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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
Department of Cell Biology (L.L., I.K.), Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan 371-8512; and Department of Bioscience and Biotechnology (M.S., H.Y.), Faculty of Engineering, Okayama University, Okayama, Japan 700-8530
Address all correspondence and requests for reprints to: Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan. E-mail: ikojima{at}showa.gunma-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Then mature BTC is released by proteolytic
cleavage of the extracellular portion of the BTC precursor.
Interestingly, the BTC precursor is also biologically active and is
thought to act as a juxtacrine factor (2). The expression
of BTC is detected predominantly in the intestine and pancreas
(3). In the adult pancreas, BTC is expressed in
non-ß-cells of the pancreatic islets (4). Immunoreactive
BTC is also observed in endocrine cells in the fetal pancreas as well
as in the regenerating pancreas (4). We previously
reported that BTC, acting coordinately with activin A, converted
amylase-secreting pancreatic AR42J cells into insulin-producing cells
(5). Given that AR42J cells resemble in many respects the
endocrine precursor cells of the pancreas (6, 7), these
results raise the possibility that BTC promotes differentiation of
pancreatic ß-cells. Consistent with this notion, treatment with BTC
induced the expression of the insulin gene in an -cell line
transfected with pancreatic and duodenal homeobox gene-1 (PDX-1)
(8). Furthermore, BTC stimulates proliferation of INS-1
cells, a mature insulinoma cell line (9), and endocrine
cells of the human fetal pancreas (10). Huotari et
al. (9) postulated that BTC may be a ß-cell mitogen
expressed in the pancreas. BTC is an intriguing polypeptide factor that
potentially promotes growth and differentiation of pancreatic
ß-cells. Reduction of ß-cell mass is critical in the genesis of type 2 diabetes (11). The ß-cell mass in type 2 diabetic patients is reduced, compared with nondiabetic subjects (12, 13). In experimental models of type 2 diabetes, replication or differentiation of ß-cells was impaired (14, 15, 16). Therefore, agents that increase ß-cell mass would be beneficial in the prevention and treatment of type 2 diabetes. In this regard, Yamamoto et al. (17) recently reported that BTC promoted the neogenesis of ß-cells in mice treated with alloxan. In the present study, we examined whether administration of BTC affected regeneration of pancreatic ß-cells following 90% pancreatectomy in rats. The results indicate that BTC promotes regeneration of ß-cells and improves the impaired glucose metabolism in pancreatectomized rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The morning-fed plasma glucose concentration was measured every day for 7 d and then weekly for up to 4 wk. The plasma insulin concentration and body weight were measured weekly for 4 wk. On the 30th d after surgery, an ip glucose tolerance test (2 g/kg body weight) was done after fasting 14 h. Blood was collected in heparinized hematocrit tubes after 0, 30, 60, 90, and 120 min. The plasma glucose was measured with a glucose analyzer and the remainder was stored for insulin assay. Three days later, rats were killed. The remnant 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 70 C water bath, centrifuged, and the supernatant was stored at -20 C until assay.
Some rats were injected with 100 mg/kg 5-bromo-2-deoxyuridine (BrdU; Sigma, St. Louis, MO) ip and killed 6 h later 2–3 d after pancreatectomy. Remnant pancreas was 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 (5).
Immunohistochemistry and histomorphometry
The paraffin sections (4 µm) were deparaffinized and
dehydrated. Endogenous peroxidase was inhibited with 1%
H2O2/methanol. After being
washed with PBS, the sections were incubated overnight at 4 C with a
guinea pig anti-porcine insulin (1:1000) provided by Dr. T. Matozaki of
Gunma University, rinsed with PBS, incubated 1 h at room
temperature with peroxidase conjugated donkey anti-guinea pig IgG
(1:500, Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA), developed with diaminobenzidine, and counterstained
with hematoxylin. These sections (four sections per rat) were
histomorphometried with the method described by Movassat et
al. (15). The ß-cell area and the area of each
section were determined with the image analysis software (NIH image).
The ratio of ß-cell area in the remnant pancreas was calculated by
dividing the area of all insulin-positive cells in one section by the
total area of this section. The ß-cell mass was calculated by
multiplying the remnant pancreas weight by the ratio of ß-cell area.
The ß-cell size was determined on sections stained with anti-insulin
antibody by evaluating the mean cross-sectional area of individual
ß-cell. The area of ß-cell in islet was measured as described above
and the number of ß-cell nuclei in the islet was counted. Ten islets
were counted in each animal.
Growth of ß-cells was analyzed by BrdU and insulin double staining. The paraffin sections were incubated in a microwave oven in target-retrieved solutions (DAKO Corp., Glostrup, Denmark) after deparaffinizations and rehydration. The sections were then allowed to cool at room temperature, washed with PBS, inhibited the endogenous peroxidase, and washed with PBS. At the first step, BrdU staining was accomplished with a cell proliferation assay kit (Amersham Pharmacia Biotech, Little Chalfont, UK). Sections were incubated for 1 h at room temperature with a mouse anti-BrdU monoclonal antibody, washed with PBS, incubated with peroxidase-linked sheep antimouse IgG, and stained with diaminobenzidine plus substrate/intensifier containing nickel chloride and cobalt chloride. The sections were then washed with 0.2 mM glycine (pH 2.2) and washed with distilled water and PBS. For the second step of the double-staining procedure, insulin staining was done as described previously. Finally, the sections were counterstained with hematoxylin.
Insulin-positive ß-cells were seen as orange-brown cytosol, and BrdU-positive cells appear blue-black nuclei (19). BrdU/insulin-positive cells in islets were counted in each section as a marker of replication of preexisting ß-cell. At least 2000 ß-cells or 20 BrdU/insulin double-positive cells were counted in one rat. The results were expressed as the percentage of BrdU-positive ß-cells. Neogenesis of ß-cells was analyzed by measuring the number of islet cell-like clusters (ICC) (less then 8 cells across) and BrdU-positive cells in ICCs. Single insulin-positive cells and ICCs were counted in sections at 400x. Data are shown as the number of ICC per field. For analysis of BrdU-positive cells in ICC, at least 100 insulin-positive cells were counted in a rat and the number of BrdU-positive cells was expressed as percent of insulin-positive cells in ICCs.
To analyze the number of PDX-positive cells in the duct, PDX-1 and duct cell-specific cytokeratin 20 (CK) double staining was performed using cryosection (6 µm). The pancreas was fixed for 4 h at 4 C in 4% paraformaldehyde/PBS, washed in PBS, and equilibrated in a 30% sucrose/PBS solution overnight at 4 C, frozen in OCT (Sakura Finechemicals, Tokyo, Japan), sectioned, mounted, and stored at -40 C. After blocking with normal donkey serum (5%), the cryosections were incubated with rabbit anti-PDX-1 antibody (1:3000, a generous gift from Dr. Y. Kajimoto of Osaka University, Japan) (17) and monoclonal mouse anti-CK antibody (1:40, DAKO Corp.) overnight at 4 C. After washing with PBS, cy3-conjugated donkey antirabbit IgG (1:3000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and fluorescein isothiocyanate-conjugated donkey antimouse IgG (1:100, Jackson ImmunoResearch Laboratories, Inc.) were added for 1 h at room temperature. The counterstaining was done with 4',6-diamidio-2-phenylindol-HCl (Boehringer Mannheim, Mannheim, Germany). PDX-1/CK-positive cells were counted at 400x and expressed as the number of PDX-1/CK-positive cells per field. Apoptotic cells were detected by terminal deoxynucleodidyl transferase technique (TUNEL method) (20) using a apoptosis in situ detection kit (Wako Jun-yaku, Tokyo, Japan).
Statistical analysis
Results were expressed as means ± SE. For
comparisons between two groups, the unpaired t test was
used. For multiple comparisons, one-way ANOVA was used.
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Results |
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and 2A).
The effect of BTC was apparent 2 d after 90% pancreatectomy and
persisted for 4 wk after the pancreatectomy. The morning-fed plasma
insulin concentration was significantly higher in BTC-treated rats than
that in saline-treated rats, and the effect of BTC lasted for 4 wk
after the pancreatectomy. Hence, the effect of BTC was persistent for
20 d after the cessation of the BTC treatment. BTC treatment did
not affect the body weight (Fig. 2B). BTC did not cause any change in
the plasma glucose and insulin concentrations in sham-operated rats
(Figs. 1 and 2A).
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). The basal plasma insulin
concentration was significantly higher in BTC-treated rats. In
saline-treated rats, the plasma insulin levels did not change after the
administration of glucose, whereas in BTC-treated rats, the plasma
insulin levels responded to glucose loading. The plasma insulin levels
were slightly but significantly higher in BTC-treated rats at 60 min
and later.
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demonstrates
representative sections of the pancreas obtained from normal, saline-
and BTC-treated pancreatectomized rats. In pancreatectomized rats, most
of islets were larger than those in normal rats. In pancreas sections
from the BTC-treated rats, the ß-cell mass was increased because
islets were larger, and more scattered single or small clusters of
insulin-positive cells were observed. Morphometrically, the ß-cell
mass was significantly greater in BTC-treated rats (Fig. 4B).
Consistent with this observation, the insulin content of the remnant
pancreas was significantly higher in BTC-treated rats, compared with
saline-treated rats (Fig. 4C). We then measured the ß-cell
cross-sectional area of individual ß-cells as an indicator of
ß-cell size. The ß-cell cross-sectional area was 205.7 ± 4.6
µm2 in BTC-treated rats, which was the same as
that of pancreatectomized rats treated with saline (209.6 ± 6.8
µm2).
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shows a representative staining of an islet with anti-insulin and
anti-BrdU antibodies. As shown in Fig. 5B, the number of BrdU/insulin
double-positive cells in pancreatic islets significantly increased in
BTC-treated rats. Similarly, the number of BrdU/insulin double-positive
cells significantly increased in ICCs in BTC-treated rats 2 d
after pancreatectomy. Note that the number of ICC was significantly
increased in BTC-treated rats (Fig. 6).
Figure 7 shows the staining of remnant
pancreas with anti-PDX-1 and anti-cytokeratin antibodies.
PDX-1/cytokeratin double-positive cells were observed in the pancreatic
duct. The number of PDX-1-positive cells in the duct was not changed by
BTC. Thus, the numbers of PDX-1/cytokeratin double-positive cells in
BTC- and saline-treated rats 2 d after pancreatectomy were
0.084 ± 0.014 and 0.081 ± 0.012/field, respectively.
Similar results were obtained in specimen from remnant pancreas 3
d after the pancreatectomy. Note that most of the PDX-1-positive cells
in the duct was insulin negative (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Fig. 4
, treatment with BTC increased the ß-cell mass and
the insulin content of the remnant pancreas. Morphometric study
suggested that BTC increased the ß-cell number because BTC increased
the ß-cell mass without increasing the ß-cell size. In general,
increases in ß-cell number may result from prevention of ß-cell
death, increase in ß-cell formation, or both. Apoptotic cells were
not observed frequently in the remnant pancreas during the course of
the experiments (data not shown). Hence, it is unlikely that BTC
reduced death of ß-cells in the remnant pancreas. The increase in
ß-cell number may be owing largely to an increase in the formation of
ß-cells. There are two pathways for the formation of ß-cells:
replication of ß-cells in islets and neogenesis from precursors
located in the pancreatic ducts (21). As shown in Fig. 5, BTC increased the number of BrdU and insulin double-positive cells in
islets at an early time point, an observation suggesting that BTC
increased replication of preexisting ß-cells in islets. BTC also
increased the number of ICCs and the number of BrdU and insulin
double-positive cells in ICCs.
Because small clusters of insulin-positive cells may represent newly differentiated ß-cells from progenitor cells locating in the pancreatic duct, these results suggest that BTC promoted formation and/or replication of newly differentiated insulin-producing cells derived from the progenitors in the pancreatic duct. At present, the exact site of BTC action during the course of differentiation is uncertain. BTC converts amylase-secreting pancreatic AR42J cells, which resemble amphicrine transitional cells in the regenerating pancreas (22), to insulin-secreting cells (5). The results suggest that BTC commits the differentiation of ß-cells from precursor cells and promotes an early differentiation step of ß-cells (5). However, the number of PDX-1-positive cells in the duct soon after the pancreatectomy was not significantly changed by BTC. Consequently, the present results do not support the notion that BTC acts on the initial step of ß-cell neogenesis (e.g. the formation of the PDX-1-positive cells in the duct). Instead, it may act on the step distal to the formation of PDX-1-positive cells. Presumably, BTC stimulates growth and possibly differentiation of already committed PDX-1-expressing cells and thereby increases the number of ICCs. Alternately, BTC may promote survival of the PDX-1-positive cells and allow them to differentiate and form ICCs. In this regard, BTC was shown to simulate proliferation of mature insulinoma cells (9). BTC may stimulate proliferation of newly formed insulin-producing cells. Collectively, BTC may increase regeneration of ß-cells by acting on multiple steps.
In 90%-pancreatectomized rats, Bonner-Weir et al.
(18) showed that ß-cell response to glucose was
impaired. In agreement with this notion, the plasma insulin
concentration did not change after glucose loading in
90%-pancreatectomized rats (Fig. 3). In contrast, a small but
significant increase in the plasma insulin concentration was observed
in BTC-treated rats. Besides increasing the ß-cell mass, BTC
treatment may have improved the ß-cell responsiveness to glucose.
Previous studies have shown that exendin-4 (23), an agonist of the glucagon-like peptide-I receptor, Reg protein (24), and islet neogenesis-associated protein (25) are effective in promoting ß-cell regeneration. The present results show that BTC is another potential ligand promoting ß-cell regeneration. Compared with exendin-4, a stable agonist of the GLP-1 receptor, BTC appears to be less effective (23). We used a single dose of BTC via a single route (sc injection). The half-life of injected BTC is shown to be relatively short (17). Hence, BTC would be more effective if higher adequate doses were administered via a more effective route. Further studies are necessary to establish an effective delivery method for administration of BTC. The present results are consistent with the recent report by Yamamoto et al. (17) that BTC promoted ß-cell neogenesis and improved the glucose intolerance in diabetic mice induced by a selective alloxan infusion. In that model, BTC increased the numbers of ICCs and islets. BTC also increased the insulin content of the pancreas. In our model, BTC significantly increased the number of ICCs and the number of BrdU-positive cells in ICCs. The effectiveness of BTC was demonstrated in two different animal models.
In summary, administration of BTC to 90%-pancreatectomized rats induced sustained improvement of hyperglycemia. BTC increased the ß-cell mass and the insulin content of the remnant pancreas. BTC accelerated regeneration of ß-cells in 90%-pancreatectomized rats.
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Acknowledgments |
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Footnotes |
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Abbreviations: BTC, Betacellulin; CK, cytokeratin; EGF, epidermal growth factor; ICC, islet cell-like cluster.
Received May 21, 2001.
Accepted for publication August 9, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TC1
clone 6 cells in the presence of betacellulin. Diabetes 45:1826–1831[Abstract]
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) and saline-treated (
) pancreatectomized
rats and BTC- (
) and saline-treated (
) sham-operated rats (n
= 5). *, P < 0.01 vs.
saline-treated.
