Betacellulin improves glucose metabolism by promoting conversion of intraislet precursor cells to {beta}-cells in streptozotocin-treated mice

Lei Li,1 Masaharu Seno,2 Hidenori Yamada,2 and Itaru Kojima1

1Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512; and 2Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama 700-8530, Japan

Submitted 20 March 2003 ; accepted in final form 12 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Betacellulin (BTC) induces differentiation of pancreatic {beta}-cells and promotes regeneration of {beta}-cells in experimental diabetes. The present study was conducted to determine if BTC improved glucose metabolism in severe diabetes induced by a high dose of streptozotocin (STZ) in mice. Male ICR mice were injected with 200 µg/g ip STZ, and various doses of BTC were administered daily for 14 days. The plasma glucose concentration increased to a level of >500 mg/dl in STZ-injected mice. BTC (0.2 µg/g) significantly reduced the plasma glucose concentration, but a higher concentration was ineffective. The effect of BTC was marked by day 4 but became smaller on day 6 or later. The plasma insulin concentration and the insulin content were significantly higher in mice treated with 0.1 and 0.2 µg/g BTC. BTC treatment significantly increased the number of {beta}-cells in each islet as well as the number of insulin-positive islets. Within islets, the numbers of 5-bromo-2-deoxyuridine/somatostatin-positive cells and pancreatic duodenal homeobox-1/somatostatin-positive cells were significantly increased by BTC. These results indicate that BTC improved hyperglycemia induced by a high dose of STZ by promoting neoformation of {beta}-cells, mainly from somatostatin-positive islet cells.

pancreas


BETACELLULIN (BTC), A MEMBER of the epidermal growth factor (EGF) family, was originally isolated from the insulinoma cell line (11). BTC is synthesized as a membrane-spanning precursor that contains a single EGF motif, and the extracellular portion of the BTC precursor is then cleaved by proteolysis (11). Interestingly, the BTC precursor itself also possesses a bioactivity, and, therefore, BTC may also exert its action through a juxtacrine mechanism (12). BTC was expected to be an angiogenic factor but was without such activity. Instead, it stimulated DNA synthesis in fibroblasts and vascular smooth muscle cells (14). Regarding its expression, BTC is predominantly found in the pancreas and the intestine. In particular, BTC is expressed in endocrine precursor cells of the fetal pancreas and also in insulin-secreting cells found in patients with nesidioblastosis (9). These data suggest that BTC plays a role in regulating growth and/or differentiation of endocrine precursor cells of the fetal pancreas. In agreement with this notion, BTC was found to convert amylase-secreting pancreatic AR42J cells into insulin-producing cells (8) and to have a mitogenic effect in human undifferentiated pancreatic epithelial cells (2).

BTC is thought to elicit growth-promoting action via the EGF receptor ErbB1 and also binds to ErbB4 (1, 14). Regarding the differentiation-inducing activity, the effect of BTC is not reproduced by the EGF receptor ligands in pancreatic AR42J cells, and we postulated that BTC may induce differentiation of {beta}-cells by acting on other unique BTC receptors (6).

BTC is intriguing in that it promotes {beta}-cell regeneration when administered in vivo. For example, BTC was shown to stimulate regeneration of pancreatic {beta}-cells in animal models of diabetes (7, 15). Accordingly, Yamamoto et al. (15) showed that BTC augmented regeneration of {beta}-cells in a unique alloxanperfused mouse pancreas. In this model, BTC promoted growth of cytokeratin-positive ductal cells and also increased the number of islet-like cell clusters (ICC). Glucose intolerance was ameliorated in BTC-treated mice. Using 90% pancreatectomized rats, we (7) showed that BTC stimulated regeneration of {beta}-cells. BTC promoted growth of {beta}-cells in the remnant pancreas and also induced neogenesis of {beta}-cells from progenitors located in or by the pancreatic duct. BTC increased the {beta}-cell mass, and the insulin content of the remnant pancreas and glucose intolerance was improved. These results suggest that BTC has a therapeutic potential in treating diabetes.

There is an alternate route of {beta}-cell regeneration, namely differentiation of precursor cells located in pancreatic islets. This is best demonstrated in mice treated with the {beta}-cell toxin streptozotocin (STZ). In this model, STZ causes significant damage in pancreatic {beta}-cells, and, when a high dose of STZ is injected, most {beta}-cells are destroyed within a short period of time and severe diabetes is induced. Even in this condition, however, regeneration of {beta}-cells takes place. Fernandes et al. (3) showed that the {beta}-cell regeneration process occurs mainly in islets. They postulated that cells expressing pancreatic duodenal homeobox-1 (PDX-1) and somatostatin serve as precursors of {beta}-cells in STZ-injected mice. Either {delta}-cells dedifferentiate to PDX-1-positive cells or preexisting PDX-1/somatostatin-positive cells proliferate, which leads to an increase in the number of PDX-1/somatostatin-positive cells. These cells then convert to insulin/somatostatin-positive cells and eventually to {beta}-cells. Although the regeneration takes place actively, the {beta}-cell mass further decreases with time. This is due to the damage to newly formed {beta}-cells caused by severe hyperglycemia (4). In the present study, we examined whether BTC could induce {beta}-cell regeneration in this severe form of experimental diabetes by promoting conversion of precursor cells in islets.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals and tissue processing. Four-week-old male ICR mice were obtained from Charles River (Ibaraki, Japan). Mice were injected in with 200 µg/g STZ (Wako, Tokyo, Japan) freshly dissolved in 0.1 M citrate buffer (pH 4.5). Recombinant BTC (0.05, 0.1, 0.2, 0.5, and 1 µg/g) or PBS was then injected daily for 14 days. At least six mice per group were investigated.

The morning-fed plasma glucose concentration and body weight were measured daily. The plasma insulin concentration was measured on day 7 and day 14. On day 15, an intraperitoneal glucose tolerance test (2 g/kg body wt) was taken after 14 h of fasting. Two days later, mice were killed. Each pancreas was excised, weighed, and divided into two parts. One portion was fixed in 4% paraformaldehyde-PBS overnight at room temperature and embedded in paraffin for histochemistry. The other was homogenized in cold acid-ethanol, heated for 5 min in a 70°C water bath, and centrifuged, and the supernatant was then stored at -20°C until assay. At least three mice in each group were examined for histochemistry.

On days 2, 3, and 7, some mice were in injected with 100 mg/kg 5-bromo-2-deoxyuridine (BrdU; Sigma, St. Louis, MO) and killed 4 h later. The pancreas was then excised and fixed as described above. The experimental protocol was approved by the Animal Care Committee of Gunma University. The insulin concentration was determined by a time-resolved immunofluorometric assay as described previously (8).

Immunohistochemistry. The methods for immunohistochemistry were described previously (7). Primary antibodies were used at the following dilutions: guinea pig anti-porcine insulin, 1:1,000 (gift from Dr. T. Matozaki of Gunma University); rabbit anti-human glucagon, 1:2,000 (Peptide Institute, Osaka, Japan); rabbit anti-human somatostatin, 1:500 (DAKO, Glostrop, Denmark); rabbit anti-human pancreatic polypeptide (PP), 1:1,000 (Chemicon, Temecula, CA); and rabbit anti-human PDX-1, 1:3,000 (a generous gift from Dr. Y. Kajimoto of Osaka University). The following immuno-staining systems were used: peroxidase-conjugated donkey anti-guinea pig IgG, 1:500 (Jackson Immunoresearch Laboratories, West Grove, PA); horseradish peroxidase-based visualization system (Envision+; DAKO); and rabbit alkaline phosphatase-conjugated Vectastain ABC kit, rabbit (Vector Laboratories, Burlingame, CA). The BrdU incorporation assay was accomplished with a cell proliferation assay kit (Amersham Pharmacia Biotech, Little Chalfont, UK).

{delta}-Cell replication was analyzed by BrdU and somatostatin double staining. The results were expressed as the percentage of BrdU-positive {delta}-cells. PDX-1-positive {delta}-cells were analyzed by PDX-1 and somatostatin double staining. At least 500 {delta}-cells per mouse were counted, and three mice were examined. The neogenesis of the {beta}-cell was analyzed by measuring the number of ICCs (those <8 cells across). Single insulin-positive cells and ICCs were counted in sections at x200 and confirmed at x400. At least five sections (cut at intervals of 200 µm) per mouse were examined. Data are presented as the number of ICCs per field.

We examined the number of {beta}-cells per islet and the islet number per unit square. At least 30 islets were analyzed in each mouse for the determination of the number of {beta}-cells per islet. The data were expressed as the number of {beta}-cells per islet. For determination of the number of islets, five sections were examined in each mouse at x200, and the structure of each islet was confirmed at x400. The data were presented as the number of islets per field.

To examine the replication of the duct, immunofluorescence double staining was performed with mouse anti-BrdU (1:100) and rabbit anti-bovine keratin for side-spectrum screening that cross-reacts with mouse keratin (15), 1:1,000 (DAKO). The paraffin sections were deparaffinized and rehydrated, washed with Tween plus Tris-buffered saline (TTBS), and blocked with DAKO protein block solution. The sections were incubated overnight at 4°C with a mixture of primary antibody (mouse anti-BrdU and rabbit anti-bovine keratin antibodies), washed with TTBS, and incubated for 1 h at room temperature with a mixture of secondary antibody (goat Alexa Fluor 488-conjugated anti-mouse IgG, 1:500; and goat Alexa Fluor 568-conjugated anti-rabbit IgG, 1:1,000; Molecular Probes, Eugene, OR). The counterstaining was done with 4',6-diamidino-2-phenylindol · HCl (Boehringer Mannheim, Mannheim, Germany). At least 1,000 ductal cells were counted in each mouse, and four mice were examined.

Statistical analysis. Results were expressed as means ± SE. For comparisons between the two groups, the unpaired t-test was used. P < 0.05 was considered to be significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effect of BTC on the plasma glucose concentration in STZ-injected mice. We injected 200 µg/g STZ intraperitoneally (day 0), and various doses of BTC were administered daily starting immediately after the STZ injection. The plasma glucose concentration in STZ-injected mice was >400 mg/dl on day 1 and >500 mg/dl on day 2. On day 4, the plasma glucose concentration reached its peak value and remained at that level thereafter (Fig. 1A). Daily administration of BTC significantly reduced the plasma glucose concentration at a dose of 0.2 µg/g. The effect of BTC was not observed at lower or higher doses. This effect was already apparent on day 1 and continued to be significant up until day 14. The effect of BTC on the plasma glucose was largest on day 4 and became less at later time points. In the STZ-injected mice, the body weight was reduced significantly on day 4 (P < 0.05 vs. day 0) and remained decreased until day 14 (Fig. 1B). In BTC-treated mice, the body weight was significantly higher than that in control after 8 days of STZ injection. BTC had an effect on body weight at the doses of 0.1 and 0.2 µg/g.



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Fig. 1. Effects of betacellulin (BTC) on plasma glucose concentration (A) and body weight (B) in streptozotocin (STZ)-injected mice. STZ (200 µg/g) was injected on day 0, and various concentrations of BTC were injected daily starting immediately after STZ injection. The plasma glucose concentration and body weight were measured. Values are means ± SE for 6 mice. {circ}, No BTC; {blacksquare}, 0.1 µg/g BTC; {triangleup}, 0.2 µg/g BTC; {bullet}, 0.5 µg/g BTC. *P < 0.05 vs. no BTC.

 

Effect of BTC on the insulin content and the plasma insulin concentration. We measured the insulin content of the pancreas 17 days after the STZ injection. It was significantly higher in the mice treated with 0.1 and 0.2 µg/g BTC compared with the mice not receiving BTC (Fig. 2A), but the levels of the insulin content were low compared with those of normal mice. Again, administration of 0.5 µg/g BTC was ineffective in increasing the insulin content. Consistent with this observation, the plasma insulin concentration on day 7 was significantly higher in mice treated with 0.1 and 0.2 µg/g BTC than in the control mice (Fig. 2B). BTC was not effective at the dose of 0.5 µg/g. On day 14, the plasma insulin concentration was undetectable (<0.1 ng/ml) in the control mice, whereas in the mice treated with 0.2 µg/g BTC it was 0.212 ± 0.08 ng/ml (n = 6). The plasma glucose concentrations in response to intraperitoneal glucose were lower in BTC-treated mice, but the changes were not statistically significant (data not shown).



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Fig. 2. Effects of BTC on the plasma insulin concentration and insulin content. STZ-injected mice were treated with various doses of BTC for 14 days, and the plasma insulin content (A) and concentration (B) on day 17 were measured. Values are means ± SE for 6 mice. The insulin content and the plasma insulin concentration in normal mice were 77.0 ± 6.8 µg/g (n = 6) and 1.67 ± 0.51 ng/ml (n = 10), respectively. *P < 0.05 vs. saline-treated control.

 

Effect of BTC on the morphology of islets. In the STZ-treated mouse pancreas, islets were severely damaged morphologically. Figure 3A, a, shows an islet of the STZ-treated mouse on day 1. {beta}-Cells were stained in brown with anti-insulin antibody. Non-{beta}-cells were stained in red by a cocktail of anti-glucagon, anti-PP, and anti-somatostatin antibodies. As depicted, most of the {beta}-cells were severely damaged by STZ (Fig. 3A). Figure 3A, b, shows the PDX-1 staining of the STZ-treated islets on day 1. Non-{beta}-cells are stained in red. Most of the {beta}-cells were lost, and only a small number of PDX-1-positive {beta}-cells remained in the islet. The number of PDX-1-positive {beta}-cells remaining in the islet in the control and BTC-treated mice was not changed significantly on day 1 (Figs. 3A, b and 4A).



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Fig. 3. Morphology of the islet in an STZ-injected mouse. A: pancreatic sections were obtained from mice injected with STZ on day 1. A section (a) was double stained with anti-insulin (brown) and a cocktail of anti-glucagon and anti-somatostatin (red) antibodies. Another section (B) was double stained with anti-PDX-1 (brown) and a cocktail of anti-glucagon and anti-somatostatin (red) antibodies. B: pancreatic sections obtained from saline-treated (a, c) and 0.2 µg/g BTC-treated (b, d) mice on day 3 (a, b) and day 17 (c, d) were stained with anti-insulin antibody.

 


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Fig. 4. Effects of BTC on the number of {beta}-cells and islets in STZ-injected mice. A: changes in the number of {beta}-cells per islet in saline-treated (open bars) and 0.2 µg/g BTC-treated (closed bars) mice were determined. Note that the number of {beta}-cells per islet on day 0 was 76 ± 4.4 (n = 3). Values are means ± SE for 4 mice. *P < 0.05 vs. control. B: number of islets containing insulin-positive cells was counted in pancreatic sections obtained from saline-treated (open bars) and 0.2 µg/g BTC-treated (closed bars) mice. Values are means ± SE for 4 mice. *P < 0.05 vs. control.

 

Since the effect of BTC on the plasma glucose was significant at a dose of 0.2 µg/g, an immunohistochemical analysis was done in mice treated with saline or 0.2 µg/g BTC. Figure 3B shows the insulin staining of islets in BTC-treated and saline-treated mice on day 3 and on day 17. As can be seen, the number of insulin-positive cells in the islet was significantly greater in BTC-treated mice on day 3. Note that the number of BrdU/insulin-positive cells was very scarce on days 2 and 3, and BTC did not affect the number of these cells (data not shown). On day 17, the number of insulin-positive cells in the islets in BTC-treated mice was slightly increased, but the change was not statistically significant (P = 0.05) (Fig. 4A).

Figure 4B shows the changes in the number of islets in STZ-injected mice. We counted the number of islets containing insulin-positive cells. As depicted, the number of insulin-positive islets decreased as a function of time in saline-treated mice. In BTC-treated mice, the number of insulin-positive islets was the same as that of saline-treated mice on day 1. On days 3 and 17, the number of insulin-positive islets in BTC-treated mice was significantly higher than that in saline-treated mice. To assess whether or not this effect of BTC resulted from improved hyperglycemia, we examined the changes in the number of insulin-positive islets in mice treated with 0.1 µg/g BTC. This dose of BTC did not reduce the plasma glucose concentration. In mice treated with 0.1 µg/g BTC, the number of insulin-positive islets was 0.059 ± 0.002/field on day 17, which was significantly (P < 0.05) greater than that in saline-treated mice (0.039 ± 0.005; n = 3).

It was shown previously that in STZ-treated mice the number of PDX-1/somatostatin-positive cells was increased. These cells then differentiated into {beta}-cells (3, 4). We therefore examined the changes in {delta}-cells in STZ-injected mice. As shown in Fig. 5A, BrdU-positive {delta}-cells were observed after STZ injection. The number of BrdU-positive {delta}-cells was ~4% on day 2 and decreased thereafter. Administration of BTC significantly increased the number of BrdU-positive {delta}-cells (Fig. 5B), and BTC induced significant effects on days 2 and 3. In addition, the number of PDX-1-positive {delta}-cells was markedly increased after STZ injection (Fig. 6). Treatment with BTC also increased the number of PDX-1-positive {delta}-cells on days 2 and 3 (Fig. 6B).



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Fig. 5. Effect of BTC on DNA synthesis of {delta}-cells A: pancreatic section obtained from STZ-injected mouse on day 2 was stained with anti-5-bromo-2-deoxyuridine (BrdU; brown) and anti-somatostatin antibody (red). Arrows indicate BrdU-positive {delta}-cells. B: BrdU-positive {delta}-cells were counted in saline-treated (open bars) and 0.2 µg/g BTC-treated (closed bars) mouse pancreases. Values are means ± SE for 3 mice. *P < 0.05 vs. control.

 


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Fig. 6. Effect of BTC on number of PDX-1-positive {delta}-cells A: pancreatic section obtained from STZ-treated mouse on day 2 and stained with anti-PDX-1 (brown) and anti-somatostatin antibody (red). Arrows indicate PDX-1-positive {delta}-cells. Inset is a higher magnification of the framed area. B: PDX-1-positive {delta}-cells were counted in saline-treated (open bars) and 0.2 µg/g BTC-treated (closed bars) mouse pancreas. Values are means ± SE for 3 mice. *P < 0.05 vs. control.

 

It is shown that {beta}-cell progenitors also express PP (5). We therefore examined the changes in the number of PP-positive cells in islets after the STZ treatment. As shown in Fig. 7, the number of PP-positive cells was increased 2 days after the STZ treatment. In normal mice, the number of PP-positive cells was 3.4 ± 0.4/islet, whereas it was 5.8 ± 1.3/islet 2 days after STZ treatment (P < 0.05). On day 3, the number of PP-positive cells declined to normal levels. BTC did not affect the number of PP-positive cells.



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Fig. 7. Changes in number of pancreatic polypeptide (PP)-expressing cells in islet pancreatic sections obtained from mice before (A) and 2 days after (B) STZ treatment and stained with anti-PP antibody are shown.

 

In STZ-injected mice, the proliferation of ductal cells took place, as evidenced by the existence of BrdU/cytokeratin-positive cells (Fig. 8A). BTC affected the ductal cells and increased the number of BrdU/cytokeratin-positive cells. This effect of BTC was significant on day 3 (Fig. 8B). BTC also increased the number of ICCs in the pancreas (Fig. 8C).



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Fig. 8. Effect of BTC on numbers of BrdU-positive ductal cells and islet-like cell clusters (ICCs). A: pancreatic section obtained from STZ-injected mouse on day 2 was stained with anti-BrdU (green) and anti-cytokeratin (CK) (red) antibodies. Nuclei were stained with 4',6-diamidin-2-phenylindol · HCl (DAPI; blue). B: number of BrdU-positive ductal cells was counted in pancreatic sections obtained from saline-treated (open bars) and 0.2 µg/g BTC-treated (closed bars) mice. Values are means ± SE for 4 mice. *P < 0.05 vs. control. C: number of ICCs was counted in pancreatic sections obtained from saline-treated (open bars) and 0.2 µg/g BTC-treated (closed bars) mice. Values are means ± SE for 4 mice. *P < 0.05 vs. control.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In the present study, we examined whether or not BTC improved glucose metabolism in severe diabetes induced by a high dose of STZ. This dose of STZ causes severe damage to {beta}-cells, and the plasma glucose level elevates soon after the injection of STZ. As shown in Fig. 3A, b, most of the pancreatic {beta}-cells, except for those in the periphery, were destroyed within a day, and the plasma glucose concentration was >400 mg/dl on day 1 and >500 mg/dl on day 2. Daily administration of BTC significantly reduced the plasma glucose levels at a dose of 0.2 µg/g. A higher dose of BTC was, however, ineffective. BTC also prevented the reduction of body weight at doses of 0.1 and 0.2 µg/g. Again, a higher dose of BTC was ineffective. Given that changes in body weight reflect the metabolic derangements, BTC was effective in improving glucose metabolism at doses of 0.1-0.2 µg/g. Consistent with this notion, the plasma insulin concentration and the insulin content of the pancreas 2 wk after the STZ injection were significantly higher in mice treated with 0.1 and 0.2 µg/g BTC. Hence, BTC dose-dependently improved hyperglycemia in STZ-injected mice by preserving production and secretion of insulin. Although BTC significantly increased the insulin content and the plasma insulin concentration, the absolute values were much lower than those in normal mice. This explains why the glucose tolerance was not improved significantly in BTC-treated mice.

The reason a high dose of BTC was ineffective is not totally clear at present. Yamamoto et al. (15) showed that 1 µg/g BTC improved glucose intolerance caused by selective alloxan perfusion in mice. In their study, BTC increased proliferation of ductal cells and formation of ICCs. The main site of the BTC action may be ductal cells or progenitors around the duct. These cells reexpress PDX-1 and may serve as precursors of newly formed {beta}-cells in regenerating pancreas (10), which form ICC, migrate, and eventually form islets. Since TGF-{alpha} induces proliferation of these cells (13), the effect of BTC on ductal cell proliferation may be exerted through the EGF receptor. In our model, contribution of ductal cells is thought to be small (3), and intraislet precursors play a major role as a source of newly formed {beta}-cells (3).

It is known that BTC also acts on receptors other than the EGF receptor (1, 6). Specifically, we have suggested that differentiation-inducing activity of BTC is exerted through a unique BTC receptor distinct from either the EGF receptor ErbB1 or ErbB4 in pancreatic AR42J cells (6). The molecular nature of this unique BTC receptor is, however, not clear at present. If this unique BTC receptor participates in the conversion of intraislet precursors, the ineffectiveness of the high dose of BTC in our model may result from a particular feature of the binding properties of the unique BTC receptor expressed in intraislet precursor cells. Alternately, higher doses of BTC may have adverse effects on residual or newly formed {beta}-cells. For example, if a higher dose of BTC elicits inappropriately strong growth signals in residual or newly formed {beta}-cells, it may hamper the maintenance of differentiated functions. In any event, the results suggest that appropriate doses of BTC are needed to produce a beneficial effect on glucose metabolism.

BTC improves the glucose metabolism in STZ-injected mice presumably by acting in multiple steps. STZ causes profound damage to {beta}-cells, but BTC does not appear to prevent immediate {beta}-cell death induced by STZ because the number of remaining {beta}-cells in each islet was not changed by BTC on day 1. On day 3, however, the number of insulin-positive cells per islet was significantly greater in BTC-treated mice. This may result mainly from the formation of new {beta}-cells. Fernandes et al. (3) postulated that somatostatin-expressing cells are the major sources of newly formed {beta}-cells in STZ-injected mouse islets. They showed that PDX-1/somatostatin-positive cells as well as somatostatin/insulin-positive cells were increased in STZ-injected mice (3). PDX-1/somatostatin-positive cells were derived from either proliferation of PDX-1/somatostatin double-positive cells found in a small fraction of {delta}-cells in normal islets or reexpression of PDX-1 in {delta}-cells (dedifferentiation). The present finding that the number of PDX-1/somatostatin-positive cells was markedly increased in STZ-injected mice confirmed their conclusion. The present results further show that treatment with BTC significantly increased the number of PDX-1/somatostatin-positive cells. As mentioned above, these cells may be derived from two sources (3): proliferation of preexisting PDX-1-positive {delta}-cells and reexpression of PDX-1 in PDX-1-negative {delta}-cells. It should also be mentioned that the number of BrdU-positive cells was only ~6% of the {delta}-cells (Fig. 5B), whereas more than half of the {delta}-cells became PDX-1 positive in BTC-treated mice on day 2 (Fig. 6B). The proliferation of preexisting PDX-1-positive cells cannot explain this drastic increase. Therefore, the increase in the number of PDX-1/somatostatin-positive cells is not only due to the proliferation of preexisting PDX-1/somatostatin-positive cells but also to reexpression of PDX-1 in PDX-1-negative {delta}-cells, even though contribution of the latter is indeed significant.

Together, the data suggest that BTC promoted neoformation of {beta}-cells by increasing the precursor pool in islets. Although our results do not address the differentiation of precursor cells to {beta}-cells, it is possible that BTC also promotes this differentiation. In addition to the intraislet events, BTC also increased the number of proliferating ductal cells and ICCs (Fig. 8). As in 90% pancreatectomized rats (6), BTC augments neogenesis of islets from precursor cells located in or by the pancreatic duct. Yet, because frequency of islet neogenesis from the pancreatic duct is relatively low (7), the contribution of islet neogenesis from the duct may be slight in the effect of BTC.

BTC may have another site of action. As shown in Fig. 4C, the number of islets containing insulin-positive cells reduced as a function of time in STZ-injected mice. This is perhaps due to the glucose toxicity induced by severe hyperglycemia as suggested by Guz et al. (4). In BTC-treated mice, the number of insulin-positive islets was greater than that in control mice. This may have resulted from the direct and indirect protective effects of BTC. As shown in Fig. 1A, 0.2 µg/g BTC improved hyperglycemia in STZ-injected mice. The decrease in the plasma glucose concentration may have reduced the glucose toxicity and thereby slowed down the disappearance of the insulin-positive islets. Direct protective action of BTC may also have been a factor. The protective effect of BTC was suggested by the data obtained on day 1. Although BTC decreased the plasma glucose concentration, it had no effect on the number of {beta}-cells. A similar protective effect was observed in the 90% pancreatectomized rat (7).

Taking these results together, BTC improved the glucose metabolism by stimulating regeneration of pancreatic {beta}-cells. BTC promoted neogenesis of {beta}-cells from the precursor pool in the islets and around the pancreatic duct. BTC also protected {beta}-cells from glucose toxicity. Since the half-life of administered BTC is very short (15), a more appropriate method to deliver BTC would increase its therapeutic potential. Further studies are needed to solve this issue.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by a Grant-in-Aid for Science Research from the Ministry of Education, Science, Sports and Culture of Japan. Lei Li is a postdoctoral research fellow supported by the Japanese Society for the Promotion of Science.


    ACKNOWLEDGMENTS
 
We are grateful to Mayumi Odagiri for technical and secretarial assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: I. Kojima, Institute for Molecular and Cellular Regulation, Gunma Univ., Maebashi 371-8512, Japan (E-mail: ikojima{at}showa.gunma-u.ac.jp).

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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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