
In summary, the abrogation of PC2 activity had multiple effects on islet cell growth and differentiation. One consequence of the inactivation of the proconvertase was an increase in -cell proliferation and decreased apoptosis (4), which lead to the appearance of -proGLU cell hyperplasia in early postnatal life. The mutation also delayed the initiation of insulin synthesis during development and resulted in the presence of -ProGLU cells in postnatal and adult mice that expressed a combination of transcription factors characteristic of -cells of early embryos. Pancreata of postnatal and adult KO mice were also characterized by islet neogenesis and the presence of islets containing cells coexpressing IN and SOM, a cell type normally found only during development. Identification of the mechanisms mediating the multiple effects of the abrogation of PC2 will provide critical insight into the processes regulating islet cell differentiation, maturation, and growth.
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Footnotes |
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Received January 17, 2003.
Accepted for publication May 29, 2003.
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cell hyperplasia in prohormone convertase 2 knockout mice. Diabetes 51:398–405 and ß cells from neurogenin 3 expressing precursors. Diabetes 49:163–176[Abstract]
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