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Lawson Research Institute, St. Joseph’s Health Center (D.J.H., B.S., E.A., S.C.), London, Ontario, Canada N6A 4V2; the Departments of Physiology (D.J.H.), Medicine (D.J.H.), and Pediatrics (D.J.H.), University of Western Ontario, London, Ontario, Canada N6A 5A5; and the Department of Zoology, University of Oxford (S.Z., C.F.G.), Oxford, United Kingdom OX1 3PS
Address all correspondence and requests for reprints to: Dr. D. J. Hill, Lawson Research Institute, St. Joseph’s Health Center, 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 and circulating insulin levels were not changed.
Islet cell DNA synthesis was increased in transgenic mice on days 13
and 16. The total islet number per section did not alter. The results
show that a persistent presence of circulating IGF-II postnatally
alters endocrine pancreatic ontogeny in the mouse and largely prevents
the wave of developmental apoptosis that precipitates ß-cell turnover
in neonatal life. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is substantial evidence that ongoing ß-cell proliferation in early life is dependent on the insulin-like growth factor (IGF) axis. Exogenous IGF-I or -II promotes DNA synthesis in isolated islets or ß-cells through the type 1 IGF receptor (8, 9, 10, 11). The mitogenic effects of IGFs are modulated by locally produced IGF-binding proteins (12). IGF-II is expressed mostly in pancreas during fetal and neonatal life, and this is localized primarily to the islets and ductal epithelium (13). We demonstrated that an overexpression of IGF-II within multiple tissues of embryonic and fetal transgenic mice, including pancreas, resulted in a 5-fold increase in mean islet size at birth, without a change in islet number (14). This suggests that IGF-II is unlikely to function primarily as an islet neogenic factor, but, rather, functions as a potent growth factor for existing islets. It is also possible that the developmental ß-cell apoptosis seen in the neonatal rat is linked to IGF-II availability. We showed that ß-cell apoptosis was temporally associated with a rapid decline in islet IGF-II expression (4, 13, 15), and that endogenous or exogenous IGF-II was cytoprotective against cytokine-induced ß-cell apoptosis (4). Protein deprivation in early life caused an increase in developmental ß-cell apoptosis accompanied by a reduction in pancreatic IGF-II expression (6). This is in agreement with the established ability of IGF-I and -II to prevent apoptosis in multiple cell types (16, 17, 18).
To obtain functional proof that changes in IGF-II availability within the pancreas in early life regulate ß-cell ontogeny and are involved in the timing or magnitude of developmental ß-cell apoptosis, we used a transgenic model in which the Igf2 gene driven by a keratin 10 promoter is selectively overexpressed in mice in skin, gut, and uterus (19). This results in an increased circulating IGF-II, which, unlike that in normal animals, does not decline postnatally to disappear by weaning (20). Our hypothesis, therefore, was that a maintenance of IGF-II availability to the pancreas postnatally would suppress or abolish the wave of neonatal ß-cell apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were maintained on a 14-h light, 10-h dark cycle and fed Porton Combined Diet (PCD.MOD., Special Diet Services, Witham, UK). The mice in Oxford were killed by cervical dislocation at all ages. The work was performed under license from the Home Office (United Kingdom), and the procedures were performed with ethical approval of the animal care committee of the University of Western Ontario and in accordance with the guidelines of the Canadian Council for Animal Care. After death, animals were assessed for the presence of the Igf2 transgene, and body weight and wet weights of the pancreas were recorded. Blood was collected from some animals under Avertin (Winthrop Laboratories, Surbiton, Surrey, UK) anesthesia by direct puncture of the heart into a heparinized syringe, and serum was prepared for measurement of glucose, insulin, and IGF-I and -II. The pancreas was immediately removed from each animal and placed in 5 ml sterile, ice-cold HBSS, pH 7.5 (Life Technologies, Inc., Burlington, 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 four washes at 4 C in PBS. Fixed tissues were dehydrated in 50% ethanol (vol/vol; twice, 10 min each time) followed by 70% ethanol and embedded in paraffin. Mice carrying the transgene were compared with their nontransgenic littermates. No differences were noted between males and females for any parameter measured in this study.
Immunohistochemistry
Sections of pancreas for histology (5 µm) were cut from
paraffin blocks with a rotary microtome and mounted on SuperFrost Plus
glass slides (Fisher Scientific, Nepean, Canada).
Immunohistochemistry was performed to localize IGF-II, IGF-I,
proliferating cell nuclear antigen (PCNA), insulin, and glucagon within
islets by a modified avidin-biotin peroxidase method (24) as described
by us previously for pancreas (13). Sections were deparaffinized in
xylene, rehydrated in a descending alcohol series (100%, 90%, and
70%, vol/vol), and washed in PBS. Sections were then incubated in 1%
(vol/vol) hydrogen peroxide to block endogenous peroxidase activity,
followed by a 15-min incubation in 5% (wt/vol) BSA and 0.01% (wt/vol)
sodium azide in PBS to reduce nonspecific binding. Slides were
incubated with rabbit antihuman IGF-II (1:300 dilution; GroPep Pty. Ltd., Adelaide, Australia), rabbit antihuman IGF-I (1:500
dilution; GroPep Pty. Ltd.), anti-PCNA (1:750 dilution;
Sigma, St. Louis, MO), guinea pig antiinsulin (1:15
dilution; provided by Dr. T. J. McDonald, University of Western
Ontario, London, Canada), or rabbit antiporcine glucagon (1:100
dilution; C-terminal specific O4A antiserum, provided by Dr. R. Ungar,
Dallas, TX). All antisera were diluted in 0.01 M PBS (pH
7.5) containing 1% (wt/vol) BSA and 0.02% (wt/vol) sodium azide (100
µl/slide), and incubations were performed at 4 C. All subsequent
incubations were performed at room temperature. Biotinylated antirabbit
IgG (1:30 dilution), antimouse IgG (1:100 dilution), and antiguinea pig
IgG (1:500 dilution; Vector Laboratories, Inc.,
Burlingame, CA) were diluted in the same buffer and applied to the
tissue for 2 h in a humidified chamber. The slides were then
washed in PBS and incubated with ExtrAvidin (1:30 dilution;
Sigma) for 1 h. Peptide immunoreactivity was
visualized by incubation with diaminobenzidine tetrahydrochloride (Fast
DAB tablets, Sigma) for 2 min. Tissue sections were
counterstained with Carrazi’s hematoxylin, dehydrated in ascending
series of alcohols (50%, 70%, 90%, and 100%, vol/vol), cleared in
xylene, and mounted under glass coverslips with Eukitt (Ruth Wagener
Enterprises, Inc., Newmarket, Canada).
To establish specificity of staining, the primary antisera for IGF-II, IGF-I, insulin, and glucagon were preadsorbed overnight at 4 C with excess homologous antigen before application to the sections, resulting in an abolition of staining. Further controls included substitution of primary antisera with nonimmune serum and omission of the secondary antiserum. Dual staining for PCNA and insulin was undertaken by first performing immunohistochemistry for PCNA as described above using diaminobenzidine as the chromogen. Before counterstaining and dehydration, the sections were subjected to immunohistochemistry for insulin as described above, using alkaline phosphatase (blue) (alkaline phosphatase substrate kit III, Vector Laboratories, Inc.) as the chromogen. After incubation with the insulin antibody, antiguinea pig alkaline phosphatase conjugate (Sigma) was applied to the sections for 1 h, followed by incubation with alkaline phosphatase substrate for 20 min before washing and counterstaining with Mayer’s hemalum. Sections were mounted under glass coverslips with an aqueous mountant (Aquamount, Polysciences, Warrington, PA).
Visualization of apoptosis
Molecular histochemistry was performed to localize apoptotic
nuclei within histological sections of pancreas (25) using the Apoptag
in situ apoptosis detection kit (Intergen
Company, Purchase, NY), as described by us in detail (4). Staining was
performed according to the manufacturer’s protocol. Histological
sections (5 µm) were de-paraffinized in xylene, rehydrated in a
descending alcohol series (100%, 90%, 70%, vol/vol), and washed in
PBS before incubation with proteinase K (20 µg/ml;Roche Molecular Biochemical Corp., Dorval, Canada) for 15 min at room
temperature. After proteinase K digestion, sections were incubated with
2% (vol/vol) hydrogen peroxide for 5 min to quench endogenous
peroxidase activity, followed by application of the terminal
deoxynucleotidyl transferase enzyme for 1 h at 37 C. Apoptotic
nuclei were visualized with diaminobenzidine (Fast-DAB tablets,
Sigma) as described for immunohistochemistry, followed by
counterstaining with methyl green for 30 sec. Tissues were dehydrated
in butanol, cleared in xylene, and mounted under glass coverslips with
Eukitt (Ruth Wagener Enterprises Inc.).
RIAs
Insulin was measured by RIA using the Wright antiserum in a
modification of the method of Hales and Randle (26) as modified by
Herbert et al. (27) and described by us previously (28). Rat
insulin (Novo Nordisk, Mississauga, 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. IGF-I and -II RIAs were also
performed on mouse serum as previously described (29) after extraction
of IGF-binding proteins by separation on Sephadex G-50. Glucose
concentrations were measured in mouse serum using a glucose oxidase
method (Sigma).
Morphometric and statistical analysis
Morphometric analysis was performed using a Carl Zeiss transmitted light microscope (New York, NY) at a
magnification of x250 or x400. Analyses were performed with Northern
Eclipse version 2.0 morphometric analysis software (Empix Imaging Co.,
Mississauga, Canada). The islet area and number; percent
immunopositivity for IGF-I, IGF-II, insulin, or glucagon; and the
percentage of islet cells immunopositive for PCNA or demonstrating
apoptotic nuclei were calculated at each age from five sections of each
pancreas, representing predominantly the head regions adjacent to the
stomach and spleen. Sections chosen contained at least five islets, and
pancreata from four or five animals were examined for each age,
representing either control or transgenic animals. Individual cells or
the total areas of immunoreactive cells within islets were circled for
image analysis and selected by red-green-blue threshold.
Differences between mean values for variables within individual
experiments were compared statistically by two-way ANOVA, followed by
Scheffe’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In
transgenic animals serum IGF-II levels did not decline with age and
were significantly greater than those in nontransgenic mice at each age
examined. There were no differences between mean serum IGF-I values in
transgenic and control animals, and circulating insulin levels were
similar, as was blood glucose (Table 1).
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). This resulted in islets representing
a significantly greater area of the pancreas between 11 and 16 days in
mice overexpressing IGF-II (Fig. 1b). ß- and -cells were
identified within pancreatic sections by immunohistochemical staining
for insulin and glucagon, respectively. The relative area of islets
occupied by each did not differ with age or transgene expression
despite the fact that the islets in the transgenic mice were larger
from 11–16 days (insulin: control, 74 ± 5%; transgenic, 73
± 4%; glucagon: control, 19 ± 2%; transgenic, 19 ± 1%;
mean ± SEM; n = 20–25 animals). Islet number
per section of pancreas did not change with age or the presence of the
transgene (control, 15 ± 3; transgenic, 14 ± 2; n =
20–25 animals). Islets were classified as small (<10,000
µm2), intermediate (10,000–20,000
µm2), or large (>20,000
µm2). A transient increase in small islets
occurred in control mice at 11 days (day 7, 4 ± 3; day 11,
10 ± 2; P < 0.05 vs. day 7; day 16,
9 ± 2; day 26, 5 ± 2; mean ±
SD; n = 5), which was associated with less
intermediate and large islets. No such increase in small islets was
seen in IGF-II transgenic mice (day 7, 6 ± 2; day 11, 5 ±
3; day 16, 6 ± 1; day 26, 4 ± 3).
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). Dual staining for either insulin or
glucagon and PCNA showed that 73 ± 2% of PCNA-positive cells
were ß-cells (Fig. 3a), whereas 16
± 3% were cells, and this did not proportionately differ with age
or transgene presence. Hence, the increased presence of replicating
cells in transgenic animals on days 13 and 16 involved both cell
populations. Ductal epithelial cells and acinar cells also demonstrated
staining for PCNA, but no obvious changes were seen in their numbers
with age of transgene presence.
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). However, the
number of apoptotic cells was significantly lower in IGF-II transgenic
animals on days 11 and 13. Apoptotic cells were predominantly localized
to the ß-cell-rich core of the islets (Fig. 3b
). Colocalization
immunohistochemistry failed to demonstrate the presence of
immunoreactive insulin or glucagon in apoptosing cells, which may be
due to the withdrawal of cytoplasm around the apoptotic nuclei.
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- and ß-cells. No differences were found between
transgene and control cells. IGF-II immunoreactivity in both had
decreased to less than 5% of islet area by day 26. IGF-I
immunoreactivity was seen in 15 ± 7% of islet cells on day 7,
rising to 31 ± 6% on day 26 (n = 8–10). Again, no
differences were found between transgenic and nontransgenic
animals. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although there is abundant IGF-II in the circulation of the fetal rat or mouse, this declines after birth and is absent by weaning, whereas circulating IGF-I levels increase postnatally (33). In the rat, IGF-II expression declines at differing rates in various organs (20), and in the pancreatic islets this is abrupt and occurs between postnatal days 7 and 14 coincident with the wave of ß-cell apoptosis (4, 15). Although such detailed data do not exist for the mouse, we found here that the presence of IGF-II immunoreactivity in pancreatic islets of nontransgenic mice declined steadily from postnatal day 7 and was absent by day 26. IGF-I immunoreactivity in the pancreas increased with age. No Igf2 mRNA was detectable by RT-PCR by postnatal days 22–24 in either control or transgenic mouse pancreas, confirming that gene expression has normally ceased by this time and is not retained within the pancreas in transgenic animals. This suggests, firstly, that the ontogeny of the IGF axis in pancreas is similar between mouse and rat and, secondly, that the increased IGF-II available to the pancreas in the transgenic animals postnatally derives predominantly from the circulation after synthesis at other sites. It is possible that differences in Igf2 expression could exist within the pancreas between control and IGF-II transgenic animals in the embryo or neonate, but the transgene construct incorporating a bovine keratin 10 gene promoter is not expressed in pancreas.
The model of IGF-II transgenesis used leads to a targeted overexpression in the skin, gut, and uterus only, leading to local overgrowth of these tissues (19). Circulating IGF-II is retained and is in excess of 40 ng/ml at least until 6 months of age (34). Although circulating insulin and blood glucose values are no different postnatally, the IGF-II transgenic animals have 40% less fat, affecting both white and brown adipose tissue (34). As the transgene is not expressed in fat, the increased oxidation rate of lipids observed in transgenic mice must result from the increased circulating IGF-II, demonstrating that this endocrine source is available to target tissues. The present study shows that circulating IGF-II is greater in transgenic animals at each age from postnatal day 7 compared with that in controls and is likely to be available to the pancreatic islets. Circulating levels of IGF-I were not altered.
In IGF-II transgenic mice the neonatal wave of islet cell apoptosis
commencing on day 11 was almost abolished. Consequently, the mean islet
area was significantly increased at days 11–16, but was not
significantly different from that in controls by day 26. This may be
due to a higher rate of islet cell DNA synthesis on days 13 and 16,
which would partially compensate for cells lost by apoptosis. The
proportion of ß- to -cells did not significantly change throughout
this period, suggesting that the additional IGF-II availability
provided a mitogenic stimulus to both cell types. This would agree with
our previous observations that the overexpression of IGF-II within
multiple tissues in the mouse embryo, including pancreatic islets,
caused an overgrowth of the islets affecting all endocrine cell types
(14). In that study, in which additional IGF-II was available as an
autocrine or paracrine agent, the mean islet size was increased 5-fold
at birth, and islet architecture was disrupted. In the present
experiments, in which additional IGF-II was available only as an
endocrine agent, mean islet size and architecture did not differ
between transgenic and control animals on day 7 and only became
apparent once local expression by the endogenous IGF-II gene would have
declined. This may indicate that IGF-II available from the circulation
is less effective at target tissues than that produced locally or that
the increased availability of IGF-II from blood was too slight in this
model to seriously impact the growth and function of tissues in which
the endogenous gene was already expressed. Only a 2-fold increase in
serum IGF-II was seen in transgenic animals on day 7.
In the pancreas of nontransgenic mice a transient increase in the proportion of smaller islets was observed at the time of islet cell apoptosis. This is probably indicates a renewal of endocrine tissue by neogenesis from the pancreatic ducts, although this was not obviously apparent as an altered PCNA labeling index within ductal cells. In the transgenic animals, in which islet cell apoptosis was suppressed, no such increase in smaller islets occurred, which suggests that endocrine cell apoptosis in islets and the islet neogenic process are coregulated. The implications of a failure of developmental islet cell apoptosis and the associated islet neogenesis for glucose metabolism in later life are unknown and cannot readily be ascertained from this particular model due to the abnormal lipid metabolism and adipose mass (34). This may result in altered levels of circulating leptin, which has been proposed to have effects on pancreatic ß-cell function (35). Circulating leptin levels undergo a transient increase in the second postnatal week in rodents, but this is independent of fat mass (36), suggesting that it would not be a variable in the IGF-II transgenic model.
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Footnotes |
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2 Present address: University of Lund, Malmo General Hospital, 20502,
Malmo, Sweden.
Received November 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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