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Original Studies |
Whittier Institute and Department of Pediatrics (C.D., G.M.B., A.D.L., A.H.), University of California at San Diego, La Jolla, California 92037; and Federal University of Sao Paulo (C.D., S.A.D.), Sao Paulo, Brazil
Address correspondence and requests for reprints to: Dr. Alberto Hayek, The Islet Research Laboratory, Department of Pediatrics, University of California at San Diego, 9894 Genesee Avenue, La Jolla, California 92037. E-mail: ahayek{at}ucsd.edu
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Abstract |
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 Top Abstract IntroductionResearch Design and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionResearch Design and MethodsResultsDiscussionReferences |
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Activin A (Act.A), a member of the transforming
growth factor ß family was initially isolated from gonads as a
stimulator of FSH secretion. The peptide is a dimer of the ß-subunit
of inhibin, which in turn inhibits FSH secretion (1).
Inhibins consist of an 
-and either a ßA or ßB subunit linked by
disulfide bonds. Act.A is a homodimer of the ßA-subunit (ßA-ßA)
with potent activities in diverse biological systems: erythroid
differentiation (2), mesoderm induction (3),
inhibition of neural differentiation (4), and modulation
of pituitary (5) and pancreatic (6) hormone
release.
Activin signaling occurs via binding to a heterotrimeric receptor
complex with transmembrane serine/threonine kinase activity
(7). The receptor encompass two subgroups, type I (ActR I
and ActR IB) and type II (ActR II and Act IIB) receptors
(8). Transgenic mice expressing activin receptor mutants
show hypoplasia of pancreatic islets (9). Activin action
is also regulated by follistatin, a high-affinity binding protein that
neutralizes its action in various systems (10). It is
likely that activin and follistatin participate in ultra-short loop
regulation pathways in many tissues (11). Activin gene
expression has been shown in rat (12) and human fetal
pancreas (13). Moreover, immunoreactive Act.A and
follistatin have been found in rat (14) and human
pancreatic (15) and ß cells (16).
Betacellulin (BTC) was originally isolated from a mouse pancreatic ß
cell tumor line and has been shown to promote the proliferation of
epithelial and vascular smooth cells (17) as well as of
the rat insulinoma cell line INS-1 (18). It is a member of
the epidermal growth factor (EGF) family and is expressed in the human
pancreas (19). The EGF family consists of several
polypeptide growth factors (e.g. EGF, transforming growth
factor , amphiregulin, heparin-binding EGF-like growth factor, and
epiregulin) (20). They are ligands for a subfamily of
transmembrane receptor tyrosine kinases, the ErbB group, which includes
the EGF receptor (EGFR)/ErbB-1, ErbB-2, ErbB-3, and ErbB-4. It has been
shown that BTC is a ligand for EGFR and ErbB-4 (21) and
also for a heterodimer between ErbB-3 and ErbB-2, an oncogenic complex
(22).
BTC converts exocrine AR42J cells to insulin-expressing cells when combined with Act.A (23). Although this was also observed when the exocrine cells were treated with Act.A and hepatocyte growth factor (24), there are some controversies regarding the effect of these growth factors in the AR42J cell line (25).
Most of the studies concerning the effects of Act.A and or BTC in islet growth and development have been performed in rodent cell lines (17, 18, 23, 24, 25). The aim of this study was to characterize the response of human undifferentiated fetal pancreatic cells to Act.A and/or BTC. For this purpose, we used cultures of human fetal pancreas to study the effects of the growth factors on the development and proliferation of pancreatic cells in vitro.
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Research Design and Methods |
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TopAbstractIntroductionResearch Design and MethodsResultsDiscussionReferences |
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5 min and
24 h, respectively. Informed consent for tissue donation was
obtained by the procurement centers. In addition, approval for the use
of human fetal tissue was obtained from the University of California at
San Diego institutional review board. Tissue preparation and tissue culture
Extraneous material was carefully dissected away, and the
pancreases were divided into four to eight equally sized pieces that
were weighed before the digestion with 11 mg/mL Collagenase P
(Roche Molecular Biochemicals, Indianapolis, IN), as
described previously (26). Each of the digests was plated
on a separate Petri dish in RPMI 10% human serum (11.1 mmol/L
glucose), 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, and 1
µg/mL Amphotericin B. The three-dimensional cell
aggregates or islet-like cell clusters (ICCs) obtained have already
been characterized and contain mostly undifferentiated epithelial cells
and between 5% and 10% endocrine cells (mainly insulin- and
glucagon-producing cells) (26, 27). One of the dishes was
used as a control for the experimental groups. Human recombinant Act.A
(National Hormone and Pituitary Program, Harbor–UCLA Medical Center,
Torrance, CA) and human recombinant BTC (R&D Systems,
Minneapolis, MN) were used at an equimolar concentration of 4
nM, found to be maximally effective in a preliminary
dose-response experiment (Fig. 5
, A and B). Media and growth factors
were changed after 48 h. After 5 days, all well-formed ICCs were
picked and counted under a stereomicroscope, and the results were
correlated to the original tissue weight.
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After 4 days of incubation, ICCs with similar size (100 µm)
were harvested in groups of 80 into 35-mm Petri dishes in 1 mL medium
containing 1 µCi ]methyl-3H[ thymidine/mL.
After an overnight (16 h) incubation at 37 C, the ICCs were washed
twice in phosphate-buffered saline (PBS) (pH 7.4), resuspended in 250
µL distilled water, and homogenized by sonication. DNA was measured
from the sonicate fluorometrically, as described (28).
Insulin content was measured by a solid-phase RIA (Diagnostic Products Corp., Los Angeles, CA) in dilutions (1, 20) of acid ethanol extracts. Incorporation of
]3H[ thymidine was determined by trapping 10%
trichloroacetic acid precipitates of the sonicates on glass fiber
filters (Whatman GF/A, Maidstone, UK), drying, and liquid
scintillation counting in 4 mL BetaMax (ICN Radiochemicals,
Irvine, CA).
In another set of experiments, groups of 80 ICCs (100 µm in size)
were cultured in 35-mm Petri dishes in 1 mL medium collected after
24 h to determine insulin secretion. Basal and stimulated insulin
were measured by 1-h static incubations in the presence of 1.6 and 16.7
mmol/L glucose, with and without 10 mmol/L aminophylline (American
Regent Laboratories, Shirley, NY) (29). After a final
incubation of 1 h in 1.6 mmol/L glucose, the ICCs were harvested
for DNA and insulin content determinations.
Immunohistochemistry and confocal microscopy
Primary antibodies used are shown in Table 1. Control slides were incubated with a
mixture of the isotype-matched control antibodies (mouse, sheep, and/or
rabbit immunoglobulin IgGs). Secondary antibodies used for light
microscopy were biotinylated goat antirabbit or antimouse IgGs and
alkaline phosphatase- or peroxidase-conjugated streptavidin
(BioGenex Laboratories, Inc. San Ramon, CA). Secondary
antibodies used for confocal microscopy were lissamine
rhodamine-conjugated donkey antisheep, fluorescein
isothiocyanate-conjugated donkey antirabbit, or
indo-dicarbocyanine-conjugated donkey antimouse IgGs (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
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ICCs were incubated for 16 h with 0.1 mM bromodeoxyuridine (BrdU), fixed in 4% paraformaldehyde, and embedded in paraffin. Eight-micrometer sections were stained using the immunoalkaline phosphatase technique (30) for insulin, glucagon, somatostatin, and pancreatic polypeptide and the immunoperoxidase technique (31) for BrdU. Cell nuclei, which had incorporated BrdU during DNA synthesis, were identified by binding of mouse monoclonal anti-BrdU.
Morphometric analysis
The cell surface area stained for insulin or for the combination of the three other pancreatic hormones, as the percentage of the total ICC area, was quantitated with a computerized image analyzer (Oncor, San Diego, CA). The same method was used for the determination of the BrdU labeling index (32).
Analysis of ICCs by confocal microscopy
For confocal microscopy, the slides, after deparaffining and hydrating, were permeabilized in 0.3% Triton X-100 in PBS for 15 min. Then, they were placed in coplin jars filled with 0.01 mol/L citrate buffer before incubating three times for 4 min at the maximum power (750 W) in a household microwave oven (Amana-Radarange, M84TMA; Amana, IA). The sections were allowed to cool down to room temperature and were washed with PBS. Then, the slides were blocked for 1 h with 50 mmol/L glycine in PBS, 2% donkey serum, and 2% BSA. After washing, the cells were incubated in triple combinations with insulin, the proliferation marker Ki-67, and vimentin (fibroblast marker) or pan-CK (epithelial cell marker) antibodies. After a 1-h incubation, the slides were washed several times and incubated with secondary antibodies. Slides were analyzed using the confocal microscope by optical scanning at confocal planes of 0.3-µm thickness with a microscope (model Nikon Diaphot 200; Nikon, Melville, NY) equipped with a laser scanning confocal attachment (Bio-Rad MRC 1024 and Lasersharp software, Bio-Rad Laboratories, Inc., Hercules, CA). Color composite images were generated using Adobe Photoshop 4.0 (Adobe Systems, Mountain View, CA).
For quantitative analysis, each ICC preparation had enough fields examined to score at least 1000 cells.
Statistical analysis
Because of the relatively large variation among individual cultures (33), all data, except for the morphometric analysis, are presented as the percentage of changes from control ICCs obtained from the same pancreas. Data were obtained from at least three to four individual experiments, with a total of 10–15 replicates. Statistical significances of observed differences were tested with software for Macintosh (Statvie II; Abacus Concepts, Berkeley, CA). Multiple comparisons were done with one-way ANOVA and Fisher’s protected least significance difference test, with a 95% level as the limit of significance.
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Results |
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TopAbstractIntroductionResearch Design and MethodsResultsDiscussionReferences |
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After collagenase digestion the pancreatic cells formed
ICCs, which remained unattached in Petri dishes, as reported previously
(26, 32). The digested tissue stayed free floating in the
form of small cell clusters of 30–200 µm in diameter (ICCs). The
average number of ICCs harvested from control cultures was 14.5 ±
3.2 mg starting tissue. The yield per milligram of tissue was not
affected by the treatment with Act.A (Fig. 1A). In contrast, when cultured in medium
containing BTC, there was a 1.4-fold increase in the number of ICCs
(P < 0.05) (Fig. 1A). Consistent with the increase in
the number of ICCs, a 2.6-fold increase in the DNA synthesis was found,
as measured by ]3H[ thymidine incorporation at
the end of the culture (P < 0.005) (Fig. 1B). DNA
synthesis was stimulated by BTC at 2 and 4 nM
(P < 0.005), but not at a higher concentration (Fig. 5B
). BTC also induced an increase in the total DNA when compared with
control (0.45 ± 0.2 µg/mg starting tissue in control
vs. 1.14 ± 0.1 µg/mg starting tissue in BTC-treated
cultures) (P < 0.005). Morphologically, many of these
were smaller, round, and translucent as compared with the control ICCs
(Fig. 2). To minimize the size
difference, just ICCs of 100 µm were picked. The DNA content of
the individual ICCs was not affected (22.7 ± 1.3 ng/ICC in
control vs. 18.9 ± 1.1 ng/ICC in BTC-treated
cultures). The combination of Act.A and BTC resulted in a 2.3-fold
increase in DNA synthesis (P < 0.01) (Fig. 1B), but no
significant increase in the number of ICCs (Fig. 1A).
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Treatment of the fetal cells with Act.A resulted in a 1.5-fold
increase in insulin content (P < 0.005) (Fig. 3A). Act.A (4 nM)
was required for a significant effect on the insulin levels. Higher
concentration did not further increase the insulin content; in fact,
there was no significant increase at 8 nM as
compared with 4 nM (Fig. 5A). The morphometric
analysis showed a 2-fold increase in the total surface area of insulin
staining in ICC sections (P < 0.005) (Fig. 3C), but no
significant difference in the area stained for the three other
pancreatic hormones (data not shown). The hormone-positive cells were
Ki-67 negative (Fig. 4C). There
was no difference in the insulin release (Fig. 3B) or in the acute
insulin response to glucose (data not shown). There was no significant
effect of BTC on the insulin content (Fig. 3A) or in the total surface
area of insulin staining (Fig. 3C), but a 32.7% decrease in the level
of insulin release after 5 days in culture (P < 0.005)
(Fig. 3B). Consistent with a 2.6-fold increase in the DNA synthesis
(P < 0.005) (Fig. 1B), we found a 2-fold increase in
the BrdU labeling of ICCs exposed to BTC (P < 0.005)
(Fig. 3D).
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), there was a 1.7-fold increase
in the total surface area of insulin staining in ICC sections
(P < 0.05) (Fig. 3C). Whereas the significant increase
in proliferation found contributed to the lower insulin/DNA
measured by RIA, it did not affect the morphometric analysis for
insulin. Interestingly, there was a 44.5% decrease in the insulin
release compared with control (P < 0.005) (Fig. 3B). Phenotype of replicating cells in ICCs exposed to BTC.
Immunohistochemical analysis of replicating cells from ICCs
exposed to BTC is shown in Table 2. They
were negative for all pancreatic hormones. More than 60% of the
dividing cells (Ki-67-positive cells) were double-stained with KL1
(pan-cytokeratin, an epithelial cell marker), whereas 30% were
double-stained with vimentin (a fibroblast marker) (Fig. 4, E and
F).
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Discussion |
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TopAbstractIntroductionResearch Design and MethodsResultsDiscussionReferences |
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Recent evidence has been accumulated for a role of activins in pancreas development. Studies in the chick have shown that notochord can repress sonic hedgehog (shh) expression to allow for pancreas differentiation, and that may be mediated by intercellular signaling molecules, including activin ß B and fibroblast growth factor 2 (34). Moreover, follistatin, the activin-binding protein, can mimic the repressive effects of the mesenchyme on the differentiation of rat pancreatic endocrine cells (35). This, in turn, suggests that activin promotes endocrine differentiation.
Cultured ICCs represent a heterogeneous mixture of
hormone-containing and undifferentiated epithelial cells. They contain
only 3–5% insulin-positive cells and a high proportion of
undifferentiated cells. Twelve weeks after transplantation into athymic
nude mice, ICCs give rise to endocrine tissue rich in insulin- and
glucagon-containing cells (36, 37). We have previously
shown that the functional ß-cell mass obtained after transplantation
of the ICCs is due to differentiation, but not to proliferation
(38). Here, we show that Act.A promotes an increase in
insulin content and in the total surface area of insulin staining in
ICC sections. Although we show that Act.A induces an increase in the
total surface area of insulin staining in ICC sections, there was no
significant difference in the area stained for the three other
pancreatic hormones. It has been shown by immunohistochemistry that
Act.A is expressed only in glucagon-producing -cells in humans
(15). This is in agreement with other immunohistochemical
studies showing that in adult and fetal rat pancreases Act.A is
localized in -cells, whereas follistatin is present in ß-cells
(14). Although there are some conflicting data showing
Act.A in ß-cells (16), that may be explained by the use
of antibodies directed against follistatin-bound Act.A. It has been
shown that Act.A-positive pancreatic ß-cells costained with
anti-follistatin antibodies, suggesting that the activin-follistatin
complex is present in ß-cells. Because follistatin is the
Act.A-binding protein and there exists an ultra-short loop regulation
pathway between these two factors, we could speculate that -cells
are secreting Act.A with potential regulatory effects on ß-cells.
Activin/inhibins signal through transmembrane serine-threonine kinases
with two subgroups, type I and type II activin receptors
(8). Human type II receptors specific for activin/inhibin,
activin receptor II (ActRII) and activin receptor IIB (ActRIIB) have
been described (39). Moreover, the expression of activin
receptors has been shown in human adult pancreas (40) and
in midgestational human fetal pancreas (41).
In the present study, we have shown that BTC is mitogenic for
undifferentiated pancreatic epithelial cells, causing an increase in
the number of ICCs, total DNA, and DNA synthesis. The present
observations on the mitogenic action of BTC on fetal pancreatic cells
provide evidence for a role of this growth factor in islet development.
This is also based on the BTC (19) and EGFR expression in
the human pancreas, as well as in the disturbed formation of pancreatic
islets found in mice lacking EGFR (42). Moreover, BTC has
been shown to be required for insulin gene expression in clonal
-cells transfected with PDX-1 gene (43).
When Act.A and BTC were added to the medium, a combination of the effects described in ICCs exposed to each of the growth factors separately were observed. There was an increase in DNA synthesis as well as in the total surface area of insulin staining in ICC sections. Although significant, these effects were not as pronounced as when compared with those using each growth factor by its own. This could be explained by the fact that Act.A may stimulate the cells toward differentiation and BTC to proliferation, one occurring at the expense of the other. These results differ from those observed in the AR42J cells where the differentiation of insulin-producing cells was achieved just with the combination of Act.A and BTC (23), reflecting potential differences between human primary cells and rodent cell lines.
In conclusion, we have shown that Act.A induces ß-cell differentiation whereas BTC has a mitogenic effect in human undifferentiated pancreatic epithelial cells. Although these growth factors seem to be important for islet development, our results are based on in vitro experiment; thus, we can just speculate their role in normal pancreas development. Moreover, the molecular mechanisms regulating human pancreatic growth and differentiation remain to be clarified.
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Acknowledgments |
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Footnotes |
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Received February 1, 2000.
Revised May 12, 2000.
Accepted July 11, 2000.
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References |
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TopAbstractIntroductionResearch Design and MethodsResultsDiscussionReferences |
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- and ßA-subunits
of inhibin/activin in human normal endocrine cells and related tumors
of the digestive system. Virchows Arch. 434:29–36.[CrossRef][Medline]
family of
mitogens. Growth Factors. 11:235–257.[Medline]
-TC 1 clone cells in
the presence of betacellulin. Diabetes. 45:1826–1831.[Abstract]
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