Endocrinology Vol. 139, No. 6 2994-3004
Copyright © 1998 by The Endocrine Society
Apoptosis in the Pancreatic Islet Cells of the Neonatal Rat Is Associated with a Reduced Expression of Insulin-Like Growth Factor II that May Act as a Survival Factor1
J. Petrik,
E. Arany,
T. J. McDonald and
D. J. Hill
Lawson Research Institute, St. Josephs Health Center, London,
Ontario, Canada N6A 4V2; and the Departments of Physiology (J.P.,
D.J.H.), Medicine (E.A., D.J.H.), Biochemistry (T.J.M.), Pharmacology
and Toxicology (T.J.M.), and Pediatrics (J.P., D.J.H.), University of
Western Ontario, and London Health Sciences Center (T.J.M.), London,
Ontario, Canada N6A 5A5
Address all correspondence and requests for reprints to: Dr. D. J. Hill, Lawson Research Institute, St. Josephs 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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Islet cell ontogeny will define adult ß-cell mass and will consist of
a balance of islet cell birth and death. We have investigated the
ontogeny of factors that may be related to developmental apoptosis in
the islets, insulin-like growth factor II (IGF-II) and inducible nitric
oxide synthase (iNOS), in pancreata of young Wistar rats. Pancreata
were collected from rats of 21 days gestation to 29 days postnatal age.
In situ hybridization and immunohistochemistry showed
that IGF-II was expressed and present in fetal and neonatal islet
cells, but declined rapidly 2 weeks after birth. Little IGF-I was
associated with fetal or postnatal islets. Apoptosis in islet cells was
visualized by molecular histochemistry for DNA breakage in tissue
sections. Apoptosis was low in the fetus, but increased in incidence
postnatally so that 13% of islet cells were undergoing apoptosis on
postnatal day 14, with the incidence declining thereafter.
Immunohistochemistry for iNOS showed that it was expressed within
ß-cells and was most abundant 12 days after birth. When islets were
isolated from rat pancreata 2022 days after birth, islet cell
viability, DNA synthetic rate, and insulin release were reduced after
incubation with interleukin-1ß, tumor necrosis factor, or
interferon-
. An increased rate of islet cell survival was found
after simultaneous incubation with IGF-I or -II. Cytokine-mediated
islet cell death involved the induction of apoptosis. Islets isolated
from neonatal rats were not killed after exposure to these cytokines at
the same concentrations, but cytokine-induced cell death was seen when
neonatal islets were incubated with a neutralizing antibody against
IGF-II. These experiments show that a peak of islet cell apoptosis that
is maximal in the rat pancreas 14 days after birth is temporally
associated with a fall in the islet cell expression of IGF-II. IGF-II
was shown to function as an islet survival factor in
vitro. The induction of islet cell apoptosis in
vivo may involve an increased expression of iNOS within
ß-cells.
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Introduction
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IN THE RAT fetus, the cellular area
immunostained for insulin increases 2-fold over 2 days just before term
due to both ß-cell replication and recruitment, and maturation of
undifferentiated ß-cell precursors (1, 2). Endocrine cells develop
from duct-like cells in the embryo, fetus, and neonate and form
primitive islets in the mesenchyme adjacent to the ducts. Final
differentiation into glucagon-, somatostatin-, pancreatic polypeptide-,
or insulin-expressing cells probably depends on the expression of
transcription factors such as Pdx-1 (3) and on the actions
of local peptide growth factors within the surrounding mesenchyme (4).
The population growth rate of all islet cells, including ß-cells,
slowed by postnatal days 3 and 4 and continued to decline thereafter
(2). The rate of mitosis in adult pancreatic ß-cells is normally low
(3% replication rate of ß-cells/day) (5).
The change from a fetal type of ß-cell population capable of
expansion, to an adult ß-cell population that is not may occur within
a programed ontological pathway that extends into postnatal life. The
newborn rat made diabetic with streptozotocin has extensive ß-cell
destruction, but at 14 days after birth demonstrates normal glycemia,
with considerable mitotic activity apparent in the pancreatic ductal
epithelium from which the precursor ß-cells derive (6). Similar
ß-cell renewal occurs after alloxan treatment of the young rabbit
(7). Pancreatectomy (90%) of the young rat is followed by regeneration
of both exocrine and endocrine tissues (8). New ß-cells are derived
by both neogenesis and cell replication within the remaining islets.
Endocrine regeneration is also seen in association with duct epithelial
cell proliferation in transgenic mice with ß-cells expressing
interferon-
(IFN
) (9). In these mice, ß-cell destruction
leading to IDDM can be matched by new islets growing ectopically into
duct lumens. Islet regeneration occurs in the pancreas of young
diabetic patients, and in some cases, islet neogenesis is associated
with centroacinar and ductular cells, leading to the formation of large
islets consisting primarily of ß-cells (10). Islet cell regeneration
in the pancreas of recent-onset IDDM patients also occurs, particularly
in infants and small children (11, 12). Recently, it has been revealed
that the ontogeny of islet cells in early life involves a balance
between ß-cell replication and neogenesis, and programed ß-cell
death. A transient wave of apoptosis occurs in neonatal rat islets
between 12 weeks of age (5, 13). However, ß-cell mass is not
altered appreciably at this time, suggesting that a new population of
ß-cells compensates for the loss. A similar episode of ß-cell
apoptosis has recently been described in the human fetal pancreas in
third trimester (14). Although the cellular triggers involved in the
initiation of developmental ß-cell apoptosis are not known, the
apoptosis that mediates ß-cell destruction in response to cytokine
action during autoimmune diabetes involves an increased intracellular
concentration of nitric oxide (NO) and increased expression of
inducible nitric oxide synthase (iNOS) (15).
The mechanisms controlling islet cell neogenesis or apoptosis during
development are largely unknown, but these may be linked to the
relative expression of peptide growth factors within the developing
pancreas. There is considerable circumstantial evidence that the
insulin-like growth factors (IGFs) are major contributors to ß-cell
growth, maturation, and function and are expressed by ß-cells
throughout life. We previously reported that IGF-II messenger RNA
(mRNA) is most abundant in the pancreas of the fetal rat and declines
during the neonatal period (16). Conversely, IGF-I mRNA levels were
low, but detectable, in fetal life and rose to adult levels within 2
weeks after birth. Others have reported that IGF-I mRNA abundance
peaked transiently shortly after birth and then declined sharply (17),
a pattern found by us for IGF-II, but not IGF-I, mRNA. Using in
situ hybridization and immunohistochemistry, IGF-I and -II mRNAs
and peptides were shown to be present within islet cells throughout
life, including ß-cells (18). Similarly, in the midtrimester human
fetus, IGF peptides and IGF-binding protein-1 (IGFBP-1), -2, and -3
were localized by immunohistochemistry to the islets of Langerhans and
ß-cells (19). We and others showed that isolated islets from the
human (20) or rat fetus (21, 22) or neonate (23, 24) release both IGF-I
and IGF-II, that exogenous IGF-I or -II promotes increased islet cell
DNA synthesis (21, 22, 25), and that isolated
- and ß-cells from
rat islets contain the high affinity type 1 IGF signaling receptor
(26), as do pancreatic ß-cell lines (27). We also showed that
isolated adult rat islets enriched in ß-cells release IGF-I (28),
whereas others have demonstrated a retention of IGF-II mRNA expression
within the ß-cells of the pancreas in both rat and man (29, 30), and
high levels of IGF-II are expressed by the rat ß-cell line, INS-1
(31).
The transient wave of developmental apoptosis responsible for a
reduction in ß-cell number after 12 weeks of postnatal life in the
rat (13) coincides temporally with our demonstration of a diminished
pancreatic expression of IGF-II at this time, whereas pancreatic
expression of IGF-I has not yet achieved adult values (16). A nadir in
total IGF availability may therefore exist in pancreas when apoptosis
is transiently high. IGFs inhibit apoptosis in mammary carcinoma cells,
cerebellar granule neurons, ovarian preovulatory follicles, human
erythroid colony-forming cells, and hematopoietic cells (32, 33, 34, 35).
However, IGF-I inhibits iNOS induction in some tissues (36) and may
interfere with cytokine-stimulated NO synthesis. The ability of IGF-I
to limit neuronal damage elicited by experimental hypoxia-ischemia
suggests an ability of IGF-I to limit free radical generation (37).
In this study we have examined the precise anatomical pattern of
expression of IGF-I and -II in the pancreas of the rat during early
life, the relationship between developmental ß-cell apoptosis and the
presence of iNOS, and present functional evidence that IGFs can
influence islet ß-cell survival in the neonatal rat.
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Materials and Methods
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Animals
Pregnant Wistar rats (Charles River, Montreal, Canada) were
killed at day 21 of gestation or were allowed to deliver and the
offspring were killed on postnatal days 4, 6, 7, 12, 14, 16, 18, 22, or
29. Rat fetuses were decapitated with scissors, whereas the pregnant
females and all rats at postnatal ages were killed by CO2
asphyxiation. Pregnant and suckling rats were allowed free access to
food and water. All procedures were performed with ethical approval of
the animal care committee of the University of Western Ontario. After
death, the pancreas was immediately removed from each animal and placed
in 5 ml sterile, ice-cold Hanks Buffered Salts Solution, pH 7.5
(HBSS; Life Technologies, Burlington, Canada) if islets were to be
isolated. If pancreata were to be fixed for histology, they were placed
in ice-cold fixative (0.2% glutaraldehyde and 4% paraformaldehyde
buffered with 70 mM phosphate buffer, pH 7.4) for 16 h
at 4 C, followed by four washes at 4 C in PBS over a 48-h period. Fixed
tissues were dehydrated in 70% (vol/vol) ethanol and embedded in
paraffin.
Islet isolation
The islet isolation technique was modified from that of
Hellerstrom et al. (38). The pancreata were placed in
sterile, ice-cold HBSS, pH 7.4, containing penicillin (100 U/ml),
streptomycin (0.1 mg/ml), and fungizone (0.25 µg/ml; Life
Technologies). After removal of gut and spleen remnants, the pancreata
were transferred to small, sterile glass vials (
10 pancreata/vial
for fetal or neonatal islets, 5 pancreata/vial for older animals up to
22 days, and a single pancreas for the adult) and washed with ice-cold
HBSS. HBSS was removed from each of the vials and replaced with HBSS
containing 2 mg/ml collagenase (type V; Sigma Chemical Co., St. Louis,
MO). The vials were shaken for 45 min in a water bath (200
cycles/min) at 37 C for tissues from fetal and neonatal animals up to 6
days age and for 58 min for tissues from older rats. The digestion
was terminated by the addition of 15 ml ice-cold HBSS to each vial. The
digest was dispersed by pipette, and tissue was collected by
centrifugation after washing with ice-cold HBSS. The washed digest was
resuspended in 5 ml tissue culture medium, RPMI 1640, pH 7.4,
containing 11.1 mM glucose and 25 mM HEPES
(Life Technologies) and supplemented with antibiotics (100 U/ml
penicillin and 0.1 mg/ml streptomycin), fungizone (0.25 µg/ml), and
10% heat-inactivated, virus- and mycoplasma-free FBS (Life
Technologies). The digest was distributed in 1-ml portions onto tissue
culture petri dishes (100 mm; Nunc, Toronto, Canada) containing 14 ml
of an identical tissue culture medium. The culture plates were
incubated for 4872 h at 37 C in a humidified atmosphere of 5%
CO2 in air. Islets were counted by eye under a dissecting
microscope, detached from the culture dish, harvested using an
automatic pipette, and transferred to nontissue culture grade petri
dishes (100 mm; Falcon, Lincoln Park, NJ) that did not permit cell
attachment for the experimental period. A typical yield of islets from
10 pancreata was approximately 1500.
Islet culture
At the beginning of the treatment period, the islets were
distributed in equal batches (6080 islets/plate) onto nontissue
culture grade petri dishes (50 mm; Falcon) containing 1 ml tissue
culture medium. Culture medium consisted of glucose-free DMEM (Imperial
Laboratories, Andover, UK), pH 7.4, containing antibiotics and
fungizone (as described above) and supplemented with 2 mM
glutamine (Life Technologies) and 8.7 mM glucose. Medium
was further supplemented as required with recombinant human IGF-I or
IGF-II (100 ng/ml; GroPep, Adelaide, Australia), a monoclonal antibody
against rat IGF-II (20 µg/ml; Amano International Enzyme Co., Troy,
VA), recombinant human interleukin-1ß (IL-1ß; 2.5 ng/ml), tumor
necrosis factor-
(TNF
; 10 ng/ml), IFN
(10 ng/ml; R&D Systems,
Minneapolis, MN), or rabbit antihuman placental lactogen (Dimension
Laboratories, Mississauga, Canada), alone or in combination. Where
IGF-I or -II and cytokines or IGF-II antiserum and cytokines were
present together, the IGF or antiserum was added 5 min before the
cytokine and allowed to equilibrate with the islets at 37 C. Islets
were incubated for 24, 48, or 72 h. In some experiments,
[methyl-3H]thymidine (20 Ci/mM;
ICN Biomedicals, Irvine, CA) at 5 µCi/ml was added for the final
24 h of culture. At the end of the treatment period, conditioned
medium was removed, and the islets were washed in PBS (Life
Technologies) and assessed for either viability or DNA synthetic rate,
as described below. Separate experiments were required for each type of
analysis.
In some experiments islets were plated into 8-well chamber slides
(Lab-Tek, Nalge-Nunc International, Naperville, IL; 20 islets/well) and
allowed to attach to the glass slides overnight in a humidified
incubator at 37 C, gassed as described above, before the addition of
IGF-I or -II or cytokines, alone and in combination. After 48 h,
the culture medium was removed, and the islets were washed in PBS.
Islets attached during culture to chamber slides were dehydrated
through ascending ethanol concentrations (50%, 70%, 90%, and 100%),
and air-dried. Slides were stored with desiccant until histological
processing.
Assessment of islet viability
To assess islet cell viability after incubation in test culture
medium, the islets from each plate were resuspended in 0.5 ml PBS
containing 5 mg/ml trypan blue (Sigma). Trypan blue solution was
filtered through an ultramembrane (0.2 µm; Gelman Science, Ann Arbor,
IL) to remove particulates before use. All islets were examined
immediately under a dissecting microscope, and any islet containing one
or more cells that had taken up trypan blue was considered nonviable.
At the time of viability assessment, the recovery of islets was 92
± 5% (mean ±SD) of those initially added to each culture
dish, and this did not differ between control cultures or those that
had contained cytokines. The within-batch coefficient of variation on
assessment of islet viability after repeated measures was 3%.
Estimation of islet DNA content and synthetic rate
Islets were suspended in PBS (500 µl) and solubilized by
ultrasonication. DNA was precipitated with 1 ml ice-cold 10%
trichloroacetic acid and solubilized by overnight incubation with 1
M sodium hydroxide. After neutralization with 1
M HCl, the DNA content of islets was measured by
fluorometry using Hoechst fluorochrome 33258 (Aldrich Chemical Co.,
Milwaukee, WI) with an excitation wavelength of 375 nm and an emission
wavelength of 458 nm. Calf thymus DNA (1.524 µg/ml; Sigma) was used
for calibration. To measure the rate of DNA synthesis,
[3H]thymidine incorporation was measured by liquid
scintillation counting. Two 50-µl aliquots of homogenized islets were
removed, and the DNA was precipitated in 500 µl ice-cold 5%
trichloroacetic acid. The precipitates were collected by filtration
through a glass-fiber disk (2.5 cm; Whatman GF/A, Whatman
International, Maidstone, UK). Any remaining free isotope was removed
by washing with distilled water. The radioactivity on the filters was
determined by liquid scintillation counting (LS 5000 TD ß-counter,
Beckman, Palo Alto, CA). The incorporation of
[3H]thymidine was expressed as disintegrations per
min/µg DNA.
Insulin release
The insulin content of conditioned culture medium was measured
by RIA using Wright antiserum in a modification of the method of Hales
and Randle (39) as modified by Herbert et al. (40) and
described by us previously (41). 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.
Immunohistochemistry
Histological sections of pancreas (5 µm) were cut from
paraffin blocks with a rotary microtome and mounted on
poly-L-lysine-coated glass microscope slides.
Immunohistochemistry was performed to localize IGF-I or -II, insulin,
or iNOS within islets by a modified avidin-biotin peroxidase method
(42). Sections were deparaffinized in xylene, rehydrated in a
descending ethanol series (100%, 90%, and 70%, vol/vol), and washed
in PBS before incubation in 1% (vol/vol) hydrogen peroxide to block
endogenous peroxide activity, followed by a 15-min incubation in 5%
BSA in PBS to reduce nonspecific binding. 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), guinea pig antiinsulin
antibody (1:1000 dilution; provided by Dr. T. J. McDonald), or
mouse anti-iNOS antiserum (1:50 dilution; Transduction Laboratories,
Lexington, KY) in 0.01 M PBS (pH 7.5) containing 2%
(wt/vol) BSA and 0.01% (wt/vol) sodium azide (100 µl/slide). All
subsequent incubations were performed at room temperature. Biotinylated
goat antirabbit IgG (1:500), goat antimouse IgG (1:100), or mouse
antiguinea pig IgG (1:100) (Vector Laboratories, 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. After washing in PBS and 50
mM Tris-HCl, pH 7.5, peptide immunoreactivity was localized
by incubation in fresh 1.89 mM diaminobenzidine 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 Carazzis hematoxylin, dehydrated in an ascending
ethanol series, then cleared with xylene and mounted under glass
coverslips. To establish the specificity of staining, the primary
antisera to IGF-I or -II were preabsorbed overnight at 4 C with 100
nM homologous ligand before application to the sections. In
each case, staining was abolished. Further controls included
substitution of primary antisera with nonimmune serum and omission of
the secondary antiserum.
Dual staining for iNOS and insulin was performed by first performing
immunohistochemistry for insulin as described above, using
diaminobenzidine as the chromagen. Before counterstaining and
dehydration, the sections were subjected to immunohistochemistry for
iNOS as described, except that alkaline phosphatase (blue) was used as
the chromagen. Alkaline phosphatase substrate kit III was obtained from
Vector Laboratories. Antimouse alkaline phosphatase conjugate (Sigma)
was applied to each section for 1 h at room temperature, sections
were washed, and alkaline phosphatase substrate was applied for 20 min.
Sections were washed and counterstained with Mayers hemalum.
Visualization of apoptosis
Immunocytochemistry was performed to localize apoptotic nuclei
within either tissue sections or isolated islets (43) using the Apoptag
in situ apoptosis detection kit (Oncor, Gaithersburg, MD).
Islets attached during culture to chamber slides were rehydrated
through descending ethanol concentrations (100%, 90%, and 70%) and
incubated in PBS for 5 min. Staining was performed according to the
manufacturers protocol after incubation with proteinase K (20
µg/ml; Boehringer Mannheim, Dorval, Canada) for 15 min, washing in
distilled water, and the quenching of endogenous peroxidase by
incubation in 2% (vol/vol) hydrogen peroxide in PBS for 5 min. Color
was generated with diaminobenzidine [1.89 mM activated
with 0.03% (vol/vol) hydrogen peroxide for 2 min], and the tissue was
counterstained with methyl green for 1 min. Sections were dehydrated in
alcohols, cleared in xylene, and mounted with Permount (Fisher
Scientific, Fairlawn, NJ) under glass coverslips. Dual staining for
insulin was performed by incubation with antiinsulin antiserum after
detection of apoptosis and before dehydration. In this case the
chromagen used for insulin immunoreactivity was alkaline phosphatase
(as described above).
Northern blot analysis
Total RNA was extracted from isolated islets as previously
described (16). Before hybridization, the integrity and relative
amounts of RNA from each islet batch were assessed by size separation
on urea-agarose gels by analytical electrophoresis, followed by
ethidium bromide staining. Islets in which ribosomal RNA showed
degradation were not used for experiments.
For Northern blot hybridization, between 1520 µg total RNA were
denatured for 15 min at 65 C in 6% (vol/vol) deionized formaldehyde
(Fluka Chemicals, Georgetown, Canada) and 50% (vol/vol) deionized
formamide (Fluka) in 1 x hybridization buffer [20 mM
MOPS (morpholinopropanesulfonic acid), 5 mM sodium acetate,
and 5 mM EDTA, pH 7.0] and separated by size on 1%
agarose gels containing 6% deionized formaldehyde. The separated RNA
was then transferred to Zeta-Probe membranes (Bio-Rad, Richmond, CA) by
the capillary transfer technique (44). The blots were
prehybridized for 2 h in hybridization buffer containing 1 x
SSPE (150 mM NaCl, 10 mM sodium phosphate
monobasic, and 1 mM EDTA), 7% (wt/vol) SDS, 100 µg/ml
salmon sperm DNA, and 50% deionized formamide. The blots were then
hybridized in the same buffer at 42 C overnight in a shaking water bath
with 2 x 106 cpm/ml radiolabeled complementary DNA
(cDNA) probe for IGF-I or -II or with 1 x 106 cpm/ml
for a cDNA encoding 18S ribosomal RNA. After hybridization, the blots
were washed successively with 1 x SSC (standard saline citrate;
0.75 M NaCl and 0.075 M sodium citrate) with
0.1% SDS (wt/vol) once for 30 min at 22 C, followed by 30 min at 42 C.
Two final washes were performed, each for 30 min, at 42 C in 0.1
x SSC with 0.1% (wt/vol) SDS. Blots were air-dried and exposed to
x-ray film (Kodak XAR-5, Eastman Kodak, Rochester, NY) at -70 C with
intensifying screens for up to 10 days before developing. Blots were
hybridized consecutively with up to three different cDNAs. Between
consecutive hybridizations, the blots were washed with 0.01 x SSC
with 0.5% (wt/vol) SDS at 90 C for 1 h with two changes of
washing solution to remove the previous cDNA probe.
Radiolabeling of cDNA probes with
[
-32P]deoxy-CTP (ICN Biomedicals, Irvine, CA) was
carried out by random priming using a Pharmacia oligolabeling kit
(Pharmacia LKB Biotechnology, Uppsala, Sweden) to specific activities
of 12 x 109 dpm/µg. Separation of radiolabeled
cDNA from unincorporated [32P]deoxy-CTP was carried out
using Pharmacia NICK columns containing Sephadex G-50 (Pharmacia). cDNA
probes used for hybridization of Northern blots and for in
situ hybridization were provided by the following investigators. A
500-bp rat IGF-I cDNA in pGEM Blue (Promega, Madison, WI) encoding exon
3 and part of exon 4 of the recombinant IGF-I gene was provided by Dr.
L. Murphy, University of Manitoba (Winnipeg, Canada) (45). A 807-bp
mouse IGF-II in pGEM 4z was provided by Dr. G. Bell, University of
Chicago (Chicago, IL). A rat 18S ribosomal RNA cDNA was provided by Dr.
D. Denhardt, Rutgers University (Piscataway, NJ). The latter was used
to determine equality of RNA loading and transfer.
In situ hybridization
Sections of pancreas were deparaffinized in xylene, rehydrated
in a descending ethanol series (100%, 90%, and 70%, vol/vol), and
washed in PBS, then permeabilized with 10 µg/ml proteinase K
(Boehringer Mannheim) in 0.1 M Tris-HCl (pH 8.0) and 50
mM EDTA at 37 C for 30 min. After rinsing in PBS, sections
were dipped in 0.1 M triethanolamine-acetic anhydride for
10 min at room temperature, rinsed again in PBS, then dehydrated in a
graded ethanol series (70%, 90%, and 100%, vol/vol), air-dried, and
stored at -70 C until hybridization. Sections were prehybridized by
incubation at 45 C for 16 h in humidified chambers in 100 µl
1 x hybridization buffer [50% deionized formamide, 0.3
M NaCl, 20 mM Tris-HCl (pH 8.0), 1
mM EDTA, 1 x Denhardts solution, 500 µg/ml yeast
transfer RNA, 100 µg/ml denatured salmon sperm DNA, 10% (wt/vol)
dextran sulfate, 0.1% SDS, and 100 mM dithiothreitol
(DTT)]. Radiolabeled complementary RNA (cRNA) probes (106
cpm) were diluted with 25 µl water, denatured at 80 C for 2 min, and
made up to the required volume for each slide with 1.3 x
hybridization buffer. Each slide was incubated with 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 DTT
in 2 x 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 (RNase A), 1 U/ml RNase T1 in 0.5 M
NaCl, 10 mM Tris-HCl (pH 8.0), and 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 room temperature in 0.1 x SSC. To screen the extent
of RNA hybridization, slides were subjected to autoradiography with
Kodak XAR film after dehydration in an ascending ethanol series (70%,
90%, and 100%, vol/vol) and 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 Carazzis hematoxylin and dehydrated in an
ascending ethanol series (70%, 90%, and 100%, vol/vol) with a final
wash in xylene, and coverslips were applied with Permount (Fisher
Scientific, Jersey City, NJ). Slides used for in situ
hybridization were viewed under dark- and lightfield microscopy.
The restriction enzymes (BRL, Burlington, Canada) and RNA polymerases
(Promega) used to linearize the plasmids containing these cDNAs and to
generate 35S-radiolabeled riboprobes are as follows:
antisense rat IGF-I, HindIII/T7; sense rat IGF-I,
PvuII/SP6; antisense mouse IGF-II, HindIII/TSP6;
and sense mouse IGF-II, EcoRI/T7. Radiolabeled cRNA probes
were synthesized by adding the following to a sterile microfuge tube: 4
µl 5 x transcription buffer [200 mM Tris-HCl (pH
7.5), 30 mM MgCl2, 10 mM
spermidine, and 50 mM NaCl]; 2 µl 100 mM
DTT; 0.8 µl (20 U) RNase inhibitor; 4 µl 2.5 mM GTP,
ATP, UTP, or CTP; 2.4 µl 100 µM CTP or UTP; 1 µl (200
ng) linearized riboprobe DNA; 5 µl
-thio-[35S]UTP
(400 Ci/mmol; New England Nuclear, Billerica, MA), 0.3 µl sterile
water, and 1 µl (510 U) SP6, T3, or T7 RNA polymerase. Incubation
was performed at 37 C for 1 h. To remove template DNA, 1 µl
deoxyribonuclease I containing no RNase activity (1 U/µl; BRL,
Gaithersburg, MD) was added, and the reaction was further incubated at
37 C for 15 min. Yeast transfer RNA was added (50 µg), and the
reaction volume was increased to 450 µl with sterile water, then
extracted in sequence with equal volumes of phenol-chloroform and
chloroform-isoamyl alcohol. After ethanol precipitation with 0.1 vol 3
M sodium acetate (pH 5.2) and 2.5 vol cold ethanol, RNA was
pelleted by centrifugation, washed in 70% (vol/vol) ethanol, dried,
and resuspended in 500 µl 0.2% (wt/vol) SDS, 2 mM EDTA,
0.5 M sodium acetate. 35S-Labeled riboprobes
were precipitated with ethanol as described above, dried, and dissolved
in 200 µl 20 mM DTT. The quality and size of radiolabeled
cRNA transcripts were determined by urea-agarose gel electrophoresis of
10,000 cpm cRNA probe and autoradiography. To improve cRNA probe
penetration of tissue sections, limited alkaline hydrolysis was used to
reduce transcript size to about 150 bases.
Morphometric and statistical analysis
Morphometric analysis was performed using a Zeiss transmitted
light microscope and Incident-light fluorescence Axioskope (Carl Zeiss
Canada, Ltd., North York, Ontario, Canada) at a magnification of x250.
Analyses were performed with Northern Eclipse version 2.0 morphometric
analysis software (Empix Imaging Co., Mississauga, Canada). The
percentage of islet cells immunopositive for iNOS, IGF-I, or IGF-II or
demonstrating apoptotic nuclei was calculated at each age from three
sections of each pancreas, representing both head and tail regions.
Sections chosen contained at least five islets, and pancreata from at
least five animals were examined for each age.
Islet cultures were performed with three or four replicate culture
plates for each variable within an individual experiment derived from a
pool of islets. Each experiment was repeated from three to five times
using pools of islets derived from separate animals. Representative
experiments are shown as the mean ± SEM. Differences
between mean values for variables within individual experiments were
compared for statistical difference by ANOVA. For histological analyses
of the presence of IGF-I or -II, at least five separate pancreata were
examined, and representative views are shown.
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Results
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|---|
Sections of pancreas were stained for the presence of apoptotic
cells determined by DNA breakage to establish the ontogeny of islet
cell apoptosis. Apoptotic nuclei within condensed islet cells with
little cytoplasm were seen at all ages examined (Fig. 1
). The prevalence of apoptotic nuclei in
endocrine islet cells of fetuses at 21 days gestation was less than
2%, but this increased postnatally to 9% on postnatal day 12 and was
maximal at 13% on postnatal day 14 (Table 1
). At 22 days postnatal age, the
incidence of islet cell apoptosis had decreased to 2%. The majority of
apoptotic cells were located centrally within the islets, suggesting
that they were ß-cells. However, dual staining immunohistochemistry
for both insulin and apoptosis failed to confirm this, as staining for
insulin could not be conclusively demonstrated within the condensed,
apoptosing cells. The occurrence of islet cell apoptosis was a feature
of 85% of the islets examined on postnatal day 14.

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Figure 1. Immunohistochemical localization of apoptosing
cells (arrows) using molecular histochemistry in a
pancreatic islet from a 14-day-old rat. The number of condensed,
apoptotic cells is greatest at this time. Magnification
bar = 10 µm.
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Table 1. Incidence of apoptotic cells in rat pancreatic
islets (percentage of islet cells demonstrating apoptotic nuclei) in
animals from 21 days gestation until adulthood
|
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As the induction of apoptosis in pancreatic ß-cells by cytokines has
been shown to be mediated in part by an increase in intracellular NO
mediated by an increased expression of iNOS, we investigated the
presence of iNOS during developmental apoptosis in islets. Using
immunohistochemistry, islet cells, predominantly within the central
area of the islets, were found to contain cytoplasmic staining for iNOS
at all ages of the animal (Fig. 2A
). An
occasional presence of iNOS was also seen in ductal acinar cells. Dual
staining immunohistochemistry showed that at each age studied, 95% of
islet cells immunopositive for iNOS also contained insulin (Fig. 2B
).
When pancreata from various ages of rat were compared, the percentage
of islet cells containing immunoreactive iNOS was greatest at almost
10% on postnatal day 12 and declined thereafter to negligible levels
by weaning (Table 2
). Immunoreactive iNOS
could not be demonstrated in apoptotic islet cells, but the compacted
nature of these cells rendered any cytoplasmic localization of ligands
unlikely.

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Figure 2. A, Immunohistochemical localization of iNOS
(arrows) in pancreatic islets from a 12-day-old rat. B,
Costaining for insulin (brown) identified most of the
iNOS-positive cells (blue) as ß-cells. Magnification
bar = 10 µm.
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Table 2. Incidence (percentage of immunopositive cells) of
cells in rat pancreatic islets demonstrating the presence of
immunoreactive iNOS in animals from 21 days gestation until adulthood
|
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IGF-I and -II have been shown to prevent the experimental induction of
apoptosis in a variety of cell types (32, 33, 34, 35). We have previously
demonstrated a fall in the whole pancreatic expression of IGF-II in
neonatal life (16), suggesting that this may be functionally related to
the developmental apoptosis of neonatal islets. To determine the
anatomical location and ontogeny of IGF-I and -II expression, we
localized these mRNAs in pancreatic sections using in situ
hybridization and the distribution of the peptides by
immunohistochemistry. In situ hybridization for IGF-II mRNA
showed a relatively high signal intensity within islet endocrine cells
on postnatal day 7 (Fig. 3A
) in both the
central and peripheral regions of the islets. Hybridization signal was
also present in the ductal epithelial cells, but with relatively less
abundance than in islets. Hybridization with a sense strand IGF-II cDNA
showed little signal, demonstrating its specificity in pancreatic
tissues (not shown). A similar hybridization pattern was seen on
postnatal days 4 and 6. By postnatal day 29, no specific hybridization
signal for IGF-II mRNA remained within pancreatic islets (Fig. 3
, B and
D). Immunohistochemistry showed a widespread distribution of IGF-II
peptide in all islet cells in late gestation and postnatal day 7, but
this was less intense on postnatal days 12 and 14 and was absent by
postnatal day 22 (Fig. 4
). IGF-II was
also visualized in a minority of ductal epithelial cells in early life.
Conversely, no specific hybridization signal for IGF-I mRNA was seen in
the fetal or neonatal pancreas (Fig. 3C
), and only a low level of mRNA
was detected in islets after weaning (Fig. 3D
). However, on postnatal
day 29, IGF-I mRNA was seen within ductal epithelial cells. After
immunohistochemistry for IGF-I peptide, little immunoreactivity was
seen in the fetal or neonatal islets or acinar tissue (Fig. 4
). By
postnatal day 22, immunoreactivity for IGF-I was distributed throughout
the islet cells, but staining was not intense. Additionally,
immunoreactivity was located in the ductal epithelial cells. These
studies confirmed that IGF-II expression in the pancreas declined
substantially in neonatal life and showed that the major sites of
IGF-II expression were the pancreatic islets.

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Figure 3. In situ hybridization to visualize
IGF-II (A and B) or IGF-I (C and D) mRNAs in sections of rat pancreas
from animals 7 days (A and C) or 29 days (B and D) of age. Is, Islets;
E, exocrine tissue; Mes, mesenchymal connective tissue. Magnification
bar = 10 µm.
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Figure 4. Immunohistochemistry to visualize immunoreactive
IGF-II (A and B) or IGF-I (C and D) in sections of rat pancreas from
animals 7 days (A and C) or 22 days (B and D) of age. Is, Islets; E,
exocrine tissue; Mes, mesenchymal connective tissue. Magnification
bar = 10 µm.
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We performed experiments with rat islets in vitro to
determine whether either IGF-I or IGF-II could protect islet cells from
cell death induced by exposure to cytokines, which has been
demonstrated previously to involve the initiation of apoptosis (15).
Islets isolated from rats on postnatal days 2022 days or from adult
female rats were incubated in the presence of single concentrations of
IL-1ß (2.5 ng/ml), TNF
(10 ng/ml), or IFN
(10 ng/ml), with or
without IGF-I or -II (100 ng/ml), for 24 or 48 h. After 24-h
incubation, the mean viability of islets from rats 2022 days of age
incubated in the presence of each cytokine alone was significantly
lower than that in control cultures for TNF
and IFN
, but not for
IL-1ß. After incubation for 48 h, cell viability in control
cultures was 90 ± 3% (mean ± SEM; three
separate experiments), demonstrating that the islet isolation and
culture were associated with a low incidence of cell death. However, in
the presence of IL-1ß, TNF
, or IFN
, each cytokine alone caused
a 6090% significant reduction in islet cell viability after 48
h (Table 3
). Coincubation with either
IGF-I or IGF-II significantly reduced cell mortality in the presence of
IL-1ß or TNF
, but not in the presence of IFN
. Analysis of the
DNA synthetic rate showed that under control conditions there was
incorporation of [3H]thymidine into islet cells of rats
at 2022 days postnatal age, suggesting a potential for mitogenesis
(Table 4
). Exposure to IL-1ß, TNF
,
or IFN
alone each caused a significant reduction in
[3H]thymidine incorporation per µg DNA. Coincubation
with either IGF-I or -II (100 ng/ml) significantly reversed the
decrease in radiothymidine incorporation seen with IL-1ß or TNF
,
but not with IFN
. Similar results were found when
[3H]thymidine incorporation was expressed per number of
islets (not shown). The release of insulin into conditioned culture
medium was substantially reduced in response to each of the cytokines
alone, and this was not reversed by the presence of IGF-I or -II (Table 5
). Islets isolated from adult rats had
similarly reduced viability in response to cytokines, which was
reversed by coincubation with IGFs (not shown).
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Table 3. Viability of isolated rat pancreatic islets
(percentage of islets containing only viable cells) from rats 46 or
2022 days of age after exposure for 24 or 48 h to IL-1ß (2.5
ng/ml), TNF (10 ng/ml), or IFN (10 ng/ml) without or with IGF-I
or IGF-II (100 ng/ml)
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Table 4. DNA synthetic rate ([3H]thymidine
incorporation, disintegrations per min/µg DNA) of pancreatic islets
from rats 46 or 2022 days of age after exposure for 24 or 48 h
to IL-1ß (2.5 ng/ml), TNF (10 ng/ml), or IFN (10 ng/ml) without
or with IGF-I or -II (100 ng/ml)
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Table 5. Release of insulin (microunits per ml) by isolated
rat pancreatic islets from rats 46 or 2022 days of age after
exposure for 48 h to IL-1ß (2.5 ng/ml), TNF (10 ng/ml), or
IFN (10 ng/ml) without or with IGF-I or -II (100 ng/ml)
|
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Islets from rats 2022 days of age were grown in chamber slides and
exposed to IL-1ß for 48 h, with or without IGF-I or -II (100
ng/ml). Islets were then fixed, and apoptotic nuclei were visualized by
the staining for DNA breakage. Exposure to IL-1ß increased the
incidence of apoptotic cells, and this was significantly reduced after
coculture with IGFs (Fig. 5
and Table 6
). This demonstrated that the cytotoxic
effects of IL-1ß were likely to be mediated at least in part by
apoptosis, and that this could be limited by IGF-I or -II.

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Figure 5. Demonstration of endocrine cell apoptosis
(arrows) using molecular histochemistry in islets
isolated from rats 2022 days age in control medium (A), after
incubation with IL-1ß (2.5 ng/ml; B), or after incubation with
IL-1ß and IGF-I (100 ng/ml; C).
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Table 6. Incidence of islet cells containing apoptotic nuclei
(percentage) in isolated rat pancreatic islets from rats 2022 days of
age after incubation for 48 h with IL-1ß (2.5 ng/ml) without or
with IGF-I or -II (100 ng/ml)
|
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Experiments were repeated with islets isolated from rats on days 46
of postnatal life. Exposure to cytokines alone at the same
concentrations as those stated above did not significantly alter either
islet cell viability or DNA synthetic rate compared with those in
control incubations after 24 or 48 h (Tables 3
and 4
). No further
change in either parameter was seen when incubations included either
IGF-I or IGF-II. Exposure of neonatal islets to cytokines with or
without IGFs did not cause a significant change in insulin release
compared with that in control incubations (Table 5
).
The resistance of islets isolated from neonatal rats to
cytokine-induced cell death may be related to the relatively
greater expression of IGF-II seen in the neonatal rat pancreas than at
older ages. To test this hypothesis, we first established that isolated
islets from neonatal rats continued to express IGF-II after incubation
in vitro for 48 h. Total mRNA was isolated from
approximately 500 islets derived from rats either 14 or 2022 days
old, and IGF-II mRNA was visualized by Northern blot hybridization. A
major transcript of IGF-II mRNA of 4.8 kb was seen for neonatal islets,
which was no longer present at 2022 days of age (Fig. 6
). mRNA transcripts for IGF-I were not
abundant in islets isolated at either age. The presence of endogenous
IGF-II released from neonatal islets was then functionally removed by
incubation with an IGF-II antiserum, and the viability of the islets
was assessed after exposure to cytokines. Islets were isolated from
neonatal rats at 5 days of age and incubated for 48 h in the
presence of IL-1ß (2.5 ng/ml), TNF
(10 ng/ml), or IFN
(10
ng/ml), with or without antiserum against IGF-II or a nonspecific
antibody raised against human placental lactogen. Exposure to each
cytokine alone did not alter islet cell viability (Table 7
). A significant reduction in viability
was seen in response to IL-1ß, TNF
, and IFN
after coincubation
with IGF-II antiserum, but not in the presence of the control
antiserum. These results suggest that an endogenous release of IGF-II
contributed to the resistance of neonatal islets to cytokine-induced
cell death in vitro.

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Figure 6. Northern blot hybridization to demonstrate the
presence of mRNA for IGF-II. Total RNA was isolated from islets of
Langerhans taken from rats 4 or 22 days age. A major transcript of 4.8
kb was found in islets from rats on day 4, but this was undetectable by
day 22. Equivalency of loading, transfer, and RNA integrity is shown by
the abundance of 18S ribosomal RNA.
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Table 7. Viability of isolated rat pancreatic islets
(percentage of islets containing only viable cells) from rats 5 days of
age after exposure for 48 h to IL-1ß (2.5 ng/ml), TNF (10
ng/ml), or IFN (10 ng/ml) without or with antiserum against IGF-II
or control antiserum
|
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Discussion
|
|---|
The fetal and neonatal endocrine pancreas in the rat has a high
degree of plasticity, as ß-cell mass increases in proportion to the
rapid growth rate of the animal. However, only recently has it been
appreciated that substantial remodeling of the endocrine pancreas
occurs in neonatal life, in which ß-cells are deleted by apoptosis,
and ß-cell mass is maintained predominantly by the generation of new
islets from the ductal epithelium. The process of islet cell apoptosis
was first predicted on mathematical grounds (5) and was recently
confirmed by experimental observation (13). A similar phenomenon is
seen in the rat postpartum (46), where the large increase in pancreatic
ß-cells that occurs during pregnancy is reduced by ß-cell apoptosis
postpartum. Clearly, the endocrine pancreas has a much greater capacity
for functional plasticity than was previously thought, at least in
rodents. Our studies confirm those of Scaglia et al. (13) in
showing a peak of cell apoptosis in the rat islets of Langerhans 1317
days after birth. We were unable to positively identify the apoptotic
cells as once being ß-cells because they could not be shown to
contain immunoreactive insulin. This may well have been destroyed
before the terminal events in apoptosis, DNA breakage, which was the
detection criterium used here. The central location of most apoptotic
cells within the islets is consistent with them having once been
functional ß-cells. Apoptotic cells are rapidly removed from tissues
in vivo (47). Our findings that by 14 days after birth over
13% of islet cells were undergoing apoptosis may be an underestimate.
As the incidence of apoptosis increases steadily from postnatal day 6
and begins to fall only after day 14, at least a third of the islet
cells may be lost in this way in neonatal life.
NO production, driven by increased expression of iNOS, has been
implicated in the autoimmune destruction induced by cytokines such as
IL-1ß (14, 48). Other intracellular mechanisms may also exist that
lead to ß-cell apoptosis, including the expression of fas
(49) and the generation of oxygen free radicals (50). In an attempt to
identify possible intracellular mechanisms responsible for the
induction of developmental apoptosis in islets, we examined the
distribution of iNOS. Immunohistochemistry showed the presence of iNOS
in pancreatic islets, with a mean of 10% of cells having
immunopositive cytoplasm on postnatal day 12. The peak incidence of
iNOS presence in islet cells was 2 days before the maximal incidence of
apoptosis, suggesting a possible mechanistic relationship. Dual
staining for insulin and iNOS showed that almost all iNOS-positive
cells were ß-cells, located within the central areas of the islets.
The appearance of iNOS and the occurrence of apoptosis were present in
most islets 1214 days after birth. The events that lead to an
increased distribution of iNOS and increased incidence of apoptosis at
this time are not known. However, it is at this time in the
corresponding ontogeny of the mouse pancreas that infiltration of the
islets with T cells has been shown to occur (51). It is possible that
colonization of the islets with monocytes, macrophages, or T cells in
early postnatal life may lead to rises in the local concentrations of
cytokines such as IL-1ß, leading to a controlled ß-cell deletion by
apoptosis. However, it is also possible that these events require the
removal of a survival factor for ß-cells, and this may be the local
availability of IGF-II.
Exogenous IGFs have been demonstrated to prevent induced apoptosis in a
variety of differing cell types (32, 33, 34, 35) and have been implicated in
the modulation of iNOS activity (37). IGF-I may also prevent apoptosis
by a suppression of IL-1ß-converting enzyme, thus limiting the
availability of bioactive IL-1ß (36). IGF-II has been shown to act as
an autocrine survival factor for differentiating myoblasts (52). We
have previously shown that there is an abrupt decline in the pancreatic
expression of IGF-II mRNA 23 weeks after birth in the rat (16), which
would correlate temporally with the increased incidence of apoptosis.
In the present experiments he have found, by in situ
hybridization and immunohistochemistry, that IGF-II mRNA and peptide
are predominantly associated with the islets in fetal and neonatal life
and disappear with the same ontogeny as total pancreatic IGF-II mRNA.
Although IGF-I mRNA did appear postnatally by day 29, it was
predominantly associated with the ductal epithelium of the pancreas,
rather than islet cells. IGF-I immunoreactivity was, however,
associated with the endocrine pancreas. This may relate to the
sequestration of IGF-I by IGF-binding proteins (IGFBPs). The IGFs are
usually found in association with up to six high affinity binding
proteins (IGFBP-1 to -6) in biological fluids (17), and these are
widely expressed within human and rat fetal tissues. IGFBP-1 mRNA is
undetectable, and IGFBP-2 mRNA is very low in the fetal and neonatal
pancreas (16). Both IGFBP-1 and -2 mRNAs transiently appear in the
pancreas between postnatal weeks 23 and decline in the adult. IGFBP-3
and -4 mRNAs are detected in the pancreas throughout development,
whereas IGFBP-5 or -6 mRNAs are undetectable (16). It is likely that
locally produced IGF-II is more relevant to ß-cell apoptosis than is
circulating IGF-II, as the latter does not fall greatly in the rat
until weaning, coincident with a loss of hepatic IGF-II expression
(53).
Our studies show that IGF-I and IGF-II were both able to reduce the
islet cell death induced by autoimmune cytokines such as IL-1ß and
TNF
in islets from rats at 2022 days of age that no longer
expressed IGF-II mRNA, and this is likely to involve protection against
the induction of apoptosis. Exposure to cytokines also caused a
reduction in the DNA synthetic rate of islets, as reported previously
(54), and a decreased release of insulin. This would be expected in
part because of the induction of cell death. However, the DNA synthetic
rate was decreased 5-fold within 24 h of exposure to cytokines,
which could not be accounted for simply by a reduced cell viability at
this time. IGFs were also able to protect islets from a decrease in the
DNA synthetic rate in response to IL-1ß or TNF
, but did not
reverse the decrease in insulin release. This is in agreement with
previous reports of an inhibitory action of IGFs on insulin release
from isolated islets (55, 56). Islets from rats 45 days of age, which
expressed IGF-II mRNA, showed no reduction in viability in response to
the same concentrations of cytokines, but were susceptible once
endogenous IGF-II had been immunoneutralized. This provides evidence
that endogenous IGF-II can protect neonatal islet cells against
cytokine-induced apoptosis at least in vitro. Although it is
not known whether the developmental apoptosis seen in islet cells
in vivo in the rat after birth involves cytokines, the
experiments at least provide proof of the principle that IGF-II has the
capability to act as a survival factor. Similar effects have been shown
for transforming growth factor-ß (57).
In summary these studies provide a temporal linkage among the decline
in the local expression of IGF-II in pancreatic islets in the rat 23
weeks after birth, the appearance of iNOS in ß-cells, and the peak
incidence of islet cell apoptosis. Further, we provide evidence that
IGF-II is capable of functioning as a survival factor for islets
isolated at this time. It is possible that the developmental apoptosis
of ß-cells represents the deletion of cells with insulin release
characteristics suited for intrauterine life, and their replacement by
new ß-cells with pharmacokinetics of insulin release suited for
postnatal metabolism.
 |
Acknowledgments
|
|---|
We are grateful to Dr. Joanna Hogg for assistance with in
situ hybridization, and to Dr. Dianne Finegood for intellectual
input.
 |
Footnotes
|
|---|
1 This work was supported by the Juvenile Diabetes Foundation, the
Canadian Diabetes Association, and the Medical Research Council of
Canada. 
Received November 6, 1997.
 |
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