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Endocrinology Vol. 139, No. 6 2994-3004
Copyright © 1998 by The Endocrine Society

ARTICLES

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. Joseph’s 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. Joseph’s Health Center, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: dhill{at}lri.stjosephs.london.on.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 20–22 days after birth, islet cell viability, DNA synthetic rate, and insulin release were reduced after incubation with interleukin-1ß, tumor necrosis factor, or interferon-{gamma}. 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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-{gamma} (IFN{gamma}) (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 1–2 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 {alpha}- 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 1–2 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 4–5 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 5–8 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 48–72 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 (60–80 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-{alpha} (TNF{alpha}; 10 ng/ml), IFN{gamma} (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 (1.5–24 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 Carazzi’s 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 Mayer’s 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 manufacturer’s 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 15–20 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 [{alpha}-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 1–2 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 Denhardt’s 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 Carazzi’s 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 {alpha}-thio-[35S]UTP (400 Ci/mmol; New England Nuclear, Billerica, MA), 0.3 µl sterile water, and 1 µl (5–10 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1Go). 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 1Go). 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

 
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. 2AGo). 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. 2BGo). 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 2Go). 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

 
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. 3AGo) 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. 3Go, 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. 4Go). 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. 3CGo), and only a low level of mRNA was detected in islets after weaning (Fig. 3DGo). 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. 4Go). 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.

 
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 20–22 days or from adult female rats were incubated in the presence of single concentrations of IL-1ß (2.5 ng/ml), TNF{alpha} (10 ng/ml), or IFN{gamma} (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 20–22 days of age incubated in the presence of each cytokine alone was significantly lower than that in control cultures for TNF{alpha} and IFN{gamma}, 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{alpha}, or IFN{gamma}, each cytokine alone caused a 60–90% significant reduction in islet cell viability after 48 h (Table 3Go). Coincubation with either IGF-I or IGF-II significantly reduced cell mortality in the presence of IL-1ß or TNF{alpha}, but not in the presence of IFN{gamma}. Analysis of the DNA synthetic rate showed that under control conditions there was incorporation of [3H]thymidine into islet cells of rats at 20–22 days postnatal age, suggesting a potential for mitogenesis (Table 4Go). Exposure to IL-1ß, TNF{alpha}, or IFN{gamma} 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{alpha}, but not with IFN{gamma}. 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 5Go). 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 4–6 or 20–22 days of age after exposure for 24 or 48 h to IL-1ß (2.5 ng/ml), TNF{alpha} (10 ng/ml), or IFN{gamma} (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 4–6 or 20–22 days of age after exposure for 24 or 48 h to IL-1ß (2.5 ng/ml), TNF{alpha} (10 ng/ml), or IFN{gamma} (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 4–6 or 20–22 days of age after exposure for 48 h to IL-1ß (2.5 ng/ml), TNF{alpha} (10 ng/ml), or IFN{gamma} (10 ng/ml) without or with IGF-I or -II (100 ng/ml)

 
Islets from rats 20–22 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. 5Go and Table 6Go). 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 20–22 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 20–22 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)

 
Experiments were repeated with islets isolated from rats on days 4–6 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 3Go and 4Go). 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 5Go).

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 1–4 or 20–22 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 20–22 days of age (Fig. 6Go). 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{alpha} (10 ng/ml), or IFN{gamma} (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 7Go). A significant reduction in viability was seen in response to IL-1ß, TNF{alpha}, and IFN{gamma} 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{alpha} (10 ng/ml), or IFN{gamma} (10 ng/ml) without or with antiserum against IGF-II or control antiserum

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 13–17 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 12–14 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 2–3 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 2–3 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{alpha} in islets from rats at 20–22 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{alpha}, 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 4–5 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 2–3 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. Back

Received November 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hill DJ, Hogg J 1991 Growth factor control of pancreatic ß cell hyperplasia. In: Herington A (ed) Clinical Endocrinology and Metabolism. Bailliere Tindall, London, pp 689–698
  2. Kaung HL 1994 Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat. Dev Dyn 200:163–175[Medline]
  3. Fernandes A, King LC, Guz Y, Stein R, Wright CVE, Teitelman G 1997 Differentiation of new insulin-producing cells is induced by injury to adult pancreatic islets. Endocrinology 138:1750–1762[Abstract/Free Full Text]
  4. Dudek RW, Lawrence IE 1988 Morphologic evidence of interaction between adult ductal epithelium of pancreas and fetal foregut mesenchyme. Diabetes 37:891–900[Abstract]
  5. Finegood DT, Scaglia L, Bonner-Weir S 1995 Dynamics of ß cell mass in the growing rat pancreas. Diabetes 44:249–256[Abstract]
  6. Cantenys D, Portha B, Dutrillaux MC, Hollande E, Roze C, Picon L 1981 Histogenesis of the endocrine pancreas in newborn rats after destruction by streptozotocin. Virchows Arch Cell Pathol 35:109–122
  7. Bencosme SA 1955 Cytology of the islet cells in alloxan diabetic rabbits. Am J Pathol 31:1149–1164
  8. Brockenbrough JS, Weir GC, Bonner-Weir S 1988 Discordance of exocrine and endocrine growth after 90% pancreatectomy in rats. Diabetes 37:232–236[Abstract]
  9. Gu D, Sarvetnick N 1993 Epithelial cell proliferation and islet neogenesis in IFN-{gamma} transgenic mice. Development 118:33–46[Abstract]
  10. Cecil RL 1911 On hypertrophy and regeneration of the islands of Langerhans. J Exp Med 14:500–519[Abstract]
  11. Gepts W, de Mey J 1978 Islet cell survival determined by morphology. An immunocytochemical study of the islet of Langerhans in juvenile diabetes mellitus. Diabetes [Suppl 1] 27:251–261
  12. Volk BW, Wellman KF 1985 The pancreas in idiopathic diabetes. In: Volk BW, Arquilla ER (eds) The Diabetic Pancreas, ed 2. Plenum Press, New York, p 353
  13. Scaglia L, Cahill CJ, Finegood DT, Bonner-Weir S 1997 Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal rat. Endocrinology 138:1736–1741[Abstract/Free Full Text]
  14. Tornehave D, Larsson L-I 1997 Presence of Bcl-Xl during development of the human fetal and rat neonatal endocrine pancreas: correlation to programmed cell death. Exp Clin Endocrinol Diabetes 105:A27
  15. Corbett JA, McDaniel ML 1995 Intraislet release of interleukin-1 inhibits ß cell function by inducing ß cell expression of inducible nitric oxide synthase. J Exp Med 181:559–568[Abstract/Free Full Text]
  16. Hogg J, Hill DJ, Han VKM 1994 The ontogeny of insulin-like growth factor (IGF) and IGF binding protein gene expression in the rat pancreas. J Mol Endocrinol 13:49–58[Abstract]
  17. Calvo EL, Bernatchez G, Pelletier G, Iovanna JL, Morisset J 1997 Downregulation of IGF-I mRNA expression during postnatal pancreatic development and overexpression after subtotal pancreatectomy and acute pancreatitis in the rat pancreas. J Mol Endocrinol 18:233–242[Abstract]
  18. Hill DJ, Hogg J 1992 Expression of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) during pancreatic development in rat, and modulation of IGF actions on rat islet DNA synthesis by IGF BPs. In: Vinik A (ed) Pancreatic Islet Cell Regeneration and Growth. Plenum Press, New York, pp 113–120
  19. Hill DJ, Clemmons DR 1992 Similar distribution of insulin-like growth factor binding proteins-1, -2 and -3 in human fetal tissues. Growth Factors 6:315–326[Medline]
  20. Hill DJ, Frazer A, Swenne I, Wirdnam PK, Milner RDG 1987 Somatomedin-C in the human fetal pancreas: cellular localization and release during organ culture. Diabetes 36:465–471[Abstract]
  21. Hogg J, Han VKM, Clemmons DR, Hill DJ 1993 Interactions of glucose, insulin-like growth factors (IGFs) and IGF binding proteins in the regulation of DNA synthesis by isolated fetal rat islets of Langerhans. J Endocrinol 138:401–412[Abstract]
  22. Swenne I, Hill DJ, Strain AJ, Milner RDG 1987 Growth hormone regulation of somatomedin-C/insulin-like growth factor I production and DNA replication in fetal rat islets in tissue culture. Diabetes 36:288–294[Abstract]
  23. Scharfmann R, Corvol M, Czernichow P 1989 Characterization of IGF I produced by fetal rat pancreatic cells. Diabetes 38:686–690[Abstract]
  24. Romanus JA, Rabinovitch A, Rechler MM 1985 Neonatal rat islet cell cultures synthesize insulin-like growth factor I. Diabetes 34:696–702[Abstract]
  25. Rabinovitch A, Quigley C, Russel T, Patel Y, Mintz DH 1982 Insulin and multiplication stimulating activity monolayer cultures. Diabetes 31:160–164[Abstract]
  26. Van Schravendijk CF, Foriers A, Van Den Brande JL, Pipeleers DG 1987 Evidence for the presence of type I insulin-like growth factor receptors on rat pancreatic A and B cells. Endocrinology 121:1784–1788[Abstract]
  27. Fehmann HC, Jehle P, Markus U, Goke B 1996 Functional receptors for insulin-like growth factors-I (IGF-I) and IGF-II on insulin-, glucagon-, and somatostatin-producing cells. Metabolism 45:759–766[CrossRef][Medline]
  28. Swenne I, Hill DJ 1989 Growth hormone regulation of DNA replication, but not insulin production is mediated by somatomedin-C/insulin-like growth factor I in isolated pancreatic islets from adult rats. Diabetologia 32:191–197[CrossRef][Medline]
  29. Hoog A, Grimelius L, Falkmer S, Sara VR 1993 A high molecular weight IGF-2 immunoreactive peptide (pro-IGF-2?) in the insulin cells of the islets of Langerhans in pancreas of man and rat. Regul Pept 47:275–283[CrossRef][Medline]
  30. Maake C, Reinecke M 1993 Immunohistochemical localization of insulin-like growth factor 1 and 2 in the endocrine pancreas of rat, dog, and man, and their coexistance with classical islet hormones. Cell Tissue Res 273:249–259[CrossRef][Medline]
  31. Asfari M, Wei D, Noel M, Holthuizen PE, Czernichow P 1995 IGF-II gene expression in a rat insulin-producing ß-cell line (INS-1) is regulated by glucose. Diabetologia 38:927–935[Medline]
  32. Geier A, Haimshon M, Beery R, Lunenfeld B 1992 Insulin-like growth factor-I inhibits cell death induced by cycloheximide in MCF-7 cells - a model system for analyzing control of cell death. In Vitro Cell Dev Biol Anim 28A:725–729
  33. Galli C, Meucci O, Scorziello A, Werge TM, Calissano P, Schettini G 1995 Apoptosis in cerebellar granule cells is blocked by high KCl, forskolin, and IGF-I through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis. J Neurosci 15:1172–1179[Abstract]
  34. Chun SY, Billig H, Tilly JL, Furuta I, Tsafriri A, Hsueh AJW 1994 Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor I. Endocrinology 135:1845–1853[Abstract]
  35. Muta K, Krantz SB 1993 Apoptosis of human erythroid colony-forming cells is decreased by stem cell factor and insulin-like growth factor I as well as erythropooetin. J Cell Physiol 156:264–271[CrossRef][Medline]
  36. Jung Y, Miura M, Yuan J 1996 Suppression of IL-1ß converting enzyme-mediated cell death by insulin-like growth factor. J Biol Chem 271:5112–5117[Abstract/Free Full Text]
  37. Guan J, Williams C, Gunning M, Mallard C, Gluckman P 1993 The effects of IGF-I treatment after hypoxic-ischaemic brain injury in adult rats. J Cerebr Blood Flow Metab 13:609–616[Medline]
  38. Hellerstrom C. Lewis NJ, Borg H, Johnson R, Freinkel N 1979 Method for large scale isolation of pancreatic islets by tissue culture of fetal rat pancreas. Diabetes 28:769–776[Medline]
  39. Hales CN, Randle PJ 1963 Immunoassay of insulin with insulin antibody precipitate. Biochem J 88:137–146[Medline]
  40. Herbert V, Lau K, Gottlieb CW, Bleicher SJ 1965 Coated charcoal immunoassay of insulin. J Clin Endocrinol 25:1375–1384[Medline]
  41. Schnuerer EM, Rökaeus Å, Carlquist M, Bergman T, Dupré J, McDonald TJ 1990 Rat and porcine galanin are equipotent in inhibiting insulin responses to glucose in the anesthetized rat. Pancreas 5:70–74[Medline]
  42. Hsu SM, Raine L, Fanger H 1981 Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29:577–580[Abstract]
  43. Wijsman JH, Jonker RR, Keijzer R, Van de Velde CJ, Cornelisse CJ, Van Dierendonck JH 1993 A new method to detect apoptosis in paraffin sections: in situ end labelling of fragmented DNA. J Histochem Cytochem 41:7–12[Abstract]
  44. Southern EM 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–507[CrossRef][Medline]
  45. Murphy LJ, Tachibana K, Friesen HG 1988 Stimulation of hepatic insulin-like growth factor-I gene expression by ovine prolactin: evidence for intrinsic somatogenic activity in the rat. Endocrinology 122:2027–2033[Abstract]
  46. Scaglia L, Smith FE, Bonner-Weir S 1995 Apoptosis contributes to the involution of ß cell mass in the postpartum rat pancreas. Endocrinology 136:5461–5468[Abstract]
  47. Bursch W, Kleine L, Tenniswood M 1990 Biochemistry of cell death by apoptosis. Biochem Cell Biol 68:1071–1074[Medline]
  48. Kenato H, Fujii J, Seo HG, Suzuki K, Matsuoka T, Nakamura M, Tatsumi H, Yamasaki Y, Kamada T, Taniguchi N 1995 Apoptotic cell death triggered by nitric oxide in pancreatic ß-cells. Diabetes 44:733–738[Abstract]
  49. Yamada K, Takane-Gyotuku N, Yuan X, Ichikawa F, Inada C, Nonaka K 1996 Mouse islet cell lysis mediated by interleukin-1-induced Fas. Diabetologia 39:1306–1312[CrossRef][Medline]
  50. Rabinovitch A, Suarez WL, Thomas PD, Strynadka K, Simpson I 1992 Cytotoxic effects of cytokines on rat islets: evidence for involvement of free radicals and lipid peroxidation. Diabetologia 35:409–413[CrossRef][Medline]
  51. Yang Y, Charlton, B, Shimada A, dal Canto RD, Fathman G 1996 Monoclonal T cells identified in early NOD islet infiltrates. Immunity 4:189–194[CrossRef][Medline]
  52. Stewart CE, Rotwein P 1996 Insulin-like growth factor-II is an autocrine survival factor for differentiating myoblasts. J Biol Chem 271:11330–11338[Abstract/Free Full Text]
  53. Brown AL, Graham DE, Nissley SP, Hill DJ, Strain AJ, Rechler MM 1986 Developmental regulation of insulin-like growth factor II mRNA in different rat tissues. J Biol Chem 261:13144–13150[Abstract/Free Full Text]
  54. Mandrup-Poulsen T, Bendtzen K, Nerup J, Dinarello CA, Svenson M, Nielsen JH 1986 Affinity purified human interleukin 1 is cytotoxic to islet cells of Langerhans. Diabetologia 29:63–67[CrossRef][Medline]
  55. Van Schravendijk CFH, Heylen L, Van Den Brande JL, Pipeleers DG 1990 Direct effect of insulin and insulin-like growth factor-I on the secretory activity of rat pancreatic beta cells. Diabetologia 33:649–653[CrossRef][Medline]
  56. Hill DJ, Sedran RJ, Brenner SL, McDonald TJ 1997 Insulin-like growth factor-I (IGF-I) has a dual effect on insulin release from isolated, perifused adult rat islets of Langerhans. J Endocrinol 153:15–25[Abstract]
  57. Cunningham JM, Mabley JG, Mwebe B, Kovani E, Green IC 1994 Suppression of interleukin-1ß-induced nitric oxide synthase activity by transforming growth factor-ß in the insulin-secreting cell line RINm5F. J Endocrinol [Suppl] 140:37



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