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
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ABSTRACT |
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pancreas
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 -cells by acting on other unique BTC receptors
(6).
BTC is intriguing in that it promotes -cell regeneration when
administered in vivo. For example, BTC was shown to stimulate regeneration of
pancreatic
-cells in animal models of diabetes
(7,
15). Accordingly, Yamamoto et
al. (15) showed that BTC
augmented regeneration of
-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
-cells. BTC promoted growth of
-cells
in the remnant pancreas and also induced neogenesis of
-cells from
progenitors located in or by the pancreatic duct. BTC increased the
-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 -cell regeneration, namely
differentiation of precursor cells located in pancreatic islets. This is best
demonstrated in mice treated with the
-cell toxin streptozotocin (STZ).
In this model, STZ causes significant damage in pancreatic
-cells, and,
when a high dose of STZ is injected, most
-cells are destroyed within a
short period of time and severe diabetes is induced. Even in this condition,
however, regeneration of
-cells takes place. Fernandes et al.
(3) showed that the
-cell
regeneration process occurs mainly in islets. They postulated that cells
expressing pancreatic duodenal homeobox-1 (PDX-1) and somatostatin serve as
precursors of
-cells in STZ-injected mice. Either
-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
-cells. Although
the regeneration takes place actively, the
-cell mass further decreases
with time. This is due to the damage to newly formed
-cells caused by
severe hyperglycemia (4). In
the present study, we examined whether BTC could induce
-cell
regeneration in this severe form of experimental diabetes by promoting
conversion of precursor cells in islets.
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MATERIALS AND METHODS |
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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).
-Cell replication was analyzed by BrdU and somatostatin double
staining. The results were expressed as the percentage of BrdU-positive
-cells. PDX-1-positive
-cells were analyzed by PDX-1 and
somatostatin double staining. At least 500
-cells per mouse were
counted, and three mice were examined. The neogenesis of the
-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 -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
-cells per islet. The data were expressed
as the number of
-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.
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RESULTS |
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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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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. -Cells were
stained in brown with anti-insulin antibody. Non-
-cells were stained in
red by a cocktail of anti-glucagon, anti-PP, and anti-somatostatin antibodies.
As depicted, most of the
-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-
-cells are stained in red. Most of the
-cells were lost, and
only a small number of PDX-1-positive
-cells remained in the islet. The
number of PDX-1-positive
-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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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 -cells
(3,
4). We therefore examined the
changes in
-cells in STZ-injected mice. As shown in
Fig. 5A, BrdU-positive
-cells were observed after STZ injection. The number of BrdU-positive
-cells was
4% on day 2 and decreased thereafter.
Administration of BTC significantly increased the number of BrdU-positive
-cells (Fig.
5B), and BTC induced significant effects on days
2 and 3. In addition, the number of PDX-1-positive
-cells
was markedly increased after STZ injection
(Fig. 6). Treatment with BTC
also increased the number of PDX-1-positive
-cells on days 2
and 3 (Fig.
6B).
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It is shown that -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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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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DISCUSSION |
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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 -cells in regenerating pancreas
(10), which form ICC, migrate,
and eventually form islets. Since TGF-
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
-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
-cells. For example, if a higher dose of BTC elicits inappropriately
strong growth signals in residual or newly formed
-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 -cells, but BTC
does not appear to prevent immediate
-cell death induced by STZ because
the number of remaining
-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
-cells. Fernandes et al.
(3) postulated that
somatostatin-expressing cells are the major sources of newly formed
-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
-cells in normal islets or reexpression of PDX-1 in
-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
-cells and reexpression of PDX-1 in
PDX-1-negative
-cells. It should also be mentioned that the number of
BrdU-positive cells was only
6% of the
-cells
(Fig. 5B), whereas
more than half of the
-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
-cells, even though
contribution of the latter is indeed significant.
Together, the data suggest that BTC promoted neoformation of -cells
by increasing the precursor pool in islets. Although our results do not
address the differentiation of precursor cells to
-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
-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 -cells. BTC promoted neogenesis
of
-cells from the precursor pool in the islets and around the
pancreatic duct. BTC also protected
-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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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