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Lawson Research Institute (J.P., E.A., C.C., D.J.H.), St. Joseph’s Health Centre, London, Ontario N6A 4V2, Canada; Departments of Physiology (J.P., C.C., D.J.H.), Medicine (E.A., D.J.H.), and Paediatrics (J.P., D.J.H.), University of Western Ontario, London, Ontario N6A 5A5, Canada; and Laboratoire de Biologie Cellulaire (B.R., C.R., J.J.H.), World Health Organization Collaborating Centre for the Development of the Biology of the Endocrine Pancreas, Université Catholique de Louvain, B-1348 Louvain-La-Neuve, Belgium
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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Introduction |
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Potentially important events in pancreatic islet ontogeny have been recognized in the neonatal rat, which may explain why this is a period of sensitivity to nutritional insult. In the rat fetus islet cell, mass increases rapidly due to both ß cell replication and recruitment, and maturation of undifferentiated ß cell precursors within the pancreatic ducts (9, 10). Following birth, the growth rate of all islet cells, including ß cells, declines within 3–4 days and continues to decline thereafter (10), so that the rate of mitosis in adult pancreatic ß cells is low (11). A wave of apoptosis occurs in neonatal rat islets between 1–2 weeks of age (12), although because the total pancreatic ß cell mass is not substantially changed this suggests that a new population of ß cells derived from replication or neogenesis compensates for the loss. A similar ß cell apoptosis has recently been described in the human fetal pancreas in third trimester (13). This ontogeny of ß cells may signal a change from a population suited to fetal life in which acute insulin release is not necessary within a stable nutritional environment, to ß cells with altered glucose thresholds and the ability to rapidly release insulin under nutritional, endocrine and neural control characteristic of adult metabolic control.
We recently showed that the timing of neonatal ß cell apoptosis in the rat coincided with a loss of expression of insulin-like growth factor-II (IGF-II) within the pancreatic islets, and that IGFs can functionally act as a survival factors to prevent apoptosis in ß cells (14), a role consistent with the actions of IGFs in other cell types (15, 16). There is also considerable evidence that the IGFs contribute to ß cell growth, maturation, and function throughout life, and that their actions can be modulated by locally produced IGF binding proteins (IGFBPs). Messenger RNA for IGF-II is abundant in the pancreas of the fetal rat, and declines rapidly within 2 weeks of birth, whereas IGF-I mRNA expression is low in the fetus, but increases to adult levels by weaning (17). Isolated islets from the human fetus (18), or rat fetus or neonate (19, 20), release IGF-I and/or -II, which are capable of increasing islet cell DNA synthesis (19, 20, 21). The mitogenic action of IGF-II in isolated fetal rat islets was potentiated by IGFBPs-1 and-2, which together with other IGFBPs are expressed there (19). Both IGF-I and -II can also rapidly modulate insulin release from adult rat islets in a biphasic manner (22, 23).
The IGF axis is highly responsive to nutritional status (24, 25). Intrauterine growth restriction in man (26, 27), and experimentally induced growth retardation in the rat fetus (28, 29, 30), including protein restriction (31), is associated with a reduction in circulating IGFs and an altered presence of IGFBPs. Using isolated islets from the fetal rat pancreas or adult hamster, the release of IGFs and IGFBP-1 and -2 were shown to be enhanced by amino acids and/or glucose (19, 32).
Because of the reorganization of the endocrine pancreas during the suckling period, and the role of amino acids in ß cell development, the purpose of this study was to, firstly, examine how LP diet might alter the balance of islet cell replication and survival in the late fetal and neonatal rat endocrine pancreas; and, secondly, to establish if alterations in the pancreatic expression of the IGF axis might be temporally associated with induced developmental changes.
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Materials and Methods |
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At the time of birth, litters from both diet groups were reduced to eight pups, and these remained with the lactating females until they were killed at up to 21 days. Pregnant rats were anesthetized with pentobarbital (55 mg/kg body weight) on days 19.5 or 21.5 gestation. The abdomen and uterus was opened, and fetal blood samples collected through the axillary vessels; the fetus remaining attached to the mother via the placenta throughout the procedure. Postnatally, pups were killed by decapitation. Because of the higher incidence of glucose intolerance in the adult female offspring of rats subjected to the LP diet during pregnancy (8), only female offspring was used in this experiment. Pregnant mothers or pups were injected sc with 5-bromo-2-deoxyuridine (BrdU, 50 mg/kg body weight in sterile saline, Roche Molecular Biochemicals Biochemica, Brussels, Belgium) 1 h before sacrifice to label newly formed DNA. At the time of death the animals were weighed and pancreas removed from each animal and either snap-frozen in liquid nitrogen and stored at -80 C, for RNA isolation, or fixed for immunohistochemistry or in situ hybridization. If pancreata were to be used for histology they were placed in ice-cold fixative (4% paraformaldehyde in 70 mM phosphate buffer, pH 7.4, containing 0.2% glutaraldehyde) for 16 h at 4 C, followed by four washes at 4 C in PBS over a 48-h period. Fixed tissues were dehydrated through a graded ethanol series, impregnated with butanol and embedded in paraffin. All procedures were performed with approval of the animal ethics committees of the Catholic University of Louvain and the University of Western Ontario, and in accordance with the guidelines of the Canadian Council on Animal Care.
Fifty microliters of blood was collected for glucose and precipitated in 500 µl HClO4 (0.33N). Glucose concentrations were measured using a glucose oxidase test kit (Sigma, St. Louis, MO). Plasma was also prepared, and the insulin content measured by RIA (INSIK-5 P2796, Sorin Biomedica, Saluggio, Italy), using rat insulin for the standard curve (Novo Nordisk, Copenhagen, Denmark). Pancreas (15–25 mg) was removed and homogenized in 5 ml acid-ethanol (0.15 M HCl in 75% [vol/vol] ethanol) and extracted overnight at 4 C. The insulin content was determined by RIA and correlated with body weight.
Northern blot analysis
Total RNA was extracted from pancreata as previously described
(17). Before hybridization, the integrity and relative amounts of RNA
from each pancreas were assessed by size separation on 10% agarose TAE
(Tris HCl-EDTA) gels with ethydium bromide. Pancreata in which
ribosomal RNA showed degradation were not used for experiments.
Northern blot hybridization was performed as described previously (17) using between 15 and 20 µg of total RNA following separation by size on agarose gels. Hybridization was performed with 2 x 106 cpm/ml radiolabeled complementary DNA (cDNA) probe for IGF-I or -II, or 1 x 106 cpm/ml for a cDNA encoding 18S ribosomal RNA. Blots were exposed to x-ray film (Kodak XAR-5, Eastman Kodak Co. Inc., Rochester, NY) at -70 C with intensifying screens for up to 10 days before developing, and were hybridized consecutively with cDNAs for IGF-II followed by 18S ribosomal RNA. Between consecutive hybridizations, the blots were stripped with 0.01 x SSC with 0.5% (wt/vol) SDS at 90 C for 1 h to remove the previous labeled cDNA probe.
Radiolabeling of cDNA probes with 
(32P)-dCTP (ICN Biomedicals, Inc., Irvine, CA) was carried out by random priming
using a Pharmacia oligolabelling kit (Pharmacia LKB
Biotechnology, Uppsala, Sweden), to specific activities of 1–2 x
109 dpm/µg. Separation of radiolabelled cDNA from
unincorporated (32P)-dCTP was carried out using
ProbeQuant G-50 microcolumns (Pharmacia). Complimentary DNA probes
used for hybridization of Northern blots and for in situ
hybridization were kindly provided by the following investigators: a
500 bp rat IGF-I cDNA in pGEM Blue (Promega Corp.,
Madison, WI) encoding exon 3 and part of exon 4 of the rIGF-I gene was
provided by Dr. L. Murphy, University of Manitoba, Winnipeg, MT (33); a
807bp mouse IGF-II in pGEM 4z by Dr. G. Bell, University of Chicago,
Chicago, IL; and a rat 18S ribosomal RNA cDNA by Dr. D. Denhardt,
Rutgers University, Piscataway, NJ The latter was used to determine
equality of RNA loading and transfer.
In situ hybridization
We performed in situ hybridization of IGF-I and -II
mRNAs using histological paraffin sections of pancreas as described in
detail by us previously (14). 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 counter-stained 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. Where comparisons were made between pancreata from animals
of different ages all sections were included in the same hybridization
reactions to remove between procedure error.
Antisense riboprobes were prepared from cDNAs for rat (r) IGF-I (a gift
of Dr. L. Murphy, University of Manitoba, Canada), and mouse (m) IGF-II
(a gift of Dr. G. Bell, Univ. of Chicago). The restriction enzymes
(BRL, Burlington, ON) and RNA polymerases (Promega Corp.)
used to linearize the plasmids containing these cDNAs and to generate
[35S]-radiolabelled riboprobes were: antisense rat IGF I,
HindIII/T7; sense rat IGF I, PvuII/SP6; antisense
mouse IGF-II, HindIII/TSP6; sense mouse IGF-II
EcoRI/T7. Radiolabelled cRNA probes were synthesised using
linearized riboprobe DNA, -thio [35S]-UTP, and SP6, T3
or T7 RNA polymerase as described previously (14). To improve cRNA
probe penetration of tissue sections, limited alkaline hydrolysis was
used to reduce transcript size to about 150 bases.
Immunohistochemistry
Histological sections of pancreas (5 µm) were cut from
paraffin blocks and mounted on glass microscope slides (Superfrost
Plus, Fischer Scientific, Nepeon, ON, Canada).
Immunohistochemistry was performed to localize IGF-I or -II, IGFBPs,
inducible nitric oxide synthase (iNOS), BrdU, proliferating cell
nuclear antigen (PCNA), insulin, glucagon, somatostatin, cyclin D1 and
NEK2 within islets by a modified avidin-biotin peroxidase method (34)
as described by us previously for pancreas (14). Slides were incubated
for 48 h at 4 4]C with either rabbit antihuman IGF-I or IGF-II
(1:2000 dilution) (GroPep Pty. Ltd. Ltd., Adelaide,
Australia); rabbit antihuman IGFBP-1, -2, -3, -4 and -5 (all at 1:100
dilution), or monoclonal antibody against human IGFBP-6 (1:500
dilution) (Austral Biologicals, San Remon, CA); guinea pig antiinsulin
antibody (1:500 dilution) (provided by Dr. T. J. McDonald,
University of Western Ontario, London, Ontario, Canada); rabbit
antiporcine glucagon (1:100 dilution) (C-terminal specific 04A
antiserum kindly provided by Dr. R. Ungar, Dallas, TX); rabbit antirat
somatostatin (1:100) (DAKO Corp. Laboratories,
Mississauga, Ontario, Canada); mouse anti-iNOS antiserum (1:50
dilution) (Transduction Laboratories, Inc., Lexington,
KY); mouse antiproliferating cell nuclear antigen (PCNA) (1:750
dilution) (Sigma, St. Louis, MO); mouse anti-BrdU
(labeling kit supplied by Zymed Laboratories, Inc., South
San Francisco, CA); rabbit anti-NEK2 (1 µg/ml dilution) (Zymed Laboratories, Inc.); and mouse anticyclin D1 (1:500 dilution)
(Zymed Laboratories, Inc.). 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). Biotinylated goat
antirabbit IgG (1:100), goat antimouse IgG (1:100), or mouse antiguinea
pig IgG (1:500) (Vector Laboratories, Inc. Burlingame,
CA), were used as secondary antibodies. Peptide immunoreactivity was
localised by incubation in fresh diaminobenzidine tetrahydrochloride
(DAB tablets, 10 mg, Sigma) with 0.03% (vol/vol) hydrogen
peroxide for 2 min and the reaction quenched in excess 50
mM Tris.HCl pH 7.5. Tissue sections were counter-stained
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). All antisera were found to be specific as stated by the supplier with the exception of antiserum against IGFBP-4, which showed cross-reactivity with IGFBP-2. Further controls included substitution of primary antisera with nonimmune serum and omission of the secondary antiserum.
Dual staining for BrdU, PCNA, cyclin D1, or NEK2, 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 then subjected to immunohistochemistry for the second antigen as described with the exception that alkaline phosphatase (blue) was used as the chromogen. Alkaline phosphatase substrate kit III was obtained from Vector Laboratories, Inc. Antimouse alkaline phosphatase conjugate (Sigma) was applied to each section for 1 h at room temperature, sections washed, and alkaline phosphatase substrate applied for 20 min. Sections were washed and counterstained with Mayer’s haemalum.
Visualization of apoptosis
Immunohistochemistry was performed to localize apoptotic nuclei
within histological sections of pancreas (35) using the Apoptag
in situ apoptosis detection kit (Oncor Inc., Gaithersburg,
MD), as described in detail by us previously (14). Staining was
performed according to the manufacturer’s protocol, following
incubation with proteinase K (20 µg/ml; Roche Molecular Biochemicals, Dorval, Québec, 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 as described for
immunohistochemistry, and the tissue counterstained with Carazzi’s
hematoxylin for 1 min. Sections were dehydrated in butanol, cleared in
xylene and mounted with Permount under glass coverslips. Duel staining
for apoptosis and insulin was performed as described above.
Morphometric and statistical analysis
Morphometric analysis was performed using a Carl Zeiss transmitted light microscope at a magnification of x250
or x400. Analyses were performed with Northern Eclipse version 2.0
morphometric analysis software (Empix Imaging Co., Mississauga,
Ontario, Canada). The percentage of islet cells, or the islet cell
area, immunopositive for IGF-I or IGF-II, IGFBPs, insulin, glucagon,
somatostatin, BrdU, PCNA, cyclin D1, NEK2 or iNOS; or demonstrating
apoptotic nuclei, was calculated at each age from up to five sections
of each pancreas representing predominantly the head regions. This was
because we have previously shown that the impact of LP diet on islet
size is greater in the head than in the tail of the pancreas (5).
Sections chosen contained at least five islets, and pancreata from up
to 10 animals were examined for each age. Individual cell area, and
total areas of immunoreactive cells within islets were circled for
image analysis and selected by gray-level threshhold. Pancreatic ß
cell mass was calculated following immunohistochemistry for insulin on
sections obtained throughout pancreata of known weight. The mean ß
cell volume was found to be 650 µm3. ß cell mass was
estimated by calculating the mean percentage area of tissue containing
cells immunoreactive for insulin per sectional area of pancreas (five
to six sections per organ). This was then expressed as mg ß cell mass
based on the total pancreatic wet weight for individual animals. For
Northern blot analysis the ratio of hybridization signal for IGF-II
compared with 18S rRNA was calculated at each age for each of three
separate pancreata following scanning densitometry. Differences between
mean values for variables within individual experiments were compared
statistically by two way ANOVA, followed by a Scheffé’s
test.
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Results |
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). Blood
glucose levels in fetuses of both LP and control fed rats did not
differ significantly at any age. At gestational age 19.5 days the mean
(±SEM) blood glucose value was 46.5 ± 3.5 mg/dl, on
the day of birth 96.8 ± 4.4 mg/dl, and on postnatal day
10, 123.6 ± 2.9 mg/dl (n = 10 per group). Thereafter,
no substantial change was seen up to 21 days of age. Mean plasma
insulin levels did not significantly differ between offspring of LP and
control rats. The combined mean insulin values decreased sharply after
birth in both diet groups, being 8.4 ± 0.7 ng/ml on day 21.5
gestation, 4.6 ± 0.6 ng/ml on postnatal day 2, and 1.8 ±
0.7 ng/ml by postnatal day 21 (n = 10 per group). At postnatal day
21 the pancreatic insulin content of LP rats was significantly less
than in control animals (control 18.3 ± 1.1 µg/pancreas, LP
13.0 ± 1.2, P < 0.05, n = 8) but this
difference was not seen if insulin content was related to body weight
(control 48.6 ± 3.6 µg/100 g body weight, LP 50.8 ±
4.2).
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). The mean islet area was significantly reduced
by more than 50% at each age in the LP group. The mean ß cell mass
per pancreas was calculated and found to be reduced by greater than
50% in LP compared with control rats at fetal and neonatal ages, and
by 70% at postnatal day 14 (Table 1).
Analysis of the abundance of endocrine cell types within the islets
showed that the relative distributions did not alter with age, but that
ß cells containing immunoreactive insulin made up a significantly
smaller area of the islets from LP rats at all ages, whereas cells
containing immunoreactive glucagon constituted a larger area
(P < 0.001) (Table 2).
Consequently, the insulin/glucagon ratio for relative immunoreactive
islet area was decreased at all ages in LP animals (P
< 0.001). The area occupied by somatostatin-containing D cells was not
significantly altered between LP and control rat islets at most ages
(Table 2). Individual endocrine cells did not differ in size between
control and LP animals, suggesting that the above results reflected
differences in relative cell number. When apoptosing cells within
islets were also considered (Fig. 3D), which did not show
immunoreactivity for any pancreatic endocrine hormone, over 93% of
islet cells could be accounted for, the balance probably reflecting
pancreatic polypeptide-containing cells that were not analyzed. Thus,
the islets in LP rats were smaller and contained a relatively smaller
proportion of ß cells compared with controls throughout fetal and
neonatal life.
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, A and B).
The overall labeling index of islet cells with BrdU was 4–5% in
control fed animals, and did not alter with age. The percent islet
cells labeled with BrdU in LP rats was significantly lower (control
4.4 ± 0.1%, LP 2.6 ± 0.1%, n = 15 representing five
animals each at gestational day 21.5 and postnatal days 14 and 21,
P < 0.001). Dual staining for insulin and BrdU showed
that most cells, which labeled with BrdU were ß cells, that this
proportion did not change with age, but that a significantly lower
percentage of cells that were BrdU-positive in LP animals were
identified as ß cells (control 78.0 ± 2.3%, LP 62.1 ±
3.5%, P < 0.001, n = 50 animals for control and
101 for LP). This suggests that the labeling of ß cells with BrdU was
in proportion to their relative abundance in the islets of both control
and LP animals.
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). When
only ß cells were considered, the percentage of ß cells that
demonstrated immunoreactivity for PCNA and insulin was also
significantly greater in islets of LP animals and did not significantly
differ with age (control 2.3 ± 0.8%, LP 3.6 ± 0.9%,
P < 0.001, n = 63 for control and 60 for LP).
Approximately 60–70% of cells containing PCNA immunoreactivity were
ß cells, and this did not significantly alter with age or with diet
(not shown).
To further delineate cell cycle events in islet cells in
situ, immunohistochemistry was performed for cyclin D1 and for
NEK2. Cyclin D1 is associated with G1 phase of the cell cycle, whereas
NEK2 has a cell cycle-dependent expression that is maximal in G2 and M.
Cyclin D1 was visualized in the cytoplasm of a minority of islet cells,
and in some acinar epithelial cells (Fig. 2, C and D). The percentage
of ß cells that demonstrated immunoreactivity for cyclin D1 did not
change with age in control-fed animals, but was significantly greater
in LP-fed animals than in the control animals in late fetal life and at
postnatal days 14 and 17 (Fig. 3B). NEK2 immunoreactivity was seen in
the nuclei of a minority of islet cells and in clusters of acinar cells
at all ages (Fig. 2, E and F). In rats given LP diet the percentage of
ß cells demonstrating NEK2 immunoreactivity was significantly reduced
between postnatal days 10 and 17 (Fig. 3C). Thus, indicators of S phase
transition and G2/M of the cell cycle for ß cells (BrdU and NEK2)
showed a decreased incidence in LP rats postnatally, while indicators
of G1 phase (PCNA and cyclin D1) demonstrated an increased
incidence.
Apoptosis
The occurrence of apoptosis within islet cells at each age was
examined by molecular histochemistry using the TUNEL method. Apoptosing
cells were restricted to the central, ß-cell rich region of the
islets (Fig. 2, G and H). In the pancreas of control animals the
proportion of islet cells undergoing apoptosis was less than 2%,
before postnatal day 10, but a transient rise in islet cell apoptosis
occurred between days 12 and 17, which was maximal at 8% on postnatal
day 14 (Fig. 3D). In offspring of rats receiving the LP diet, the
incidence of islet cell apoptosis was significantly higher at every age
examined, including the transient neonatal rise on days 12–17.
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. Because
we previously showed that there was a transient increase in the number
of ß cells containing iNOS immediately before the neonatal rise in
ß cell apoptosis (14), we compared the number of islet cells
immunopositive for iNOS between control-fed and LP rats. Approximately
1% of islet cells were immunoreactive for iNOS in late fetal life,
rising to a transient peak of 14% on postnatal day 12, which
subsequently declined to 5% on postnatal day 21. No significant
difference was found between control and LP fed rats.
IGF axis
To determine if the altered proliferation and survival of islet
cells seen in rats fed LP diet were associated with alterations to the
IGF axis in pancreas, the abundance of mRNA for IGF-II was determined
in whole pancreata using Northern blot hybridization. Three mRNA
transcripts for IGF-II were detected of 7.5 kb, 4.4 kb, and 2.4 kb in
pancreata of both control and LP fed rats. In control fed animals the
expression of all IGF-II mRNA transcripts declined postnatally and was
barely detectable after postnatal day 14 (Fig. 4). In LP fed rats, the expression IGF-II
was reduced compared with controls at all ages. IGF-II mRNA expression
was expressed relative to that of 18S ribosomal RNA in the same samples
for three additional, separate hybridizations using pancreata from
different animals and is shown in Fig. 5, A and B, for the two larger mRNA transcripts. This confirmed that the
expression of IGF-II mRNA was reduced in the pancreata of LP fed rats,
this being most pronounced for the 7.4 kb transcript. To determine
whether the sites of expression of IGF-II in pancreas were altered in
the neonatal LP fed animals, mRNA was visualized by in situ
hybridization. IGF-II mRNA was located predominently in islet cells,
and at postnatal day 2 was less abundant in pancreas sections from LP
compared with control-fed rats (Fig. 6, A
and B). IGF-I mRNA was barely detectable using in situ
hybridization in the islets of fetal or neonatal rats. By postnatal day
21 IGF-I mRNA was located within acinar cells, but did not obviously
differ between dietary groups (not shown).
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, D and E), but declined in intensity and in the number of
immunopositive cells postnatally, to become barely detectable by
postnatal day 21 (Fig. 5C). Rats fed LP diet showed a significantly
reduced area of islets containing immunoreactive IGF-II compared with
control-fed animals. Immunoreactivity for IGF-I was low in the fetal
pancreas (day 21.5 gestation, control 8.6 ± 0.4% islet cell
area, n = 12) but appeared postnatally in association with islet
cells and some acinar epithelial cells (day 21, 13.7 ± 0.2%
islet cell area, n = 20). The area of islets immunopositive for
IGF-I did not significantly differ between control and LP animals at
any age. The presence of IGFBPs in the pancreas was also investigated
using immunohistochemistry. Immunoreactive IGFBP-1 was diffusely
present in islet cells in the fetus, and abundance declined with age
(not shown). IGFBP-2 immunoreactivity was predominantly associated with
the outer mantle of islet cells, comprising mostly cells, and
vascular endothelium and did not change greatly with age.
Immunoreactivity for IGFBP-3 and -4 was seen at all ages within the
pancreas, within islets and mesenchymal tissue, but not in most acinar
cells, whereas IGFBP-5 was seen at all ages associated with the
cells of the islets and with ductal epithelial cells. No differences
were found between the area of islets immunopositive for any IGFBP at
any age between control-fed and LP-fed rats. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, and
somatostatin-secreting cells, although the ß cell population was most
severely effected. If nutritional restriction is reversed at birth or
maintained until adulthood the islet morphology remains unaltered (8).
In both situations lower insulin secretion is apparent in adult life,
although this is more severe if LP diet is continued to adulthood. This
suggests that LP diet causes a fundamental reprogramming of ß cell
phenotype with critical periods both in fetal and neonatal life. Our
results suggest some possible mechanisms as to how this may occur. We showed previously that feeding an LP diet throughout gestation resulted in a lower nuclear labeling index with [3H] thymidine in islet cells of the newborn (5), findings reproduced here with BrdU. Labeling with BrdU in ß cells within islets from control-fed animals was consistent with the fractional contribution of ß cells to these islets, demonstrating an equivalent labeling index for ß and nonß cell types. In animals fed LP diet significantly less labeling with BrdU was located in ß cells, consistent with their reduced fractional area, and suggesting that the reduction in islet size is preferentially linked to a deficit in ß cell proliferation. The fraction of cells containing immunoreactivity for PCNA, either in all islet cells or specifically in the ß cell population was, paradoxically, increased at every age up to postnatal day 17 in LP animals. PCNA is an auxiliary protein of DNA polymerase that begins to accumulate in the nucleus during G1 of the cell cycle, is most abundant in S phase and declines during G2/M (40). The observation that more ß cells contained detectable PCNA following LP diet, despite less cells being observed in S phase by incorporation of BrdU, could be explained by an altered cell cycle kinetics in which either G1 or G2/M phases were extended in LP animals. Alternatively, because PCNA is also expressed during DNA repair (41), it is possible that islets from LP-fed rats were subject to oxidative, or some other physiological stress. To distinguish between these alternatives we further examined immunoreactivity for cyclin D1 and for NEK2. Cyclin D1 is a nuclear protein that accumulates in G1 and is necessary for transition from G1 to S phase of the cell cycle (42). It associates with PCNA and cyclin-dependent kinase 2 (43). NEK2 is a serine/threonine kinase that accumulates at the G2 to M phase transition and is necessary for progression into cell mitosis (44). The fractional increase of ß cells in LP rats containing immunoreactive cyclin D1, and a decrease of NEK2 staining would be consistent with a lengthened mean cell cycle duration in ß cells within islets from LP rats, in which the G1 phase of the cycle was extended. However, direct confirmation of this would require precise cell cycle kinetic analysis using isolated ß cells from these animals.
A modulation of mean islet area by diet might involve a change in the rate of cell attrition as well as altered proliferative rate. A low rate of islet cell apoptosis exists in the fetal rat islet, but this transiently increases at 14 to 17 days postnatal life, to return to minimal levels by weaning (12, 14). The rate of islet cell apoptosis was increased at every age examined in LP rats between late fetal life and weaning, compared with control-fed animals. However, the timing of the neonatal wave of apoptosis was not altered, suggesting that this event is qualitatively independent of diet of the mother. It is not possible to directly show that the apoptozing islet cells are ß cells because they no longer contain immunoreactive insulin. As we have described previously (14), this may be due to a loss of cytoplasm rendering any immunohistochemical signal difficult to visualize. However, their predominantly central location within the islets is consistent with the majority having been ß cells. In addition, we have previously demonstrated that the majority of apoptotic islet cells express the glucose transporter protein GLUT2 on the plasma membrane, suggesting that they are derived from ß cells (45). The percent of islet cells undergoing apoptosis in late fetal and early neonatal life is lower than we reported in a previous study of ontogeny, although the ontological pattern is identical (14). This may be due to the use of a separate, inbred rat colony in the present study. We have shown previously that the neonatal wave of islet cell apoptosis is preceded by a transient increase in the presence of iNOS within ß cells (14), which may be functionally linked to the developmental apoptosis. While a similar rise in islet cell iNOS immunoreactivity was seen in the LP animals, the percentage of immunopositive islet cells did not differ between control and LP diets. This suggests that the mechanisms underlying an increased rate of islet cell apoptosis seen in LP animals are unlikely to be due to increased detectable iNOS.
The transient increase in islet cell apoptosis in the neonatal rat is temporally related to a rapid loss of expression of IGF-II mRNA from the pancreas, where it is predominantly expressed within islet cells (14, 17), and we have shown that endogenous IGF-II acts as a survival factor that prevents apoptosis in isolated islets from 5-day old rats (14). While an increasing expression of IGF-I mRNA occurs in the pancreas postnatally, this is located mainly in the acinar tissue, not in the islets of Langerhans (14). In the pancreata from LP rats, a reduced expression of IGF-II mRNA was found at fetal and neonatal ages, before the developmentally associated decline in IGF-II expression that occurs from day 14. It is not known if these changes reflect an effect of diet on gene transcription or on the stability of the mRNAs. For animals receiving both control and LP diet, the major sites of IGF-II expression in the pancreas, assessed by in situ hybridization, were the islets. The number of islet cells that were immunoreactive for IGF-II was also significantly reduced in LP animals, confirming that differences are also likely to be present at the level of the translated protein. These findings are consistent with an action of endogenous IGF-II as a survival factor for pancreatic ß cells, and suggest that the increased apoptosis seen in islet cells in LP rats may be functionally linked to a reduced expression of IGF-II. Because IGF-II will also increase islet cell DNA synthesis (19, 46), its relative paucity in the islets of LP rats may also contribute directly to reduced ß cell proliferation rate and the reduction in islet size. It has been recently proposed that amino acids, and in particular branched-chain amino acids, may promote ß cell proliferation by stimulating phosphorylation of PHAS-1 (a regulator of translation initiation during mitogenesis) and by facilitating the proliferative effects mediated by growth factors (47). Branched-chain amino acids are most severely depleted in the plasma of fetuses from LP-fed mothers (4), which could also contribute to the lower rate of ß cell proliferation in the pancreas of the LP pups.
An effect of LP diet on pancreatic IGF-II expression may be direct. We showed previously that the release of immunoreactive IGF-II from isolated fetal rat islets was potentiated by amino acids, but not by glucose, whereas the release of insulin was increased by both (19). Circulating levels of IGF-II in the rat fetus, which derive predominantly from an hepatic expression, are not substantially decreased during models of IUGR, although the much lower levels of circulating IGF-I are depressed (29, 30). This would suggest that the development of the pancreatic islets is more sensitive to changes in locally expressed IGF-II than to circulatory changes, and that a paracrine or autocrine action of IGF-II is dominant in this tissue. No significant changes were seen in the fractional area of islets demonstrating immunoreactivity for IGF-I or IGFBPs in LP-fed animals. We have shown that increased concentrations of amino acids will increase IGFBP-1, -2, and -3 release from isolated fetal rat islets (19), while the release of IGFBP-1 and -2 from islets is glucose-responsive in the fetal rat or adult hamster (19, 32). Mice expressing the human IGFBP-1 transgene have an increased pancreatic islet size with relatively more ß cells (48). It is possible that any changes in IGFBP abundance within the islets of LP rats were too subtle to be detected by immunohistochemistry alone, and an influence of diet on IGFBP synthesis and secretion from islet cells is still possible.
In summary, the experiments suggest that LP diet causes a selective deficiency of ß cells in the fetus and neonate due to the combined effects of a reduced ß cell proliferation rate, which may involve an altered cell cycle length, and an increased incidence of apoptosis. These changes are associated with a decreased expression of IGF-II within the pancreatic islets, a growth factor known to act as a ß cell mitogen and to prevent apoptosis. It is therefore possible that perturbations of prenatal or neonatal nutrition and growth will lead to inappropriate ß cell ontogeny in the preweaning period, and result in a population of ß cells that may subsequently be functionally compromised in later life, contributing to glucose intolerance.
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Received March 12, 1999.
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*, P < 0.001
vs. control diet.
