Neogenesis of ß-Cells in Adult BETA2/NeuroD-Deficient Mice

Hsiang-po Huang1, Khoi Chu, Eric Nemoz-Gaillard, Dorit Elberg and Ming-Jer Tsai

Department of Molecular and Cellular Biology (H.-p.H., K.C., E.N.-G., D.E., M.-J.T.), Medicine (M.-J.T.) and Program in Developmental Biology (M.-J.T.), Baylor College of Medicine, Houston Texas 77030

Address all correspondence and requests for reprints to: Ming-Jer Tsai, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mtsai{at}bcm.tmc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
BETA2/NeuroD, a basic helix-loop-helix transcription factor, is expressed in pancreatic endocrine cells during development and regulates insulin gene expression. We demonstrated previously that the endocrine pancreas of BETA2/NeuroD-deficient mice undergoes massive apoptosis and, consequently, animals die of diabetes shortly after birth. Here we show that a significant fraction of BETA2-deficient mice in a new genetic background can survive diabetes and live to adulthood through the process of ß-cell neogenesis. Morphometric examination indicates that pancreatic ß-, but not {alpha}-cell mass, was restored to a level comparable to that of wild-type animals. However, the newly formed islet cells cannot form mature islets of Langerhans, indicating an indispensable role of BETA2 in morphogenesis of normal islet structure. Furthermore, immunohistochemical examinations revealed that newly formed ß-cells of BETA2/NeuroD-deficient mice come from two sources: either directly budding from the pancreatic ductal tree or from the preexisting ß-cells in the residual endocrine pancreas. Our results indicate that ß-cell neogenesis in our BETA2/NeuroD-deficient mice contributes to their survival, and these mice may provide a useful model for studying the mechanism of ß-cell regeneration.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RECENT STUDIES ON pancreatic development have led to the identification of important genes involved in islet cell differentiation. Many of these genes have been found to encode transcription factors, such as BETA2/NeuroD (1), PDX-1 (2), Isl-1 (3), Pax4 (4), Pax6 (5), Nkx2.2 (6), HB9 (7), and Neurogenin3 (8). The distinct role of each of these factors in pancreas development has been characterized by gene-targeting techniques in the mouse (2, 3, 4, 5, 6, 7, 8, 9). BETA2/NeuroD, a basic helix-loop-helix transcription factor, was cloned both as a transcriptional activator of the insulin gene (1) and as a neurogenic factor in Xenopus embryos (10). Mice lacking BETA2/NeuroD were generated by us (9) and others (11); in both cases, it has been shown that almost all of the mice exhibit severe diabetes with ketoacidosis and die shortly after birth. Consistent with this phenotype, the result of a histological examination of the BETA2-deficient pancreas indicates a lack of proper morphogenesis of islets of Langerhans and a reduction in the number of endocrine cells, especially ß-cells, which is largely due to apoptosis of ß-cells starting at embryonic d 17.0 (9). Therefore, BETA2/NeuroD plays an important role both in the maintenance of islet cell differentiation and survival and in the process of islet morphogenesis. Recently, by crossing the original line of BETA2 heterozygous mice (+/-), which was in a C57BL/129Sv mix genetic background, with NEX1 (+/-; 129SvJ background) mice (12), we observed, after a few matings, the presence of growth-retarded pups with a balance defect. Upon genotyping these mice, we realized that they were BETA2 mutants (-/-), and after careful backcrossing to both C57BL/6 and 129SvJ strains, we observed surviving BETA2 mutants in the 129SvJ, but not in the C57BL/6, background (13, 14). In this genetic background approximately 40% of mutant mice survived to adulthood with a life span comparable to that of the wild-type littermates. The surviving BETA2-/- mice exhibit a series of developmental abnormalities in the hippocampus (13), inner ear (14), and the cerebellum (11), suggesting that BETA2 is also required for brain development. It has been hypothesized (13) that these neurological defects are observed when the mice can overcome the complications of diabetes and live through the neonatal stage. The remaining question is how the new line of BETA2-/- mice manages to survive diabetes. To unravel this puzzle, we analyzed the glucose homeostasis profile and pancreatic histology of the BETA2-/- mice from different postnatal stages. In this report, we provide evidence showing that pancreatic ß-cells in these BETA2-/- mice were efficiently regenerated, organized into large aggregates, and are probably responsible for the survival of BETA2-/- mice.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Neogenesis of ß-Cells Occurred in Surviving BETA2-/- Mice
When we crossed our original BETA2 mutant mice, which are in a mixed background of C57BL/6 and 129Sv, with pure 129/SvJ mice for at least three to four generations, we found that approximately 40% of the -/- mutant mice survived for many months (13, 14). Interestingly, these BETA2-/- mice did not develop ketonuria, which is common in our original line (in a mixed background or two to three generations after crossing to the C57BL/6 background) of BETA2-/- newborn mice (9). The lack of ketonuria in the new line (in the 129SvJ background) of BETA2-/- mice suggests that these mice did not suffer from ketoacidosis and may explain why these mice could survive to adulthood. To understand the pancreatic changes that resulted in the survival of these mutant mice, we compared the insulin immunostaining pattern in the pancreas of both BETA2+/+ and BETA2-/- mice from two time points; postnatal d 4 (P4) and 1 month. Consistent with what we have reported previously (9) and as shown in Fig. 1Go, we observed a major reduction of the number of ß-cells in the BETA2-/- pancreas at P4 compared with the BETA2+/+ pancreas (Fig. 1Go, A and C). However, the number of ß-cells increased subsequently and formed large aggregates throughout the pancreas in the 1-month-old BETA2-/- mice comparable to the pancreas of wild or heterozygous littermates (Fig. 1Go, B and D). This observation suggested that neogenesis of at least ß-cells has occurred in these mice between P4 and 1 month of age, which may contribute to their survival. In addition, we also noticed that the fasting blood glucose level of BETA2-/- mice dropped gradually and reached a level that is only slightly higher than that of BETA2+/+ littermates at the age of 2 months (Fig. 1EGo), suggesting that these BETA2-/- mice no longer suffered from severe diabetes. We next compared {alpha}- and ß-cell masses of both BETA2+/+ and -/- mice at different time points, to determine when the expansion of ß-cells occurred. As shown in Fig. 1FGo, we found that the BETA2-/- ß-cell mass increased greatly after d 4 and approached the wild-type level at 2 months of age. Therefore, these data are consistent with the observation in Fig. 1Go, A–D, that ß-cell population was restored. Moreover, the restoration of ß-cells can explain the gradual disappearance of hyperglycemia in the BETA2-/- mice during this period (Fig. 1EGo). In contrast, the {alpha}-cell mass of BETA2-/- mutants declined to 30% of the wild-type mice at 1 month of age (Fig. 1FGo). Although {alpha}-cell mass increases (mean {alpha}-cell mass: 18.2 µg at P4; 115.8 µg at 2 months of age; n = 5) in the BETA2-/- pancreas, its increase is not as fast as their BETA2+/+ littermates (mean {alpha}-cell mass: 29.8 µg at P4; 399.9 µg at 2 months of age; n = 6) during this period. The decrease of the {alpha}-cell mass in BETA2-/- mice compared with that of +/+ mice is most likely due to the insufficient expansion of {alpha}-cells. In addition, there was no major change in the numbers of both {delta}- and PP cells in the BETA2-/- mice as compared with those of the wild-type littermates (data not shown).



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Figure 1. Neogenesis of Pancreatic ß-Cells in 1-Month-Old BETA2-/- Mice

Pancreatic sections from 1-month and P4 BETA2+/+ (A and B) and -/- (C and D) mice were examined for insulin activity by immunoperoxidase staining with antiinsulin antibody. In the pancreas of P4 BETA2-/- mice (C), the number of ß-cells was significantly reduced when compared with BETA2+/+ (A). The residual islet cells were present as small clusters in the pancreas (C, arrows). In contrast, large aggregates of ß-cells were observed in the pancreas of 1-month-old -/- mice (D, bracket), suggesting regeneration of ß-cells has occurred. Magnification bars, 100 µm. E, Gradual reduction of the blood glucose levels in surviving BETA2-/- mice after birth. Age-matched BETA2 +/+, +/-, and -/- mice were fasted for 14–16 h, and the blood was taken from the tail vein at indicated time points. The blood glucose levels of each group over time (P7, d 14, d 30, and d 60) are expressed as mean ± SEM (n = 16 for BETA2+/+, n = 24 for BETA2+/-, n = 12 for BETA2-/-) in each group. F, Restoration of ß- but not {alpha}-cell mass in BETA2-/- mice at the age of 2 months. ß- And {alpha}-cell masses were measured by point counting morphometry on immunoperoxidase-stained pancreatic tissues from mice of different age (P4, d 14, d 30, and d 60). Results were expressed as mean ± SEM (n = 6 for +/+ mice, n = 5 for -/- mice).

 
Newly Generated Islet Cells Partially Restored the Islet Function
To determine whether the newly generated islet cells that formed the large clusters in 1-month-old BETA2-/- mice can restore the function of islets of Langerhans, we measured the fasting serum insulin level of BETA2+/+, +/-, and -/- mice at 2 wk, 1 month, and 2 months of age. As shown in Fig. 2AGo, the average insulin level of BETA2-/- mice was less than one-fifth of the wild-type and heterozygous levels at the age of 2 wk. Later, the insulin level increased gradually and reached approximately 50% of the wild-type level at the end of 2 months (Fig. 2AGo), indicating that the regenerated ß-cells could only partially restore the level of insulin. To determine whether the production or secretion of insulin can be regulated in a normal manner in BETA2-/- mice in response to hyperglycemia, we performed a glucose tolerance test on both BETA2+/+ and -/- mice at 2 months of age. After ip injection with 20% glucose (2 g/kg body weight), the glucose levels of BETA2+/+ and +/- mice increased by about 4- to 5-fold within 30 min and subsequently declined gradually to the fasting level in 2 h. In contrast, the blood glucose level of BETA2-/- mice showed a 5- to 6-fold increase in 30 min but remain at a high level 2 h after the injection (Fig. 2BGo). The results clearly indicated that the BETA2-/- mice have impaired glucose tolerance. In addition, there was no significant difference in blood glucose level between BETA2+/+ and +/- mice during the test, suggesting that BETA2+/- mice did not have impaired glucose tolerance (Fig. 2BGo). Taken together, although these results indicate that the regenerated islet cells in the BETA2-/- pancreas could restore the fasting insulin concentration to approximately 50% of wild-type level, they still have a defect in sensing or responding to the abrupt elevation of blood glucose levels by producing or secreting the proper amount of insulin.



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Figure 2. Analysis of Insulin and Blood Glucose Concentration in BETA2 Mice

A, Partial restoration of serum insulin level in BETA2-/- mice. The fasting insulin level over time was measured using an ELISA kit. Results are expressed as mean ± SEM (at d 14, n = 15 for +/+ mice, n = 21 for +/-, n = 11 for -/-; at d 30, n = 13 for +/+, n = 25 for +/-, n = 19 for -/-; at d 60, n = 14 for +/+, n = 19 for +/-, n = 11 for -/-). The mean serum insulin level of BETA2-/- mice at 14 d was significantly lower than those of BETA2+/+ and +/- mice. However, at 60 d, the mean insulin level of BETA2-/- mice reached approximately 50% of the wild-type levels. Note a lack of significant difference between the serum insulin levels of BETA2+/- mice and those of +/+ mice. B, Impaired glucose tolerance of surviving BETA2-/- mice. The 2-month-old BETA2+/+, +/-, and -/- mice were fasted and then injected with 20% glucose (2 g/kg body weight) ip. Blood was taken from the mouse tail vein, and the glucose level was measured with a glucometer. Mean blood glucose levels over time (15 min, 30 min, 60 min, and 120 min) are shown with SEM (n = 12 for wild-type mice, n = 15 for BETA2+/- mice, n = 10 for BETA2-/- mice). The blood glucose levels of the BETA2-/- mice did not return to the baseline 2 h after the injection.

 
Newly Expanded ß-Cells Did Not Form Mature Islets
Because proper morphogenesis of islets is disrupted in our original genetic background (9), the appearance of large aggregates of islet cells in the BETA2-/- pancreas of the surviving animals prompted us to determine whether the newly generated islet cells could form the characteristic morphology of islets of Langerhans. Using double immunofluorescence microscopy with both antiinsulin and antiglucagon antibodies, we found that the restored islet cell population could not form the proper structure of islets of Langerhans, with ß-cells in the center core and non-ß-cells at the periphery (Fig. 3Go). Instead, {alpha}- and ß-cells mixed with each other randomly to form a disorganized islet. Thus, BETA2 is critical for the proper morphogenesis of islets even in the new genetic background. The observed structural defect of the islet may also contribute to the impaired glucose tolerance of BETA2-/- mice (Fig. 2BGo).



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Figure 3. The Large Islet Cell Aggregate in the 2-Month-Old BETA2-/- Pancreas Did Not Form the Typical Structure of Mature Islets of Langerhans

The pancreatic tissues of 2-month-old BETA2+/+ (A) and BETA2-/- (B) mice were stained for insulin and glucagon using double immunofluorescence microscopy with antiinsulin (green; visualized by FITC-conjugated secondary antibody) and antiglucagon (red; visualized by Cy3-conjugated secondary antibody). The {alpha}- and ß-cells in a large aggregate (B) of BETA2-/- pancreas were mixed randomly. The number of {alpha}-cells in the large aggregate (B) is also fewer than that in the BETA2+/+ islet (A). Magnification, 200x.

 
The Pancreatic Duct as One of the Sources of ß-Cell Neogenesis
To investigate the origin of ß-cell neogenesis in our system, we analyzed the location and distribution of all islet cells in the BETA2-/- mice by X-gal histochemistry. This is made possible because the LacZ gene has been knocked in to replace the BETA2 coding sequence, and thus its expression is under the control of the BETA2 promoter (9). Because the BETA2-LacZ gene is expressed exclusively in endocrine cells of the BETA2+/- pancreas and in residual pancreatic endocrine cells in BETA2-/- mice, LacZ staining is very useful in detecting the location of islet cells in the surviving BETA2-/- mice. Starting from 3 wk of age, we detected an unusually close association of endocrine cells with the pancreatic ducts of various diameters in the BETA2-/- mice (Fig. 4Go), suggesting that newly formed endocrine cells could arise from the pancreatic ductal system. A remarkable example is shown in Fig. 4AGo, revealing a cross-section of a major pancreatic duct, from which endocrine cells (X-gal-stained) seemed to bud from the ductal epithelium, migrate out, and aggregate into clusters in the muscular layers. Similarly, endocrine cells also budded from medium-sized interlobular pancreatic duct and from smaller intralobular ducts (Fig. 4Go, B–D). In total, approximately 10% of pancreatic ducts in BETA2-/- mice were undergoing budding to generate new islet cells. It should be emphasized that the budding of endocrine cells from pancreatic ducts, especially from the larger ones, was rarely found in the pancreas of the BETA2+/+ or +/- littermates of the same age (data not shown). Occasionally a single islet cell can be identified in pancreatic ducts of BETA2+/+ and +/- mice. This observation indicates that it is a unique phenomenon of the BETA2-/- pancreas. In addition, we observed that many of the islet cell-associated pancreatic ducts were markedly dilated and had intraluminal stasis of exocrine secretion (Fig. 4EGo). Again, this was not seen in the BETA2+/+ or +/- littermates. However, the mechanism causing the dilatation is not known. To confirm that the budding X-gal-stained cells are indeed endocrine cells, we performed immunofluorescence analysis on X-gal-stained tissue sections using either antiinsulin (Fig. 4Go, F and G) or antiglucagon antibody (data not shown). Interestingly, we observed that the majority of these budding cells expressed insulin but not glucagon. This result is consistent with the previous finding (Fig. 1FGo) that a major restoration occurred in ß-cells but not in {alpha}-cells.



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Figure 4. Endocrine Cells Bud from the Pancreatic Ducts of the 3-wk-Old BETA2-/- Mice

A, A cross-section of a major pancreatic duct, which was surrounded by multiple layers of muscle and connective tissue. The aggregates of endocrine cells (blue, X-gal-stained) were located in the muscular layer; some of them were still associated with the ductal epithelium. B, A cross-section of a interlobular duct. Note that endocrine cells appeared to bud from the duct. C and D, Two smaller ducts (intralobular duct) with budding endocrine cells (blue, X-gal-stained). Magnification bars, 100 µm. E, Dilatation and secretion stasis of a pancreatic duct that gives rise to newly formed ß-cells. Immunostaining for insulin was performed using pancreatic sections from 1-month-old BETA2-/- mice. A representative picture demonstrates that many ß-cells (brown) emerge from a dilated pancreatic duct, and some of them bud from the duct to form a small aggregate (arrow). Original magnification, 50x. F and G, The budding cells from the pancreatic duct produced insulin. The X-gal-stained (F) pancreatic tissues from 3-week-old BETA2-/- mice were examined for insulin (G) using immunofluorescence microscopy with antiinsulin antibody and were visualized by FITC-conjugated secondary antibody. Most X-gal-stained budding cells expressed insulin. Magnification bars, 30 µm.

 
In a previous study, Fernandes et al. (18) proposed a model indicating that in streptozotocin-treated mice, the newly formed ß-cells come from somatostatin-producing {delta}-cells; and there is an intermediate stage wherein a considerable number of endocrine cells, coexpressing somatostatin and insulin, can be found. To determine whether ß-cell neogenesis in our BETA2-/- mice also derived from {delta}-cells, we performed double immunofluorescence microscopy using both antiinsulin and antisomatostatin antibodies on those sections with marked duct-endocrine cell complexes. As shown in Fig. 5Go, we did detect some somatostatin immunoreactivity in the budding endocrine cells (Fig. 5Go, B and C); however, the number of these cells is much fewer than that of insulin-producing cells (Fig. 5AGo). More importantly, we did not detect cells coexpressing both insulin and somatostatin in this region (Fig. 5DGo). Thus, the ß-cell regeneration observed in our system is different from that observed in the streptozotocin-treated mouse.



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Figure 5. Most Ductal-Associated Endocrine Cells in BETA2-/- Mice Did Not Coexpress Insulin and Somatostatin

The X-gal-stained pancreatic tissues from 3-wk-old BETA2-/- mice were examined for the expression of insulin and somatostatin using double immunofluorescence microscopy with both antiinsulin (A, green, visualized by FITC-conjugated secondary antibody) and antisomatostatin antibodies (B, red, visualized by Cy3-conjugated secondary antibody). An overlapped picture of panels A and B is shown in panel C. Relatively few cells in this region expressed somatostatin (B), and very few, if any, cells coexpressed insulin and somatostatin. The same view under the light field with ß-galactosidase staining is also shown (D). Magnification bars, 20 µm.

 
To investigate the possibility that the newly formed ß-cells derived from a proliferating cell population of ductal origin, we performed dual color immunohistochemistry with immunoperoxidase staining for proliferating cell nuclear antigen (PCNA), a general marker for proliferating cells, and with immunofluorescence microscopy for insulin. We observed that most of the pancreatic ducts that contained budding endocrine cells have a high proliferation activity (Fig. 6Go, B–E). Moreover, many newly formed ß-cells were localized immediately adjacent to the proliferating ductal cells (Fig. 6FGo, arrows), implying a possible lineage relationship between them. In contrast, there was only a low proliferation activity detected in pancreatic ducts of BETA2+/+ and +/- mice (data not shown).



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Figure 6. Close Association of Proliferative Ductal Cells with Insulin-Producing Cells in the BETA2-/- Mice

The pancreatic tissues from 3-wk-old BETA2-/- mice were examined for proliferation using immunoperoxidase staining with anti-PCNA antibody (B and E). The same sections were subsequently examined for the activity of insulin (A and D) using immunofluorescence microscopy with antiinsulin antibody (visualized by FITC-conjugated secondary antibody). Panel C is an overlapped picture of panels A and B; panel F is an overlapped picture of panels D and E. Focal regions of panels A, B, and C were magnified and shown in panels D, E, and F, respectively. Note that some insulin-producing cells are immediately adjacent to the PCNA-positive cells (F, arrows) in the duct. Magnification bars, 20 µm.

 
The Residual Islet Cells as the Other Source of ß-Cell Neogenesis
In addition to the pancreatic duct of the BETA2-/- mice as a source of new ß-cells, we found that the preexisting ß-cells in the residual aggregates of BETA2-/- mice were also undergoing active proliferation (Fig. 7Go, A and B). To determine whether replication of preexisting ß-cells contributes to the restoration of ß-cell mass in BETA2-/- mice, we compared the mitotic index of ß-cells in the aggregates of BETA2-/- pancreas with that in the islets of wild-type pancreas at different postnatal time points. As shown in Fig. 7CGo, significantly higher rates of PCNA immunoreactivity were found in ß-cell BETA2-/- aggregates in comparison to the wild-type islets at all three time points tested. We also compared the apoptosis rate of ß-cells in BETA2-/- and +/+ pancreas (data not shown). The apoptosis rate of ß-cells in both groups at the same time points were very low (<0.1%), and no significant difference could be found between wild-type and BETA2-/- mice. Thus, replication of preexisting ß-cells also contributes to the regeneration of the ß-cell mass of the BETA2-/- mice.



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Figure 7. Mitosis of ß-Cells in an Islet Cell Aggregate of BETA2-/- Mice

A, PCNA-positive cells in an islet cell aggregate of P30 BETA2-/- mouse. The PCNA-positive cells (nuclear staining, brown) were visualized by immunoperoxidase staining. B, The location of ß-cells in the same islet cell aggregate. The same pancreatic section in panel A, after the PCNA immunoperoxidase staining, was subjected to immuofluorescence microscopy using antiinsulin antibody. The cytoplasmic staining for insulin was visualized by Cy3-conjugated secondary antibody. A comparison of panels A and B reveals the location of those ß-cells undergoing mitosis (positive for PCNA), which are labeled by black arrows in panel A and white arrows in panel B. C, ß-Cells in BETA2-/- mice have higher PCNA-expressing rates than those in BETA2+/+ mice. The rates of ß-cells positive for PCNA over time were measured by counting cells in pancreatic sections subjected to dual-color immunohistochemistry. Each time point, 20 pairs of sections from four BETA2+/+ and BETA2-/- mice each were examined. At least 1,000 cells were counted per pancreas. Values are expressed as mean ± SEM. *, P < 0.05, between BETA2+/+ and -/- at d 14. **, P < 0.005, between BETA2+/+ and -/- at d 30 and between BETA2+/+ and -/- at d 60.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In our previous study (9), we have shown that mice lacking BETA2 develop severe diabetes after birth due to the failure of forming mature islets of Langerhans. Almost all of our original lines (with higher C57BL/6 background) of BETA2-/- mice die within 5 days of birth (9). Close examinations revealed that most of them had ketonuria, an indication of severe diabetes. In contrast, the new line of BETA2-/- mice had a lower rate of neonatal lethality, and about 40% of the BETA2-/- mice survived to adulthood. The surviving BETA2-/- mice were easily distinguished from the BETA2+/+ and +/- littermates after 1 wk of age, because they started to exhibit ataxic gait and other neuronal phenotypes that have been described elsewhere (11, 13). Although our data indicated that these surviving BETA2-/- mice still suffered from mild hyperglycemia (Fig. 1Go) and impaired glucose tolerance (Fig. 2Go), they did not develop ketonuria. These results suggest that either the defect of endocrine pancreas was less severe than those of original BETA2-/- mice, or the defect was compensated by other mechanisms, e.g. islet cell neogenesis. However, at the early postnatal stage (e.g. P4) the reduction of {alpha}- and ß-cell numbers in the new line of BETA2-/- resembles the original BETA2-/- mice (Fig. 1Go and data not shown), suggesting other mechanisms in addition to ß-cell expansion must also contribute to their lack of ketonuria.

Intriguingly, the regenerated islet cells could form large aggregates but lacked the characteristic structure of the mature islets of Langerhans. The defective islet morphogenesis could simply be due to a lack of proper expression of adhesion molecules (19) or molecules required for maintenance of the islet architecture in the absence of BETA2 gene expression. Alternatively, proper islet morphogenesis may require a specific microenvironment, which probably only exists during embryogenesis. However, at present, experimental evidence seems to favor the former. Rosenberg et al. (20) have found that newly generated islet cells can be organized into mature islets of Langerhans in adult hamster with the pancreatic duct wrapped by cellophane, suggesting that the adult pancreatic microenviroment does allow proper islet morphogenesis. Therefore, although the mechanism is still unknown, BETA2 is indispensable for proper islet morphogenesis; neither the mouse strain difference nor the timing of islet morphogenesis affects its importance in this process.

Disorganized islets may not be specific to BETA2-/- mice, because similar defects were also found in the pancreata of mice lacking Nkx2.2 (6) and mice with ß-cell-specific disruption of PDX-1 (17), indicating that process of proper islet morphogenesis can be affected by multiple factors. Interestingly, impaired glucose tolerance was also found in some of these mouse models (17). It is possible that the impaired glucose tolerance seen in our BETA2-/- mice is at least partially attributed to the lack of proper islet structure. Several research reports (21, 22) have indicated that appropriate glucose-induced insulin release depends on the islet microanatomy that allows proper communication between ß- and non-ß-cells. Therefore, it is reasonable to speculate that without proper islet morphology for islet cell-cell interactions, the newly generating ß-cells cannot respond normally to high glucose levels by secreting sufficient insulin. Nonetheless, it is equally possible that impaired glucose tolerance in our BETA2-/- mice was caused by the defect of the glucose-sensing and -responding systems in individual ß-cells. Future isolation and functional characterization of individual ß-cells from our BETA2-/- mice will help elucidate these possibilities.

It has been shown recently by Malecki et al. (23) that the development of diabetes may be associated with the heterozygous state of the BETA2 gene. They suggested that either deficient binding of BETA2 or binding of a transcriptionally inactive BETA2 protein to target promoter leads to the development of a subgroup of type 2 diabetes. In our experiment, we observed (Fig. 2Go) that BETA2+/- mice do not have impaired glucose tolerance and abnormal fasting insulin levels, which were observed in PDX-1+/- mice (17, 24). Therefore, the copy number of the BETA2 gene, at least during the early stage of mouse life (2 months), does not seem to change greatly the insulin level or glucose homeostasis. The heterozygosity effect of BETA2 on the development of diabetes observed by Malecki et al. (23) could be due to the possible dominant-negative effect of mutated BETA2 gene product.

Our morphometric studies revealed that only ß-, but not {alpha}-cell mass, was significantly restored in our BETA2-/- mice (Fig. 1Go). Although there was a slight increase of {alpha}-cell mass in the BETA2-/- mice from P4 to 2 months, the magnitude was much smaller than that in the BETA2+/+ mice. This is consistent with our observation that very few {alpha}-cells were present in those regions in which regenerating endocrine cells budded from the pancreatic ducts. Selective neogenesis of ß-cells has been described elsewhere (25); however, the underlying mechanism for the selection is still unclear.

In our system, we have demonstrated that newly formed ß-cells come from many locations of the pancreatic ductal tree and from preexisting residual islet cells. The pancreatic ductal tree as a source of islet cell neogenesis is especially intriguing, although it has been recognized in several other models, including the ductal ligation model (26), the partial pancreatic duct obstruction model (27), and the 90% pancreatectomy model (28). One interesting distinction between our model and other models is that, in other models, new islet cells were reported to arise from duct-like tubular complexes, small intralobular ducts, and proliferating small ducts, but not directly from larger ducts as seen in our system. In the 90% pancreatectomy model, Bonner-Weir et al. (28) reported that ductal proliferation was seen first in the common pancreatic duct and sequentially in smaller ducts of the duct tree. However, the direct budding of islet cells from larger ducts has not been described in that model. The reason for these differences remains unclear. One possible explanation is that different triggering events may lead to different regeneration mechanisms for islet cell neogenesis. The triggering events in most of the previous models involved diffuse inflammation or tissue damage. The only exception is the partial pancreatic duct obstruction model (29), in which damage to the ductal epithelium is only minor due to intraluminal stasis of exocrine secretion. In contrast, BETA2 null mice exhibit significant reduction of ß-cell population caused by apoptosis (9), which does not involve inflammation because no infiltration of inflammatory cells was observed.

Little is known about the exact factors initiating islet cell neogenesis from the pancreatic duct in our model after apoptosis of islet cells during late gestation. Hyperglycemia may play a role in it, because hyperglycemia itself has been shown to stimulate islet cell neogenesis to a certain degree (30). In addition, although not well characterized, ductal dilatation and intraluminal stasis of exocrine secretion, which were seen in both our surviving BETA2-/- mice (Fig. 4Go) and the partial pancreatic duct obstruction model (29), may somehow contribute to trigger ß-cell neogenesis from the pancreatic duct. Glucagon-like peptide-1 (GLP-1) is a peptide expressed in L cells of the intestine and has been implicated in islet neogenesis in vitro and in vivo (31, 32, 33, 34, 35, 36). We have measured the levels of active GLP-1 in BETA2 mutants (3–8 wk) and did not observe any difference between +/+ and -/- mice (data not shown). Thus, the role of GLP-1 in this model of islet neogenesis appears unlikely. Identification of the initiation factors in our system may help reproduce islet cell neogenesis in vitro and in diabetic animals. Regardless of the unidentified initiating factors, our data support a hypothesis on the origin of ß-cell neogenesis that some ductal cells retain the ability to generate new ß-cells possibly via a process termed transdifferentiation, which may include multiple stages such as dedifferentiation, proliferation, and redifferentiation (37). Alternatively, these ductal cells may have the characteristics of stem cells. Interestingly, the colocalization of BETA2 promoter activity (represented as ß-galactosidase activity) in the budding and newly formed islet cells from the duct seems to suggest a role of BETA2 in a certain stage of this complex process. Although BETA2 may not be absolutely required during this process, based on the fact that islet cell neogenesis still occurs in mice lacking BETA2, it is likely that other basic helix-loop-helix factors in the pancreas could functionally compensate for the loss of BETA2. Future characterization of different stages of ß-cell neogenesis from the duct and molecules expressed in this process may reveal more details in this context.

In our model, we also found that the preexisting ß-cells in aggregates of BETA2-/- mice had higher mitotic indices than ß-cells in BETA2+/+ islets, suggesting that replication of preexisting ß-cells also contributes to the restoration of ß-cell mass. This is consistent with our observation that the residual aggregates of islet cells in BETA2-/- mice expanded the size gradually with time, even though these aggregates were distant from pancreatic ducts, which were the budding new ß-cells. Neogenesis of ß-cells from preexisting islet cells has been described in several models (18, 28). In the model using streptozotocin-treated mice (18), newly formed ß-cells seem to come from the preexisting ß-cells in residual islets. However, in the 90% pancreatectomy model (28), replication of preexisting differentiated ß-cells was suggested to be one of the two pathways by which ß-cells are regenerated. The other pathway in the 90% pancreatectomy model, which has been discussed above, involves ductal proliferation and differentiation into both exocrine and endocrine cells. Comparing our model with these two models, we found that our model shares some similarities with the 90% pancreatectomy model. The collaboration of both pathways may well explain why our BETA2-/- mice can successfully survive diabetes by restoring ß-cell mass rapidly.

Islet cell neogenesis, a fascinating process of cell renewal, which occurs mainly in adult animals, has long been thought to be an event that significantly modifies the physiology and pathology of the pancreas. Our new BETA2-/- mouse is an excellent model to study islet cell neogenesis. The advantage of this system is that it does not require administration of chemicals or surgical operations, and islet cell neogenesis can be studied in smaller animals such as mice. The system is suitable for studying the early mechanisms of islet cell neogenesis from both pancreatic ductal cells and from preexisting islet cells. In addition, this system can be used to assay the expression profiles of potential downstream genes of BETA2 in the pancreas. Finally, because we observed a strain difference in the severity of diabetes in the absence of BETA2, these mice might be useful for the identification of the genetic factors modifying the diabetic phenotype.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Generation of the original BETA2-/- mice has been described elsewhere (9). To study the effect of strain background on the diabetes phenotype, mice were maintained in either 129/SvJ background or C57BL/6 background by continuous backcrossing to the original BETA2+/- mice with pure wild-type 129/SvJ or C57BL/6 mice (13, 14). The resulting BETA2+/- mice were then crossed to generate the new BETA2-/- mice. Surviving BETA2-/- mice as well as the age-matched +/+ and +/- mice were killed at the same time points examined. Wild-type animals were obtained from The Jackson Laboratory (Bar Harbor, ME). All experiments on mice were conducted in accordance with the rules and regulations of Baylor College of Medicine and the Center for Comparative Medicine.

Measurement of Glucose, Insulin, and Glucagon-Like Peptide 1 (GLP-1) Levels
Blood was taken from the tail vein of age-matched BETA2+/+, +/-, and -/- mice after they were fasted for 14–16 h. The glucose level was measured with a One Touch Glucose Monitoring kit (LifeScan, Milpitas, CA) using 10 µl of blood. Blood glucose levels are represented as the average ± SD. The fasting insulin level was measured using an ELISA kit (Crystal Chem., Inc., Chicago, IL) using 5 µl of serum in duplicate. Insulin levels are expressed as means ± SEM. Blood for GLP-1 measurements was collected via orbital puncture. GLP-1 measurements were performed by Linco Bioanalytical Services (St. Charles, MO) on 50 µl of plasma on mice ranging in age from 3 to 8 wk.

Morphometry
Both {alpha}- and ß-cell masses were measured using the point counting method (15) on immunoperoxidase-stained pancreas sections. A grid was used to cover each section and to obtain the total number of intersections that intersect the immunostained cells or the whole pancreatic tissue. For each pancreas, at least 20 sections were counted to obtain an average value. For each age group, five or six animals were surveyed. The relative volume of islet cells (either ß- or {alpha}-cells) was calculated by dividing the intercepts over each individual type of islet cells by intercepts over the total pancreas tissue. The islet cell mass was then calculated by multiplying islet cell relative volume by the pancreas weight.

Immunohistochemistry
The pancreatic tissues for double immunohistochemistry were cut at 7 µm. For immunofluorescence microscopy on the X-gal-stained tissues, the tissue slides were treated with 1% sodium borohydrite for 1 h to reduce the autofluorescence caused by X-gal solution before going through the standard antigen blocking procedure. The detailed protocols for immunoperoxidase staining and double immunofluorescence microscopy have been described previously (9). Primary antibodies were used at the following dilution: guinea pig antiinsulin (Linco, St. Charles, MO), 1:100 for immunofluorescence and 1:1,000 for immunoperoxidase staining; rabbit antiglucagon (INCSTAR Corp., Stillwater, MN), 1:40 for immunofluorescence; and mouse monoclonal anti-PCNA (Sigma, St. Louis, MO), 1:20. Secondary antibodies were used at the following dilution: fluorescein isothiocyanate (FITC)-conjugated donkey antiguinea pig IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), 1:160; Cy3 (Indocarbo-cyanine)-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.), 1:400; biotinylated rabbit antimouse IgG (Vector Laboratories, Inc., Burlingame, CA), 1:300. Dual-color immunohistochemistry was performed as described previously (16). Immunoperoxidase staining was visualized using a Vectastain ABC kit (Vector Laboratories, Inc.). Controls for specificity included nonimmune sera, mismatched primary and secondary antisera, known positive sections, and absorption with specific and heterologous antigens.

Glucose Tolerance Test
Glucose tolerance tests were performed as described previously (17). Briefly, 2-month-old BETA2 +/+, +/-, and -/- mice were fasted for 14–16 h before the experiment. The mice were then ip injected with 20% glucose (2 g per kg of body weight). Blood samples were drawn from the tail vein at 0, 15, 30, 60, and 120 min after injection and subjected to glucose measurement as described above.

Statistical Analysis
All data were presented either as means ± SE or as means ± SEM, as described in figure legends. For comparison of two means, a two-tailed unpaired t test was used to test significance of the results.


    ACKNOWLEDGMENTS
 
We thank Drs. Debra Bramblett, Austin Cooney, David Tsai, and Sophia Y. Tsai for critical reading of the manuscript; Francesco DeMayo, Frank Naya, Fred Pereira, and Min Liu for assistance with animal dissection and immunofluorescent microscopy; and Fabrice Petit and Song-Chang Lin for helpful discussion.


    FOOTNOTES
 
This work was supported by NIH Grants HD-17397 and DK-55325 (to M.J.T.)

1 Current address: Department of Pediatrics, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan 10016 Back

Abbreviations: FITC, Fluorescein isothiocyanate; GLP-1, glucagon-like peptide 1; PCNA, proliferating cell nuclear antigen; X-gal, 5-bromo-4-chloro-3-indolyl ß-D-galactoside.

Received for publication July 2, 2001. Accepted for publication November 9, 2001.


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 MATERIALS AND METHODS
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