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Lawson Research Institute (J.P., E.A., T.J.M., D.J.H.), St. Joseph’s Health Centre, London, Ontario, N6A 4V2, Canada; Departments of Physiology (J.P., D.J.H.), Medicine (E.A., T.J.M., D.J.H.), Paediatrics (J.P., D.J.H.), Pharmacology and Toxicology (T.J.M.), and Biochemistry (T.J.M.), University of Western Ontario, London, Ontario, N6A 5A5, Canada; and Laboratory of Developmental Genetics and Imprinting (J.M.P., W.L.D., W.R.), The Babraham Institute, Cambridge CB2 4AT, United Kingdom
Address all correspondence and requests for reprints to: Dr. D. J. Hill, Lawson Research Institute, St. Joseph’s Health Centre, 268 Grosvenor Street, London, Ontario, Canada, N6A 4V2. E-mail: dhill{at}lri.stjosephs.london.on.ca
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Abstract |
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-cells was higher. Normal islet
morphology was disrupted, with -cells appearing in small groups
within the islets, as well as on the periphery, whereas ß-cells were
often seen at the edge of the islets. Twice as many islet cells (21.9%
vs. 11.4%) were involved in cell replication, detected
by the presence of immunoreactive proliferating cell nuclear antigen,
in pancreata from transgenic mice vs. controls, whereas
the number of cells undergoing apoptosis was significantly reduced.
Abundant IGF-II messenger RNA was found within the islets of
transgenic animals by in situ hybridization, and the
relative area of islets demonstrating immunoreactive IGF-II was
significantly greater. Immunoreactive IGF-I was much less abundant and
was further reduced in islets of transgenic animals. The area of islets
immunopositive for IGF binding protein-2 was unaltered. Despite the
presence of islet hyperplasia, circulating insulin levels and serum
glucose levels were not significantly different between transgenic and
control mice. These results show that an overexpression of IGF-II in
fetal life has a profound effect on islet morphology and causes islet
hyperplasia while reducing the attrition of islet cells by apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The population growth rate of all islet cells, including ß-cells, decreases postnatally, and the rate of mitosis in adult pancreatic ß-cells is normally low (<3% replication rate of ß-cells per day) (4, 8). Recently it has been shown that the ontogeny of islet cells in early life involves a balance between ß-cell replication and neogenesis, and programmed ß-cell death and that a transient wave of apoptosis occurs in neonatal rat islets between 1–2 weeks of age (9, 10). The factors which regulate the balance between islet cell generation and apoptosis are poorly understood, but maintaining pregnant rats on a reduced protein diet will decrease the rate of ß-cell DNA synthesis, while increasing the rate of apoptosis in fetal and neonatal rat islets (11). Little is known of the cellular mechanisms of developmental ß-cell apoptosis, but we have found a transient increase in the number of islet cells expressing inducible nitric oxide synthase (iNOS) in the neonatal rat just before an increase in apoptosis (10). The apoptosis which characterizes ß-cell destruction in response to cytokines in type 1 diabetes involves increased intracellular concentration of nitric oxide and increased expression of iNOS (12).
Experiments with isolated islets of Langerhans from the rat or human fetus, or using established ß-cell lines, have shown that both IGF-I and -II will promote DNA synthesis in ß-cells (13, 14, 15, 16). Using fetal rat islets isolated in late gestation, we showed that IGF-I was more potent as a mitogen than IGF-II but that immunoreactive IGF-II was released in much greater amounts than was IGF-I (13). The type-1 IGF receptor, which is primarily responsible for initiating intracellular mitogenic signaling pathways, is abundant on ß- and other islet cells (17, 18). We have also demonstrated that IGF-I or -II will promote ß-cell survival in islets from neonatal rats, by reducing the rate of cell apoptosis (10). The stability, biological availability to tissues, and actions of IGFs are modulated by at least six IGF binding proteins (IGFBPs), which are widely expressed in human and rat fetal tissues (19). Isolated fetal rat islets release IGFBPs-1 to-3, with the release of IGFBP-1 and -2 being regulated by glucose. Exogenous IGFBP-1 or -2 was able to enhance the mitogenic actions of IGF-II on isolated islets from fetal rats (13).
A role for the IGF axis in islet cell development is supported by a complex pattern of pancreatic expression. IGF-II expression is greatest in fetal rat pancreas and then declines neonatally, immediately before a wave of islet cell apoptosis (10, 20). IGF-II messenger RNA (mRNA) is expressed throughout the islets and in isolated pancreatic ductal cells (10). Conversely, IGF-I expression is low in fetal life and does not rise to adult levels until after weaning. IGF-I mRNA is barely detectable in the islets but is increasingly expressed within acinar cells with age (20). Both IGF-I and -II immunoreactivity are associated with islet cells throughout development, suggesting that IGF peptide distribution may depend on IGFBPs that are also expressed within the pancreas (20). In addition to a trophic action on ß-cells, IGF can also influence ß-cell function, at least in adult life. Both IGF-I and -II have been shown to alter glucose-stimulated insulin release by adult islet ß-cells, with a biphasic action which can be modulated by endogenous IGFBPs (21, 22).
Direct evidence for a trophic action of IGF-I or -II on islet development in vivo is lacking. Though a general increase in the birth size and organ weights of mice expressing either IGF-I or -II transgenes has been described (23, 24), only one study has specifically reported a substantial increase in pancreatic weight at birth after an overexpression of IGF-II (25). However, because the endocrine component of the pancreas is relatively small, substantial changes in islet morphology may occur without a noticeable change in pancreas size. We have examined IGF-II transgenic animals for possible changes in pancreatic islet morphology that might result from altered pancreatic expression of IGF-II or from an increase in its circulating levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-II expression constructs were introduced into embryonic stem cells, and chimeras raised, as described in detail previously (26). A native genomic configuration of the mouse IGF-II gene included the strong fetal promoters 2 and 3 within a pBluescript SK+ (Stratagene, San Diego, CA) IGF-II mes-neo construct. This consisted of a 16.5-kb EcoRI fragment containing the IGF-II locus (27), and a neomycin resistance gene under the HSV-TK promoter. A unique sequence tag (an alloenzyme of glucose phosphate isomerase not found in the background mouse strain; Gpi) was inserted into the 3' untranslated region (Mlu-I site), which provided a marker to distinguish the transgene from the endogenous gene using the ratio of Gpi A and B isoforms for both RNA and DNA analysis. The levels of transgene expression were variable among clones but were of the same order of magnitude as the endogenous IGF-II gene. Two transgenic clones were used in which IGF-II was overexpressed, and three negative clones in which the IGF-II gene was absent from the construct served as controls.
Chimeras were made from these five transgenic cell lines for analysis of pancreata. Embryonic stem cells were microinjected into F2 [F1 x F1 (C57BL/6 x CBA/Ca)] host blastocysts, and the resulting fetuses were killed at day 19 or 20 of gestation. Mouse fetuses were decapitated with scissors after asphyxiation of the pregnant females with CO2. All procedures were performed with ethical approval of the Animal Care Committee of the University of Western Ontario. After death, fetuses were assessed by Gpi analysis (28) to assess chimerism, and body weight and wet weights of the placenta, heart, kidney, brain, tongue, and liver were recorded. Blood was collected after decapitation, and serum was prepared for measurement of glucose, insulin, and IGF-II. Glucose concentrations were measured using a glucose oxidase method (Sigma Chemical Co., St Louis, MO). The pancreas was immediately removed from each animal and placed in 5 ml sterile, ice-cold HBSS, pH 7.5 (Gibco BRL, Burlington, Ontario, Canada). If pancreata were to be fixed for histology, they were placed in ice-cold fixative (4% paraformaldehyde in PBS, pH 7.4) overnight at 4 C, followed by two washes at 4 C in PBS. Fixed tissues were dehydrated in 50% (vol/vol) (2 x 10 min), followed by 70% ethanol, and embedded in paraffin.
Immunohistochemistry
Histological sections of pancreas (5 µm) were cut from
paraffin blocks with a rotary microtome and mounted on glass microscope
slides (Superfrost Plus, Fisher Scientific, Nepean,
Ontario, Canada). Immunohistochemistry was performed to localize IGF-I
or -II, IGFBPs-1 to -3, iNOS, proliferating cell nuclear antigen
(PCNA), insulin, glucagon, and SRIF within islets, by a modified
avidin-biotin peroxidase method (29), as described by us previously for
pancreas (10). Briefly, slides were incubated for 48 h at 4 C in a
humidified chamber with either rabbit antihuman IGF-I or IGF-II (1:2000
dilution) (GroPep Pty. Ltd., Adelaide, Australia); rabbit
antihuman IGFBP-1, -2, or -3 (all at 1:100 dilution) (Austral
Biologicals, San Ramon, CA); guinea-pig antiinsulin antibody (1:500
dilution) (provided by Dr. T. J. McDonald, University of Western
Ontario, London); rabbit antiporcine glucagon (1:100 dilution)
(C-terminal specific 04A antiserum, kindly provided by Dr. R. Ungar,
Dallas, TX); rabbit antirat SRIF (1:100) (DAKO Corp. Laboratories, Mississauga, Ontario, Canada); mouse anti-iNOS
antiserum (1:50 dilution) (Transduction Laboratories, Inc.., Lexington, KY); and mouse anti-PCNA (1:750 dilution)
(Sigma Chemical Co.). All antisera were diluted in
0.01 M PBS (pH 7.5) containing 2% (wt/vol) BSA and 0.01%
(wt/vol) sodium azide (100 µl per slide). All subsequent incubations
were at room temperature. Biotinylated goat antirabbit IgG (1:100),
goat antimouse IgG (1:100), or mouse antiguinea pig IgG (1:500)
(Vector Laboratories, Inc., Burlingame, CA) were diluted
in the same buffer and applied for 2 h; then the slides were
washed in PBS and incubated with avidin and biotinylated horseradish
peroxidase for 1 h. Peptide immunoreactivity was localized by
incubation in fresh diaminobenzidine tetrahydrochloride (DBS tablets,
10 mg, Sigma Chemical Co.) with 0.03% (vol/vol) hydrogen
peroxide for 2 min, and the reaction was quenched in excess 50
mM Tris.HCl, pH 7.5. Tissue sections were counterstained
with Carazzi’s hematoxylin. To establish specificity of staining, the
primary antisera for IGF-I or -II were preadsorbed overnight at 4 C
with 100 nM homologous antigen before application to the
sections. In each case, staining was abolished. Antisera against IGFBPs
were preabsorbed with 100 nM of homologous or heterologous
IGFBP proteins (Austral). Further controls included substitution of
primary antisera with nonimmune serum and omission of the secondary
antiserum.
Dual staining for PCNA and insulin or glucagon, or iNOS and insulin, was undertaken by first performing immunohistochemistry for insulin or glucagon, as described above, using alkaline phosphatase (blue) as the chromagen. Alkaline phosphatase substrate kit III was obtained from Vector Laboratories, Inc. Antimouse alkaline phosphatase conjugate (Sigma Chemical Co.) was applied to each section for 1 h at room temperature, sections were washed, and alkaline phosphatase substrate was applied for 20 min. Before counterstaining and dehydration, the sections were then subjected to immunohistochemistry for PCNA or iNOS, as described above, using diaminobenzidine as the chromagen. Sections were washed and counterstained with Mayer’s hemalum.
Immunofluorescent microscopy was employed to visualize the distribution of the transcription factor, Pdx-1, in sections of pancreas. The primary antiserum used (1:1500 dilution) was kindly provided by Dr. Christopher Wright (Vanderbilt University, Nashville, TN). A secondary antibody of donkey antirabbit IgG was conjugated to C43 red fluorochrome (1:100, ML Grade, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
Visualization of apoptosis
Immunocytochemistry was performed to localize apoptotic nuclei
within pancreatic tissue sections (30) using the Apoptag in
situ apoptosis detection kit (Oncor Inc., Gaithersburg, MD) as
described previously (10). Staining was performed according to the
manufacturer’s protocol, after incubation with proteinase K (20
µg/ml; Boehringer Mannheim, Dorval, Québec,
Canada) for 15 min, washing in distilled water, and quenching of
endogenous peroxidase by incubation in 2% (vol/vol) hydrogen peroxide
in PBS for 5 min. Color was generated with diaminobenzidine, as
described for immunohistochemistry, and the tissue was counterstained
with methyl green for 1 min. Sections were dehydrated in butanol,
cleared in xylene, and mounted with Permount under glass
coverslips.
In situ hybridization
We performed in situ hybridization of IGF-I and -II
mRNAs using histological paraffin sections of pancreas (10). Each slide
was incubated with complementary RNA (cRNA) probe under glass
coverslips in a humidified chamber at 50 C for 16 h, the
coverslips were removed by soaking the slides in 10 mM
dithiothreitol in 2 x saline-sodium citrate (SSC), and (after a
further incubation at 55 C for 10 min in 1 x hybridization
buffer) the sections were treated with 20 µg/ml ribonuclease A, 1
U/ml ribonuclease T1 in 0.5 M NaCl, 10 mM
Tris.HCl (pH 8.0), 1 mM EDTA at 37 C for 30 min. Sections
were washed as follows: twice for 30 min at room temperature in 2
x SSC; twice for 30 min at 55 C, then twice for 15 min at 55 C in
0.1 x SSC. To screen the extent of RNA hybridization, slides were
subjected to autoradiography with Kodak XAR film (Eastman Kodak, Rochester, NY) after dehydration in ethanol and were air
dried. Kodak NTB-3 photoemulsion, diluted 1:1 with water, was applied
subsequently to all sections and exposed for up to 14 days at 4 C; then
slides were developed in Kodak D19, rinsed in water, and fixed in
Kodafix. Sections were counterstained with hematoxylin and eosin.
Slides were viewed under dark- and lightfield microscopy. As controls
for nonspecific hybridization, hybridization was also carried out using
sense strand cRNA probes.
Antisense riboprobes were prepared from cDNAs for mouse IGF-II (a gift
of Dr. G. Bell, Howard Hughes Medical Institute University of
Chicago). The restriction enzymes (Gibco BRL) and
RNA polymerases (Promega Corp., Madison, WI) used
to linearize the plasmid containing the IGF-II cDNA and to generate
[35S]-radiolabeled riboprobe were
HindIII/TSP6 for antisense mouse IGF-II, and
EcoRI/T7 for the sense strand. Radiolabeled cRNA probes were
synthesized using linearized riboprobe DNA; -thio
[35S]-uridine 5'-triphosphate; and SP6, T3, or T7
RNA polymerase, as described previously (10).
Hormone assay
The insulin content of mouse serum was measured by RIA using the
Wright antiserum in a modification of the method of Hales and Randle
(31), as modified by Herbert et al. (32) and described by us
previously (22). Rat insulin (Novo Nordisk Pharma Ltd., Mississauga, Ontario, Canada) was used for the
standard curve. The within-assay coefficient of variation was 6.5%,
and the between-assay coefficient of variation was 9%. The minimum
level of detection was 2 fmol/ml. There was no detectable
cross-reactivity in the insulin assay with IGF-I or -II. An IGF-II RIA
was also performed on mouse serum (33) after extraction of IGFBPs by
separation on Sehadex G50.
Morphometric and statistical analysis
Morphometric analysis was performed using a Carl Zeiss transmitted-light microscope at a magnification of x250.
Analyses were performed with the Northern Eclipse version 2.0
morphometric analysis software (Empix Imaging Co., Mississauga,
Ontario, Canada). The number and area of islets; the size of individual
endocrine cells; and the percent of islet cells immunopositive for
insulin, glucagon, SRIF, IGF-I, IGF-II, IGFBPs, PCNA, iNOS, or
demonstrating apoptotic nuclei, was calculated from five sections of
each pancreas, representing the head region. Sections chosen contained
at least five islets. Differences between mean values for each variable
were compared for statistical difference (by ANOVA). Changes in body
and organ weight, and circulating levels of insulin, IGF-II, and
glucose were compared, in relation to the degree of expression of the
IGF-II transgene, using multiple linear regression analysis (by the
least-squares method).
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Results |
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). The mean wet
weights of the heart, kidney, brain, tongue, and liver were also
significantly greater in IGF-II transgenic mice, although the changes
in mean weight of the brain were small. The mean placental weight
tended to be higher in transgenic animals, but it did not reach
statistical significance. Mean serum levels of insulin and glucose did
not differ between transgenic and control mice, but animals carrying
the transgene had a 65% increase in the levels of circulating IGF-II
(Table 1). To determine whether the changes in body and organ size and
in serum IGF-II were dose-related to the expression of the IGF-II
transgene, the animals were divided into: those with a relatively low
(5–35% chimeras), intermediate (35–50% chimeras), or high (50–80%
chimeras) levels of chimerism. ANOVA showed that the increases in body
weight and the weights of the heart, kidney, and liver were
significantly related to dosage of the transgene but not changes in the
weight of the brain or tongue (Table 2).
Circulating IGF-II increased with the degree of chimerism. Mean serum
levels of glucose and insulin did not significantly differ with the
level of chimerism, but there was considerable variability between
animals.
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). Fibrous inclusions were often visible
within the islets. Immunohistochemical staining for insulin revealed
that ß-cells were located throughout the islets, unlike the central
location of ß-cells seen within the rounded islets of control
animals. Staining for insulin was less intense in islets of IGF-II
transgenic animals than in those of controls. Immunohistochemical
staining for glucagon showed that the distribution of -cells was
also disrupted in islets of animals transgenic for IGF-II (Fig. 1),
these being often seen as a single-cell-deep rim on the outer edge of
the islets, with occasional centrally-located cells. This contrasts to
the regularly shaped outer mantle of -cells seen in islets of
control mice. Morphometric analysis showed that the mean area of islets
in IGF-II transgene-bearing mice was over 5-times greater than in
controls (Fig. 2), although the number of
islets per section was not significantly different. The relative
surface area of pancreas occupied by endocrine cells, compared with
nonendocrine tissue, was 4.5 ± 1.6% (mean ±
SEM) in control mice, compared with 11.6 ± 1.3% in
transgenic animals (50–80% IGF-II chimerism, estimated for each from
15–18 sections representing five animals, P < 0.005).
The total mean area of pancreas per section tended to be greater in
transgenic than in control animals, but this did not reach statistical
significance. To establish whether there were changes in the relative
abundance of endocrine cell types within the islets, the area occupied
by insulin, glucagon, or SRIF immunoreactive cells was quantified. In
animals with high-level chimerism (50–80%), there was a relative
decrease in the area occupied by ß-cells and an increase in the
-cell area, compared with controls (Table 3). The fractional area occupied by
SRIF-containing D cells was not changed. The mean size (area) of
individual endocrine cells within the islets of transgenic animals did
not significantly differ from those in control mice (-cells: control
66 ± 10 µm2, transgenic 65 ± 10
µm2; ß-cells: control 77 ± 7 µm2,
transgenic 89 ± 9 µm2; mean ± SEM
using 15–20 sections from five animals).
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). The occurrence of Pdx-1
immunoreactivity outside of structured islets did not differ between
IGF-II transgenic and control mice.
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and Fig. 4). To determine the number of - or
ß-cells within islets that were undergoing cell proliferation, dual
staining was performed for PCNA and for either glucagon or insulin
(Fig. 5). In islets from control mice,
20.2 ± 5.8% of cells containing immunoreactive glucagon also
stained positive for PCNA. This was significantly greater in islets of
transgenic mice (33.4 ± 0.7%; P < 0.05
vs. control, estimated for each from 15 sections
representing 3 mice). The percentage of insulin-staining cells within
islets that costained for PCNA was also significantly greater for
transgenic mice, compared with controls (control: 10.1 ± 0.5%;
transgenic: 24.4 ± 0.9%; P < 0.01). Because
intracellular nitric oxide can contribute to the onset of islet
ß-cell apoptosis, we examined the presence of immunoreactivity for
iNOS. The number of islet cells containing immunoreactive iNOS was
significantly less in islets from IGF-II transgenic mice than for
controls (Table 4). When only ß-cells were considered, the percentage
of cells costaining for i-NOS and insulin was also significantly lower
in transgenic animals (1.3 ± 0.2%) than in controls (4.4 ±
0.4%; P < 0.001, estimated for each from 15 sections
representing 3 mice). Similarly, the percentage of cells within islets
that were undergoing apoptosis was significantly decreased in
transgenic (compared with control) mice (Table 4 and Fig. 4).
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A, and B). Immunohistochemistry showed
that the relative mean islet area containing immunoreactive IGF-II was
significantly higher in transgenic animals (Fig. 6, D and E). IGF-I
immunoreactivity in the pancreas was much less than that for IGF-II and
was limited to some islet cells and to isolated acinar or ductal
cells. The relative mean area of islets containing immunoreactive IGF-I
was significantly lower in IGF-II transgenic mice than in controls.
Because the cellular distribution of IGF peptides can be determined by
a sequestration by cell-associated IGFBPs, as well as by in
situ expression, we also examined the presence of immunoreactivity
for IGFBPs-1 to -3. Each of these IGFBPs could be detected by
immunohistochemistry in the mouse pancreas, predominantly in islet
cells, although clusters of acinar cells were also immunoreactive (not
shown). Immunoreactivity for IGFBPs-2 and -3 was more intense in the
peripheral, -cell-rich portion of the islets in control animals but
was more generally distributed in islets from IGF-II transgenic mice.
The relative mean islet area immunopositive for IGFBP-2 tended to be
greater in transgenic animals than in controls, but this was not
statistically significant (Table 3
), and no differences were found in
the relative presence of immunoreactivity for IGFBPs-1 or -3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wet weight of the fetal pancreas is particularly hard to evaluate
accurately, because it is difficult to remove all of the mass of the
organ during dissection. We did not consider pancreatic weight, but we
examined in detail the morphology of the endocrine component. Animals
carrying the IGF-II transgene had over a 4-fold increase in mean islet
area with a disruption of normal islet architecture. Consequently,
there was an increased contribution of endocrine cells to the total
area of pancreatic tissue. The increased size of the islets in
transgenic mice was not caused by hypertrophy of individual - or
ß-cells, which did not alter in size from controls. The ß-cell
compartment of the islets is normally located centrally, with a
surrounding rim of -cells. In islets of transgenic animals,
glucagon-containing -cells were present only as a discontinuous
layer, often only a single cell deep, around the periphery of the
islets, with isolated clumps of -cells in the interior of the islet.
The ß-cell compartment often reached to the extremities of the
islets, and fibrous tissue inclusions frequently disrupted the
ß-cell-rich core. Assessment of the relative areas occupied by -
and ß-cells showed a significant increase in the fractional area
occupied by -cells in islets of transgenic mice, whereas that for
ß-cells was decreased. However, given the islet cell hyperplasia, the
total number of ß-cells per islet was substantially increased in
transgenic animals. Because the percentage area of islets occupied by D
cells was maintained, the islet cell hyperplasia affected all of the
major endocrine cell types. It is not known whether the increased mass
of -cells resulted in an increased level of circulating glucagon,
because this was not measured.
The mechanisms underlying the islet cell hyperplasia seen in IGF-II
transgenic animals are likely to be a combination of increased cell
replication coupled with enhanced survival. An increased percentage of
islet cells within the replicative cycle, assessed by the presence of
immunoreactive PCNA, was found in transgenic mice. When - and
ß-cells were considered individually, after identification from the
immunohistochemical localization of glucagon or insulin, respectively,
the percentages of both cell types that were undergoing cell
replication was greater in islets from transgenic animals. This is
consistent with the ability of IGF-II to promote DNA synthesis in fetal
rat islets in vitro (13). Because type 1 IGF receptors are
present on -, ß-, and D cells (18), it seems likely that the
increased presence of IGF-II in transgenic animals provided a mitogenic
stimulus to each of the endocrine cell populations, although cells
expressing pancreatic polypeptide were not investigated. Conversely,
the number of islet cells undergoing apoptosis was reduced. It is
difficult to positively identify the phenotypes of apoptotic cells,
because little cytoplasm remains, and immunohistochemistry for
endocrine hormones is no longer possible. However, we have previously
shown, during rat development, that most apoptosis occurs within
ß-cells, based on the central location of such cells within the
islets (10). The IGFs have been shown to prevent apoptosis in a variety
of cell types (37, 38), including isolated rat islets (10), by
mechanisms that include an inhibition of caspases (39). We have
previously shown that an increase in developmental islet cell
apoptosis, which occurs after birth in the rat, is preceded by an
increase in the number of cells containing iNOS, a known inducer of
islet cell apoptosis during type 1 diabetes (10, 12). Islets from
IGF-II transgenic mice showed a decrease in the percentage of cells
containing iNOS, and this was so also when only the ß-cell population
was considered. However, it cannot be ascertained, within the present
experimental design, whether cells that expressed iNOS later progressed
to apoptosis.
The number of islets visible per section of pancreas was not different between IGF-II transgenic mice and controls, suggesting that although IGF-II is likely to function as an islet cell mitogen, it may not be an important factor in the generation of new islets from the pancreatic ductal epithelium by a process of neogenesis. This conclusion is supported by the distribution of immunofluorescence for Pdx-1, a transcription factor expressed initially in all differentiating endocrine cells during islet neogenesis but becoming increasingly limited to the ß-cell component during islet maturation (6). Pdx-1 was seen predominantly within the ß-cell population of existing islets, with little presence in isolated clusters of endocrine cells indicative of neogenesis. However, the number of isolated cells immunopositive for insulin per tissue section was increased in pancreata from transgenic animals, suggesting that a limited degree of neogenesis was occurring. A role for IGF-II as primarily a mitogenic factor for existing endocrine cells is consistent with its ability to increase DNA synthesis within isolated islet-like cell clusters, which contained ß-cells, from human fetal pancreas, but its relatively poor ability to increase the numbers of these structures compared with hepatocyte growth factor of fibroblast growth factor-7 (40, 41).
It is not clear whether the generation of islet cell hyperplasia was
primarily caused by an increased local expression of IGF-II within the
pancreas of the transgenic animals or resulted from exposure to the
increased circulating levels. IGF-II mRNA and peptide were abundant in
the islets of transgenic mouse pancreata, but this could be a
reflection of increased mean islet size, as well as a cause. A low
level of immunoreactivity for IGF-I was also associated with the
islets, and this was reduced in IGF-II transgenic mice. Because the
IGF-I gene is weakly expressed in the fetal rodent pancreas (20), the
reduced number of islet cells containing immunoreactivity may represent
a relative displacement of IGF-I from IGFBPs by the increased presence
of IGF-II. This could lead to a loss of IGF-I from the islet cell
surfaces. We found no major differences in the presence of
immunoreactivity for IGFBPs-1 to -3, which are the predominant forms
expressed in the rodent pancreas (20), in mouse pancreatic islets
between transgenic and control animals, although there was a tendency
for more cells to contain immunoreactive IGFBP-2. This would suggest
that although IGFBPs-1 and -2 can modulate the actions of IGF-II as a
mitogen for islets, both in vitro (13), and islet cell
hyperplasia was seen in mice carrying the human IGFBP-1 transgene (42),
changes in IGFBP presence may not contribute greatly to the islet
overgrowth seen in IGF-II transgenic mice. Despite the presence of
islet cell hyperplasia in transgenic animals, there was not an increase
in circulating insulin concentrations, and plasma glucose levels were
comparable with those of control animals. This may be caused by a
down-regulation of insulin secretion by IGF-II, as has been reported
for islet-like cell clusters from the human fetus (40) and for
perifused islets from adult rats (22). Alternatively, if glucagon
release was increased as a result of the increased pancreatic -cell
mass in the transgenic animals, this might also suppress the release of
insulin. Fetal overgrowth accompanied by elevated circulating IGF-II
and hypoglycemia are features of Beckwith-Weidemann syndrome in human
infants (43). Our findings show that an overexpression of IGF-II
leading to tissue overgrowth in fetal life is not necessarily
associated with hypoglycemia.
In summary, the overproduction of mouse IGF-II in a transgenic model resulted in overgrowth of the pancreatic islets, with an increased population of all three major endocrine cell types, but did not alter the number of mature islets. This was not accompanied by hyperinsulinemia, possibly because of a down-regulation of insulin release by IGF-II.
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Footnotes |
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Received August 5, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 M. M. Bonaventura, P. N. Catalano, A. Chamson-Reig, E. Arany, D. Hill, B. Bettler, F. Saravia, C. Libertun, and V. A. Lux-Lantos GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice Am J Physiol Endocrinol Metab, January 1, 2008; 294(1): E157 - E167. [Abstract] [Full Text] [PDF]
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 A. M Ackermann and M. Gannon Molecular regulation of pancreatic {beta}-cell mass development, maintenance, and expansion J. Mol. Endocrinol., February 1, 2007; 38(2): 193 - 206. [Abstract] [Full Text] [PDF]
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 H. Jiang, H. Zhu, X. Chen, Y. Peng, J. Wang, F. Liu, S. Shi, B. Fu, Y. Lu, Q. Hong, et al. TIMP-1 Transgenic Mice Recover From Diabetes Induced by Multiple Low-Dose Streptozotocin Diabetes, January 1, 2007; 56(1): 49 - 56. [Abstract] [Full Text] [PDF]
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 I. Giurgea, C. Sempoux, C. Bellanne-Chantelot, M. Ribeiro, L. Hubert, N. Boddaert, J.-M. Saudubray, J.-J. Robert, F. Brunelle, J. Rahier, et al. The Knudson's Two-Hit Model and Timing of Somatic Mutation May Account for the Phenotypic Diversity of Focal Congenital Hyperinsulinism J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 4118 - 4123. [Abstract] [Full Text] [PDF]
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 P. M. Vuguin, M. H. Kedees, L. Cui, Y. Guz, R. W. Gelling, M. Nejathaim, M. J. Charron, and G. Teitelman Ablation of the Glucagon Receptor Gene Increases Fetal Lethality and Produces Alterations in Islet Development and Maturation Endocrinology, September 1, 2006; 147(9): 3995 - 4006. [Abstract] [Full Text] [PDF]
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 H. Iwakura, K. Hosoda, C. Son, J. Fujikura, T. Tomita, M. Noguchi, H. Ariyasu, K. Takaya, H. Masuzaki, Y. Ogawa, et al. Analysis of Rat Insulin II Promoter-Ghrelin Transgenic Mice and Rat Glucagon Promoter-Ghrelin Transgenic Mice J. Biol. Chem., April 15, 2005; 280(15): 15247 - 15256. [Abstract] [Full Text] [PDF]
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J. A. McCann, Y. Q. Xu, R. Frechette, L. Guazzarotti, and C. Polychronakos The Insulin-Like Growth Factor-II Receptor Gene Is Associated with Type 1 Diabetes: Evidence of a Maternal Effect J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5700 - 5706. [Abstract] [Full Text] [PDF]
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E. A. Joanette, B. Reusens, E. Arany, S. Thyssen, R. C. Remacle, and D. J. Hill Low-Protein Diet during Early Life Causes a Reduction in the Frequency of Cells Immunopositive for Nestin and CD34 in Both Pancreatic Ducts and Islets in the Rat Endocrinology, June 1, 2004; 145(6): 3004 - 3013. [Abstract] [Full Text] [PDF]
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 W. R. Bennett, T. E. Crew, J. M. W. Slack, and A. Ward Structural-proliferative units and organ growth: effects of insulin-like growth factor 2 on the growth of colon and skin Development, March 15, 2003; 130(6): 1079 - 1088. [Abstract] [Full Text] [PDF]
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C. Sempoux, Y. Guiot, K. Dahan, P. Moulin, M. Stevens, V. Lambot, P. d. Lonlay, J.-C. Fournet, C. Junien, F. Jaubert, et al. The Focal Form of Persistent Hyperinsulinemic Hypoglycemia of Infancy: Morphological and Molecular Studies Show Structural and Functional Differences With Insulinoma Diabetes, March 1, 2003; 52(3): 784 - 794. [Abstract] [Full Text] [PDF]
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A. Cebrian, A. Garcia-Ocana, K. K. Takane, D. Sipula, A. F. Stewart, and R. C. Vasavada Overexpression of Parathyroid Hormone-Related Protein Inhibits Pancreatic {beta}-Cell Death In Vivo and In Vitro Diabetes, October 1, 2002; 51(10): 3003 - 3013. [Abstract] [Full Text] [PDF]
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B. Duvillie, C. Currie, T. Chrones, D. Bucchini, J. Jami, R. L. Joshi, and D. J. Hill Increased Islet Cell Proliferation, Decreased Apoptosis, and Greater Vascularization Leading to {beta}-Cell Hyperplasia in Mutant Mice Lacking Insulin Endocrinology, April 1, 2002; 143(4): 1530 - 1537. [Abstract] [Full Text] [PDF]
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 J. Nakae, Y. Kido, and D. Accili Distinct and Overlapping Functions of Insulin and IGF-I Receptors Endocr. Rev., December 1, 2001; 22(6): 818 - 835. [Abstract] [Full Text] [PDF]
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S. A. Kassem, I. Ariel, P. S. Thornton, K. Hussain, V. Smith, K. J. Lindley, A. Aynsley-Green, and B. Glaser p57KIP2 Expression in Normal Islet Cells and in Hyperinsulinism of Infancy Diabetes, December 1, 2001; 50(12): 2763 - 2769. [Abstract] [Full Text] [PDF]
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 C. E Bertram and M. A Hanson Animal models and programming of the metabolic syndrome: Type 2 diabetes Br. Med. Bull., November 1, 2001; 60(1): 103 - 121. [Abstract] [Full Text] [PDF]
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A. L Fowden and D. J Hill Intra-uterine programming of the endocrine pancreas Br. Med. Bull., November 1, 2001; 60(1): 123 - 142. [Abstract] [Full Text] [PDF]
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B. Tyrberg, J. Ustinov, T. Otonkoski, and A. Andersson Stimulated Endocrine Cell Proliferation and Differentiation in Transplanted Human Pancreatic Islets: Effects of the ob Gene and Compensatory Growth of the Implantation Organ Diabetes, February 1, 2001; 50(2): 301 - 307. [Abstract] [Full Text]
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D. J. Hill, B. Strutt, E. Arany, S. Zaina, S. Coukell, and C. F. Graham Increased and Persistent Circulating Insulin-Like Growth Factor II in Neonatal Transgenic Mice Suppresses Developmental Apoptosis in the Pancreatic Islets Endocrinology, March 1, 2000; 141(3): 1151 - 1157. [Abstract] [Full Text] [PDF]
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