Most cases of hyperinsulinism of infancy (HI) are caused by mutations in either the sulfonylurea receptor-1 (SUR1) or the inward rectifying K+ channel Kir6.2, two subunits of the β-cell ATP-sensitive K+ channel (KATP channel). Histologically, HI can be divided into two major subtypes. The diffuse form is recessively inherited and involves all β-cells within the pancreas. Focal HI consists of adenomatous hyperplasia within a limited region of the pancreas, and it is caused by somatic loss of heterozygosity (LOH), including maternal Ch11p15-ter in a β-cell precursor carrying a germ-line mutation in the paternal allele of SUR1 or Kir6.2. Several imprinted genes are located within this chromosomal region, some of which, including p57KIP2 and IGF-II, have been associated with the regulation of cell proliferation. Using double immunostaining, we examined p57KIP2 expression in different islet cell types, in control pancreases from different developmental stages (n = 15), and in pancreases from patients with both diffuse (n = 4) and focal HI (n = 9). Using immunofluorescence and computerized image analysis, we quantified IGF-II expression in β-cells from patients with focal HI (n = 8). Within the pancreas, p57KIP2 was specifically localized to the endocrine portion. β-Cells demonstrated the highest frequency of expression (34.9 ± 2.7%) compared with ∼1–3% in other cell types. The fraction of β-cells expressing p57KIP2 did not vary significantly during development. β-Cells within the focal lesions did not express p57KIP2, whereas IGF-II staining inside focal lesions was mildly increased compared with unaffected surrounding tissue. In conclusion, we demonstrate that p57KIP2 is expressed and is paternally imprinted in human pancreatic β-cells. Loss of expression in focal HI is caused by LOH and is associated with increased proliferation and increased IGF-II expression. Manipulation of p57KIP2 expression in β-cells may provide a mechanism by which proliferation can be modulated, and thus this gene is a potential therapeutic target for reversing the β-cell failure observed in diabetes.

Hyperinsulinism of infancy (HI) is a rare genetic disorder with a prevalence in outbred populations of ∼1/50,000 live births (1,2). An incidence as high as 1/2,500 has been reported in inbred populations (2,3). The molecular basis of the disease was recently elucidated, and most cases are caused by mutations in either the sulfonylurea receptor-1 (SUR1) gene ABCC8 or the inward rectifying K+ channel Kir6.2 gene KCNJ11, the two subunits of the β-cell ATP-sensitive K+ channel (KATP channel) (4,5,6,7). A minority of patients have glucokinase or glutamate dehydrogenase-1 mutations, whereas in 40–50% of the patients, the genetic cause of the disease is still not known (5,8,9,10). The clinical presentation of HI can be variable, ranging from mild disease to severe, life-threatening hypoglycemia that, if not adequately treated, causes irreversible neurological damage (11,12).

The histological appearance of the pancreases from affected children is heterogeneous and can be subdivided into two major forms: diffuse HI and focal HI (13,14,15). The diffuse form is more common and bears some histological characteristics of nesidioblastosis, a normal phenomenon observed in the fetus and newborn that includes poorly defined islets, small clusters of endocrine cells scattered throughout the exocrine tissue, and a high frequency of endocrine cells interposed between ductular cells (15,16,17).

Focal HI can generally be recognized as a discrete region of adenomatous hyperplasia, often too small to be identified macroscopically. Histologically, the lesion is comprised of nodules of endocrine and exocrine elements. The β-cells are pleomorphic, some having giant nuclei and abundant cytoplasm (13). The rest of the pancreas has normal endocrine architecture for age, with β-cells containing small nuclei and shrunken cytoplasm (18).

We have previously reported increased frequencies of proliferating β-cells in pancreases from HI patients and in pancreases in early stages of human development. Focal HI presented the highest proliferation frequency compared with diffuse HI and control subjects (19). The mechanisms regulating the rate of β-cell proliferation are not known; however, the genetic alteration in focal HI may provide an insight into the control of β-cell turnover.

Focal HI is caused by the somatic loss of part of the short arm of maternal chromosome 11 in a β-cell precursor of a patient carrying a mutant SUR-1 gene on the paternal allele (20,21). In all cases, it is the paternal allele that carries the mutation and the maternal allele that is somatically lost, suggesting that the gene(s) responsible for the focal proliferation is imprinted. A large number of genes are located in the lost portion of Ch11p, including p57KIP2, H19, IGF-II, and a p53-induced protein with a death domain (Pidd). Pidd is a 910–amino acid protein induced by a tumor suppressor (p53) that promotes apoptosis (22). It is not known whether this gene is imprinted or whether it is expressed in β-cells. IGF-II is imprinted, with only the paternal allele expressed, and increased expression of this gene has been associated with increased β-cell proliferation and overgrowth syndromes (23,24). Both p57KIP2 and H19 are paternally imprinted, with only the maternal allele expressed, and thus are candidate genes for enhanced cell proliferation (25,26,27,28). H19 is an untranslated RNA molecule thought to be an important regulator of IGF-II mRNA levels (29). p57KIP2 (CdkN1C) is a 1.5-kb gene encoding a 335–amino acid peptide that belongs to the cyclin-dependent kinase (Cdk) inhibitor family. It is an important inhibitor of several G1 cyclin/Cdk complexes, causing cell cycle arrest in terminally differentiated cells (26,28); loss or underexpression of p57KIP2 has been related to several malignancies (30,31,32). It is not known whether p57KIP2 is expressed or imprinted in human β-cells.

We examined pancreases from patients with diffuse and focal HI and normal pancreases from different developmental stages, using immunohistochemistry to test for p57KIP2 expression. Using immunofluorescence and computerized imaging, we developed a method to quantify IGF-II staining in β-cells.

Our observations in pancreatic tissue suggest a cell-specific localization of p57KIP2 and IGF-II in β-cells. A stable fraction of β-cells expressed p57KIP2 during different developmental stages. We demonstrated a loss of p57KIP2 inside lesions of focal HI, a finding consistent with the increased rates of proliferation previously demonstrated. IGF-II expression inside the focal lesions was mildly increased when compared with the β-cells in the unaffected surrounding tissue.

Archival tissues from 15 pancreatectomized HI patients were obtained from five clinical centers (Table 1). In all, the diagnosis of HI was made according to accepted criteria (11,12). A total of 11 male and 4 female patients, age range 2 weeks to 13 months, were included in the study. Of these patients, 11 had focal disease; for these patients, analyses compared tissue inside and outside the lesion in each patient. Four patients had the diffuse form of HI. Of the 15 subjects, 12 were previously reported (Table 1).

Controls.

A total of 15 control pancreatic samples were included in the study; 12 were obtained from autopsies carried out between 1988 and 1998 in seven male subjects and five female subjects aged 17 weeks gestation to 3 years. These samples consist of a random subgroup of the previously published control population (19). All fetuses and infants died as a result of diseases not related to the pancreas, and in all, autopsies were done for medical reasons according to accepted procedures at each individual institution. All dysmorphic subjects were excluded, as were subjects with known chromosomal abnormalities or genetic syndromes. We only included subjects in whom the autopsy was performed within 24 h of death. Adult control pancreas samples were obtained from two pancreas donors and from a patient who underwent partial pancreatectomy for insulinoma. All samples were stained with hematoxylin and eosin and screened for adequate quantity of tissue, normal morphology, and good tissue preservation.

Immunohistochemistry.

Sections (5 μm thick) were prepared from archival paraffin-embedded tissue, placed on SuperFrost Plus glass slides (Menzel-Glaser, Germany), and left to dry at 37°C overnight. Slides were deparaffinized in xylene and then rehydrated in serial concentrations of alcohol (100, 90, and 80%) and double-distilled water. Antigen retrieval was carried out as described by Cattoretti et al. (33). Briefly, slides were microwaved in 0.01 mol/l citrate buffer (pH 6) for 3 min at full power until boiling and then for 15 min at 20% power. Slides were left to cool at room temperature (RT) for 30 min. They were then blocked by nonimmune serum for 10 min at RT before application of each primary antibody.

p57KIP2 Hormone double-staining.

Slides were double-stained for p57KIP2 and each of the four major pancreatic hormones (insulin, glucagon, somatostatin, and pancreatic polypeptide). Antibodies, incubation times, detection systems, and substrates are listed in Table 2. To prevent cross-reactivity of the two detection systems, avidin-biotin blocking kit (catalogue no. 00-4303; Zymed) was used before incubation with anti-hormone antibody. As a negative control, slides underwent the same procedure but were incubated with phosphate-buffered saline without anti-p57KIP2 antibody. Each batch included a negative control.

IGF-II/insulin.

Sections were double-stained for IGF-II and insulin. Antibodies, incubation times, detection systems, and substrates are listed in Table 2. Cross-reactivity of the anti–IGF-II antibody with proinsulin or insulin was excluded by preabsorbing the antibody with the two peptides overnight, a procedure that did not affect the intensity of the stain. Pretreatment, incubation times, and conditions were similar for all slides in each step described.

Quantification

p57KIP2/Hormone.

All slides were coded, and ≥1,000 hormone-positive cells were counted under high magnification (400×). The frequency of p57KIP2/hormone-positive cells was determined and expressed as percent hormone-positive cells (mean ± SE).

IGF-II/insulin.

Altogether, 11 different fields were assessed under high magnification (400×). Two images were produced from each field, using different filters in the same settings of microscope and camera (L-600 and Coolpix 950, respectively; Nikon)

Images were analyzed using Image-Pro Plus software (Media Cybernetics). Total stained area was expressed in pixels, and total integrated optic density was expressed in arbitrary optic density units. β-Cell IGF-II protein content was expressed as a ratio of IGF-II integrated optical density (IOD) to insulin-stained area. Counting criteria and software settings were identical for all slides.

Statistical analysis.

Results for p57KIP2 expression in different age-groups were analyzed using the Kruskal-Wallis nonparametric analysis of variance test, whereas the HI groups were compared with control subjects using the Mann-Whitney U test. The Wilcoxon paired nonparametric test was used to compare IGF-II expression inside and outside the lesion in focal HI.

p57KIP2 Expression.

p57KIP2 Expression was demonstrated as dark brown nuclear staining, whereas pancreatic hormones where stained red in cell cytoplasm (Figs. 1A–H). Within the pancreas, p57KIP2 was specifically localized to the endocrine cells. Very few p57KIP2 positive cells were seen in the acinar tissue, and none were found among the duct cells (Fig. 1A). In the control pancreas, β-cells demonstrated the highest frequency of p57KIP2 expression (34.9 ± 2.7%), whereas other islet cell types stained for p57KIP2 with much lower frequency (∼1–3%) (Figs. 1B–D and 2). No significant change in p57KIP2-positive β-cell proportion was observed during the different developmental stages of the human pancreas (Figs. 1E and 3).

The percentage of p57KIP2-positive β-cells in diffuse HI was similar to that in the control subjects (Figs. 1F and 4). Complete loss of p57KIP2 staining was clearly demonstrated inside the affected area of focal HI (Figs. 1G–H). Interestingly, a tendency toward increased p57KIP2 expression was observed in β-cells outside the affected area of focal HI compared with diffuse HI and control subjects, although this did not reach statistical significance (Fig. 4).

IGF-II expression.

Because the increased proliferation previously documented in focal HI could be caused by increased expression of the maternally imprinted IGF-II gene, we quantitated the IGF-II protein content of β-cells inside and outside of the lesion in eight patients. IGF-II staining was identified exclusively in β-cell cytoplasm, both inside and outside the focal lesion (Figs. 1I–L). Outside the lesion, all cells stained with IGF-II also stained for insulin; however, only ∼27% of the insulin-stained area also stained for IGF-II. In normal β-cells from the same age group, a similar IGF-II distribution was seen (data not shown). To quantify the amount of IGF-II within the β-cell mass, the intensity of IGF-II staining was expressed as the ratio of IGF-II IOD to insulin-stained area. In focal HI, IGF-II staining within the focal lesion was slightly increased when compared with that outside of the lesion in the same patient (7.5 ± 0.9 vs. 5.7 ± 0.6 arbitrary units; P < 0.04) (Fig. 5).

We have shown that in the pancreas, p57KIP2 is expressed almost exclusively in the endocrine cells, and that within the islets, expression is primarily localized to β-cells. During development, the proportion of β-cells expressing p57KIP2 does not appear to vary, and in diffuse HI, the proportion of β-cells expressing the protein is not different from control subjects of a similar age group. In contrast, p57KIP2 is not expressed by β-cells within the focal HI lesion. IGF-II expression was also seen primarily in β-cells, and staining was increased within the lesion of focal HI when compared with β-cells in the unaffected region from the same patient.

p57KIP2 Was originally described in 1995; it is a Cdk inhibitor causing cell cycle arrest and accumulation of cells in the G1 phase. It has been shown to bind to cyclin/Cdk complexes in a cyclin-dependent manner and inhibit their activity (26,28). The gene is located within a cluster of imprinted genes in humans and mice, with the maternal allele primarily expressed (27,34).

The finding that p57KIP2 expression is limited to the endocrine portion of the pancreas explains the low gene expression reported in human whole-pancreas mRNA preparations (26,28). It is also consistent with the nature of the islet cell population, especially β-cells, in that it is postmitotic and terminally differentiated. This may partially account for the failure of β-cell regeneration after exposure to harmful factors such as hyperglycemia and hyperlipidemia, a phenomenon that may have important implications in the pathogenesis of type 2 diabetes. The low expression in other islet cells compared with β-cells suggests that the latter represent a higher differentiation stage. The very low p57KIP2 expression in acinar and ductular cells may provide a possible explanation for the proliferative capacity those cells retain (35).

The finding that p57KIP2 expression does not change during different stages of development is unexpected, because the proportion of β-cells undergoing proliferation does change during fetal development, as we previously reported (19). However, the proliferation frequency ranged from ∼5% at gestational week 17 to 0% in the adult pancreas. Because only 30–40% of β-cells are p57KIP2 positive, it is likely that the methods used are not sufficiently sensitive to detect small absolute differences in the low proportion of cells undergoing proliferation at the different developmental stages. In two samples triple-stained for insulin, p57KIP2 and Ki67 (a nuclear marker of cell proliferation), we were unable to identify any cells that expressed both nuclear antigens, suggesting that cells expressing p57KIP2 do not undergo proliferation (data not shown).

Focal HI is caused by specific loss within affected β-cells of a portion of the maternal allele of Ch11p, which contains the p57KIP2 gene (20,36). p57KIP2 Has been shown to be paternally imprinted in several tissues (27). Our finding of loss of p57KIP2 expression within the focal HI lesion suggests that the gene is also imprinted in human β-cells. This relatively simple immunohistologic stain can be used to confirm loss of heterozygosity (LOH) of the maternal allele in these lesions and may be of use in differentiating focal HI from other forms of hyperinsulinism. Similarly, the same stain may be useful in confirming LOH for this region in other tissues in diseases such as BWS and in certain tumors, such as Wilms’ tumor (37), adrenocortical tumors (31), and lung cancers (30), as long as normal expression and imprinting is confirmed for each tissue type.

The finding of loss of p57KIP2 expression in focal HI may explain the increased β-cell proliferation in the adenomatous portion compared with unaffected pancreas and diffuse HI (19,38). Other adjacent genes, some imprinted, located in the chromosomal region lost in focal HI may play an additive or synergistic role in inducing β-cell proliferation. Furthermore, other mechanisms must also regulate β-cell proliferation because even within the focal lesion, proliferation rates are only ∼6% (19).

p57KIP2-Positive cells tended to be more frequent outside the focal lesion compared with cells from control and diffuse HI subjects of the same age group, although this difference did not reach statistical significance. β-Cells outside the lesion are exposed to hypoglycemia and high insulin concentration released from the lesion. This leads to suppressed metabolic activity and decreased cytoplasmic volume (18) and may also result in decreased proliferation mediated by high p57KIP2 expression.

IGF-II is located in the same region on chromosome 11 but is maternally imprinted and has been associated with increased β-cell proliferation (23). Others have demonstrated that pancreatic IGF-II expression is limited to β-cells (39). In our samples, positive staining was also seen exclusively in β-cells, and a similar staining pattern was found in pancreases from all age groups (data not shown). To estimate the IGF-II content of affected and unaffected β-cells in focal HI, we developed a method using quantitative image analysis of immunofluorescence. By comparing IGF-II IOD to insulin area, we obtained an estimate of the quantity of IGF-II protein as a function of β-cell area. Because we previously showed that IGF-II is expressed exclusively in β-cells, this calculation defines the amount of protein within the β-cells. Insulin content (as defined by insulin IOD) was not used because this reflects the secretory state of the β-cells, which clearly differs inside and outside the lesion. β-Cells that have undergone complete degranulation were not included in this calculation; however, examination of the sections indicated that very few if any of the cells within the lesion are completely degranulated, and those outside the lesion are uniformly heavily granulated, reflecting the suppressed secretion in these functionally normal cells.

Our finding of increased IGF-II within the focal lesion, relative to outside the lesion, supports the hypothesis that IGF-II may be involved in the regulation of focal proliferation. The mechanism causing this increased IGF-II expression is unknown. Paternal disomy has been documented in focal HI (40), and this increase in gene dosage may be responsible for increased expression. Alternatively, H19, a paternally imprinted gene thought to regulate IGF-II expression (29), may play a critical role. However, H19 expression and imprinting in β-cells has not yet been proven. It is also possible that p57KIP2 may have a direct effect on IGF-II expression, although such a connection has not yet been established. Furthermore, the relatively small absolute difference raises the possibility that this may be a secondary phenomenon.

In conclusion, we have demonstrated that p57KIP2 is expressed and is paternally imprinted in human pancreatic β-cells. Levels of expression do not appear to parallel changes in rates of β-cell proliferation during development, whereas decreased expression in focal HI is associated with increased rates of proliferation and increased IGF-II expression. Manipulation of p57KIP2 expression in β-cells may provide a mechanism by which the rate of proliferation can be modulated, and thus this gene may be a potential therapeutic target for reversing the β-cell failure observed in diabetes.

FIG. 1.

A: Low-power (100×) image of adult pancreas stained for p57KIP2 (brown nuclear stain) and insulin (red cytoplasmic stain). BD: Adult islets (400×) stained for p57KIP2 (brown nuclear stain) and insulin, glucagon, and somatostatin, respectively (red cytoplasmic stain). EH: Islets stained (400×) for p57KIP2 and insulin from a 26-week-old gestation fetus (E), a 6-week-old patient with diffuse HI (F), and a 5-week-old patient with focal HI showing staining outside the lesion (G) and inside the focal lesion (H). p57KIP2-Positive nuclei are indicated with arrows. I–L: Immunofluorescent staining (400×) for insulin (I and K) and IGF-II (J and L) in focal HI outside (I and J) and inside (K and L) the lesion.

FIG. 1.

A: Low-power (100×) image of adult pancreas stained for p57KIP2 (brown nuclear stain) and insulin (red cytoplasmic stain). BD: Adult islets (400×) stained for p57KIP2 (brown nuclear stain) and insulin, glucagon, and somatostatin, respectively (red cytoplasmic stain). EH: Islets stained (400×) for p57KIP2 and insulin from a 26-week-old gestation fetus (E), a 6-week-old patient with diffuse HI (F), and a 5-week-old patient with focal HI showing staining outside the lesion (G) and inside the focal lesion (H). p57KIP2-Positive nuclei are indicated with arrows. I–L: Immunofluorescent staining (400×) for insulin (I and K) and IGF-II (J and L) in focal HI outside (I and J) and inside (K and L) the lesion.

FIG. 2.

Percentage of different islet cell types positive for p57KIP2 in control pancreases. The number of samples in each group is given above each column. Pancreatic polypeptide cells were very rare, and only in two samples was it possible to count 1,000 PP p57KIP2-positive cells.

FIG. 2.

Percentage of different islet cell types positive for p57KIP2 in control pancreases. The number of samples in each group is given above each column. Pancreatic polypeptide cells were very rare, and only in two samples was it possible to count 1,000 PP p57KIP2-positive cells.

FIG. 3.

Percentage of β-cells staining positive for p57KIP2 in different age groups. Each column represents the mean of three samples.

FIG. 3.

Percentage of β-cells staining positive for p57KIP2 in different age groups. Each column represents the mean of three samples.

FIG. 4.

Percent of β-cells staining positive for p57KIP2 in control pancreases and diffuse and focal HI pancreases. In focal disease, β-cells were evaluated separately outside and within the lesion from the same pancreas. The number of samples in each group is given above each column.

FIG. 4.

Percent of β-cells staining positive for p57KIP2 in control pancreases and diffuse and focal HI pancreases. In focal disease, β-cells were evaluated separately outside and within the lesion from the same pancreas. The number of samples in each group is given above each column.

FIG. 5.

IGF-II expression inside and outside the lesion from eight patients with focal HI, expressed as the ratio of IGF-II staining IOD to insulin-stained area. AU, arbitrary units.

FIG. 5.

IGF-II expression inside and outside the lesion from eight patients with focal HI, expressed as the ratio of IGF-II staining IOD to insulin-stained area. AU, arbitrary units.

TABLE 1

Clinical characteristics of HI patients

Patient no.SexBirth weight (kg)Age of onset (months)Age at surgery (months)Postoperative statusPaternal mutationMaternal mutation
Diffuse HI
 
      
 1 4.1 1.25 1.5 Hypoglycemic 3992–3 c to g N188S 
 2 3.6 Birth 1.6 Diabetes delcP317 delcP317 
 3 5.04 Birth 3.25 Hypoglycemic Kir Y12X Kir Y12X 
 4 4.4 Birth 13 Hypoglycemic 3992–9 g to a delF1388 
Focal HI
 
      
 5 5.36 Birth 0.5 Euglycemic 3992–9 g to a None found 
 6 3.19 Birth Euglycemic R1494Q None found 
 7 3.3 Euglycemic No DNA — 
 8 3.25 Birth 0.833 Euglycemic None found* None found 
 9 4.18 Birth 1.25 Diabetes None found None found 
 10 3.61 Birth 5.5 Euglycemic None found None found 
 11 Birth 12 — No DNA — 
 12 Birth 1.5 Diabetes None found None found 
 13 3.9 Birth Euglycemic No DNA — 
 14 3.8 Birth Diabetes No DNA — 
 15 3.63 10 11 Euglycemic A1493T None found 
Patient no.SexBirth weight (kg)Age of onset (months)Age at surgery (months)Postoperative statusPaternal mutationMaternal mutation
Diffuse HI
 
      
 1 4.1 1.25 1.5 Hypoglycemic 3992–3 c to g N188S 
 2 3.6 Birth 1.6 Diabetes delcP317 delcP317 
 3 5.04 Birth 3.25 Hypoglycemic Kir Y12X Kir Y12X 
 4 4.4 Birth 13 Hypoglycemic 3992–9 g to a delF1388 
Focal HI
 
      
 5 5.36 Birth 0.5 Euglycemic 3992–9 g to a None found 
 6 3.19 Birth Euglycemic R1494Q None found 
 7 3.3 Euglycemic No DNA — 
 8 3.25 Birth 0.833 Euglycemic None found* None found 
 9 4.18 Birth 1.25 Diabetes None found None found 
 10 3.61 Birth 5.5 Euglycemic None found None found 
 11 Birth 12 — No DNA — 
 12 Birth 1.5 Diabetes None found None found 
 13 3.9 Birth Euglycemic No DNA — 
 14 3.8 Birth Diabetes No DNA — 
 15 3.63 10 11 Euglycemic A1493T None found 

Subjects 1–13 were evaluated for p57K1P2 expression, whereas subjects 8–15 were evaluated for IGF-II expression. All patients except for nos. 12–14 were previously reported (19).

*

For samples in which no mutation was found, only part of the coding sequence of SUR1 and Kir6.2 were sequenced. It is likely, therefore, that mutations will be identified in the future.

TABLE 2

Materials and incubation details for immunohistology

Primary antibodySupplierConcentrationIncubation time and temperatureDetection systemSubstrate
p57K1P2 Expression study
 
    
 Rb anti-p57K1P2 Santa Cruz 1:500 1 h, 37°C Streptavidin biotin peroxidase DAB-black 
 GP anti-insulin Dako 1:100 1 h, 37°C Streptavidin biotin alk. phos. FR 
 Rb anti-glucagon DPC As supplied 1 h, 37°C Streptavidin biotin alk. phos. FR 
 Rb anti-SMS DPC As supplied 1 h, 37°C Streptavidin biotin alk. phos. FR 
 Rb anti-PP DPC As supplied 1 h, 37°C Streptavidin biotin alk. phos. FR 
IGF-II quantification study
 
    
 Ms anti–IGF-II Upstate 1:100 1 h, 37°C Gt anti-Ms CY5 conjugate — 
 GP anti-insulin Dako 1:100 1 h, 37°C Rb anti-GP FITC conjugate — 
Primary antibodySupplierConcentrationIncubation time and temperatureDetection systemSubstrate
p57K1P2 Expression study
 
    
 Rb anti-p57K1P2 Santa Cruz 1:500 1 h, 37°C Streptavidin biotin peroxidase DAB-black 
 GP anti-insulin Dako 1:100 1 h, 37°C Streptavidin biotin alk. phos. FR 
 Rb anti-glucagon DPC As supplied 1 h, 37°C Streptavidin biotin alk. phos. FR 
 Rb anti-SMS DPC As supplied 1 h, 37°C Streptavidin biotin alk. phos. FR 
 Rb anti-PP DPC As supplied 1 h, 37°C Streptavidin biotin alk. phos. FR 
IGF-II quantification study
 
    
 Ms anti–IGF-II Upstate 1:100 1 h, 37°C Gt anti-Ms CY5 conjugate — 
 GP anti-insulin Dako 1:100 1 h, 37°C Rb anti-GP FITC conjugate — 

Alk. phos., alkaline phosphatase; DAB, diaminobenzidine tetrachloride; DPC, Diagnostic Products Corporation; FITC, fluorescein isothiocyanate; FR, fast red; GP, guinea pig; Gt, goat; Ms, mouse; Rb, rabbit.

The authors would like to thank Drs. Feilberg Jorgensen and Claus Fenger for providing us with tissue preparations from some of their HI patients. This study was supported by Israel Science Foundation Grant 512-98 and the Hadassah Diabetes Center. Grant BMH4-CT98-3284, awarded as part of the European Commission Biomed 2 program, provided support for international collaborations. Updated information on neonatal hyperinsulinism is available on the European Network for Research into Hyperinsulinism (ENRHI) website at http://www.phhi.u-net.com.

1.
Bruining GJ: Recent advances in hyperinsulinism and the pathogenesis of diabetes mellitus.
Cur Op Pediatr
2
:
758
–765,
1990
2.
Otonkoski T, Ammala C, Huopio H, Cote GJ, Chapman J, Cosgrove K, Ashfield R, Huang E, Komulainen J, Ashcroft FM, Dunne MJ, Kere J, Thomas PM: A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland.
Diabetes
48
:
408
–415,
1999
3.
Mathew PM, Young JM, Abu OY, Mulhern BD, Hammoudi S, Hamdan JA, Sa’di AR: Persistent neonatal hyperinsulinism.
Clin Pediatr
(Phila) 
27
:
148
–151,
1988
4.
Nestorowicz A, Wilson BA, Schoor KP, Inoue H, Glaser B, Landau H, Stanley CA, Thornton PS, Clement JP IV, Bryan J, Aguilar-Bryan L, Permutt MA: Mutations in the sulfonylurea receptor gene are associated with familial hyperinsulinism in Ashkenazi Jews.
Hum Molec Genet
5
:
1813
–1822,
1996
5.
Nestorowicz A, Inagaki N, Gonol T, Schoor KP, Wilson BA, Glaser B, Landau H, Stanley CA, Thornton PS, Seino S, Permutt MA: A nonsense mutation in the inward rectifier potassium channel gene, KIR6.2, is associated with familial hyperinsulinism.
Diabetes
46
:
1743
–1748,
1997
6.
Thomas PM, Cote GJ, Hallman DM, Mathew PM: Homozygosity mapping, to chromosome 11p, of the gene for familial persistent hyperinsulinemic hypoglycemia of infancy.
Am J Hum Genet
56
:
416
–421,
1995
7.
Thomas P, Ye Y, Lightner E: Mutation of the pancreatic islet inward rectifier KIR6.2 also leads to familial persistent hyperinsulinemic hypoglycemia of infancy.
Hum Mol Genet
5
:
1809
–1812,
1996
8.
Glaser B, Kesavan P, Heyman M, Davis E, Cuesta A, Buchs A, Stanley CA, Thornton PS, Permutt MA, Matschinsky FM, Herold KC: Familial hyperinsulinism caused by an activating glucokinase mutation.
N Engl J Med
338
:
226
–230,
1998
9.
Stanley CA, Fang J, Kutyna K, Hsu BY, Ming JE, Glaser B, Poncz M, the HI/HA Contributing Investigators: Molecular basis and characterization of the hyperinsulinism/hyperammonemia syndrome: predominance of mutations in exons 11 and 12 of the glutamate dehydrogenase gene.
Diabetes
49
:
667
–673,
2000
10.
Nestorowicz A, Glaser B, Wilson BA, Shyng S-L, Nichols CG, Stanley CA, Thornton PS, Permutt MA: Genetic heterogeneity in familial hyperinsulinism.
Hum Mol Genet
7
:
1119
–1128,
1998
11.
Aynsley-Green A: Nesidioblastosis of the pancreas in infancy.
Dev Med Child Neurol
23
:
372
–379,
1981
12.
Landau H, Perlman M, Meyer S, Isacsohn M, Krausz M, Mayan H, Lijovetzky G, Schiller M: Persistent neonatal hypoglycemia due to hyperinsulinism: medical aspects.
Pediatrics
70
:
440
–446,
1982
13.
Rahier J, Falt K, Muntefering H, Becker K, Gepts W, Falkmer S: The basic structural lesion of persistent neonatal hypoglycaemia with hyperinsulinism: deficiency of pancreatic D cells or hyperactivity of B cells?
Diabetologia
26
:
282
–289,
1984
14.
Jaffe R, Hashida Y, Yunis EJ: Pancreatic pathology in hyperinsulinemic hypoglycemia of infancy.
Lab Invest
42
:
356
–365,
1980
15.
Goossens A, Gepts W, Saudubray JM, Bonnefont JP, Nihoul F, Heitz PU, Kloppel G: Diffuse and focal nesidioblastosis: a clinicopathological study of 24 patients with persistent neonatal hyperinsulinemic hypoglycemia.
Am J Surg Pathol
13
:
766
–775,
1989
16.
Jaffe R, Hashida Y, Yunis EJ: The endocrine pancreas of the neonate and infant.
Perspect Pediatr Pathol
7
:
137
–165,
1982
17.
Rahier J, Wallon J, Henquin JC: Cell populations in the endocrine pancreas of human neonates and infants.
Diabetologia
20
:
540
–546,
1981
18.
Rahier J, Sempoux C, Fournet JC, Poggi F, Brunelle F, Nihoul-Fekete C, Saudubray JM, Jaubert F: Partial or near-total pancreatectomy for persistent neonatal hyperinsulinaemic hypoglycaemia: the pathologist’s role.
Histopathology
32
:
15
–19,
1998
19.
Kassem SA, Ariel I, Thornton PS, Scheimberg I, Glaser B: β-Cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy.
Diabetes
49
:
1325
–1333,
2000
20.
Fournet JC, Verkarre V, De Lonlay P, Rahier J, Brunelle F, Robert JJ, Nihoul-Fékété C, Saudubray JM, Junien C: Loss of imprinted genes and paternal SUR1 mutations lead to hyperinsulinism in focal adenomatous hyperplasia.
Ann Endocrinol (Paris)
59
:
485
–491,
1998
21.
Ryan FD, Devaney D, Joyce C, Nestorowicz A, Permutt MA, Glaser B, Barton DE, Thornton PS: Hyperinsulinism: the molecular aetiology of focal disease.
Arch Dis Child
79
:
445
–447,
1998
22.
Lin Y, Ma W, Benchimol S: Pidd, a new death-domain-containing protein, is induced by p53 and promotes apoptosis.
Nat Genet
26
:
122
–127,
2000
23.
Petrik J, Pell JM, Arany E, McDonald TJ, Dean WL, Reik W, Hill DJ: Overexpression of insulin-like growth factor-II in transgenic mice is associated with pancreatic islet cell hyperplasia.
Endocrinology
140
:
2353
–2363,
1999
24.
Petrik J, Arany E, McDonald TJ, Hill DJ: 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 factor.
Endocrinology
139
:
2994
–3004,
1998
25.
Rachmilewitz J, Goshen R, Ariel I, Schneider T, de Groot N, Hochberg A: Parental imprinting of the human H19 gene.
FEBS Lett
309
:
25
–28,
1992
26.
Matsuoka S, Edwards MC, Bai C, Parker S, Zhang P, Baldini A, Harper JW, Elledge SJ: p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene.
Genes Dev
9
:
650
–662,
1995
27.
Matsuoka S, Thompson JS, Edwards MC, Bartletta JM, Grundy P, Kalikin LM, Harper JW, Elledge SJ, Feinberg AP: Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15.
Proc Natl Acad Sci U S A
93
:
3026
–3030,
1996
28.
Lee MH, Reynisdottir I, Massague J: Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution.
Genes Dev
9
:
639
–649,
1995
29.
Li M, Squire JA, Weksberg R: Overgrowth syndromes and genomic imprinting: from mouse to man (Review).
Clin Genet
53
:
165
–170,
1998
30.
Kondo M, Matsuoka S, Uchida K, Osada H, Nagatake M, Takagi K, Harper JW, Takahashi T, Elledge SJ: Selective maternal-allele loss in human lung cancers of the maternally expressed p57KIP2 gene at 11p15.5.
Oncogene
12
:
1365
–1368,
1996
31.
Bourcigaux N, Gaston V, Logie A, Bertagna X, Le Bouc Y, Gicquel C: High expression of cyclin E and G1 Cdk and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors.
J Clin Endocrinol Metab
85
:
322
–330,
2000
32.
Thompson JS, Reese KJ, DeBaun MR, Perlman EJ, Feinberg AP: Reduced expression of the cyclin-dependent kinase inhibitor gene p57KIP2 in Wilms’ tumor.
Cancer Res
56
:
5723
–5727,
1996
33.
Cattoretti G, Becker MH, Key G, Duchrow M, Schlüter C, Galle J, Gerdes J: Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections.
J Pathol
168
:
357
–363,
1992
34.
Hatada I, Mukai T: Genomic imprinting of p57KIP2, a cyclin-dependent kinase inhibitor, in mouse.
Nat Genet
11
:
204
–206,
1995
35.
Bouwens L: Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas.
Microsc Res Tech
43
:
332
–336,
1998
36.
Fournet JC, Mayaud C, de Lonlay P, Verkarre V, Rahier J, Brunelle F, Robert JJ, Nihoul-Fekete C, Saudubray JM, Junien C: Loss of imprinted genes and paternal SUR1 mutations lead to focal form of congenital hyperinsulinism.
Horm Res
53
:
2
–6,
2000
37.
Hatada I, Inazawa J, Abe T, Nakayama M, Kaneko Y, Jinno Y, Niikawa N, Ohashi H, Fukushima Y, Iida K, Yutani C, Takahashi S, Chiba Y, Ohishi S, Mukai T: Genomic imprinting of human p57KIP2 and its reduced expression in Wilms’ tumors.
Hum Mol Genet
5
:
783
–788,
1996
38.
Sempoux C, Guiot Y, Dubois D, Nollevaux MC, Saudubray JM, Nihoul-Fekete C, Rahier J: Pancreatic B-cell proliferation in persistent hyperinsulinemic hypoglycemia of infancy: an immunohistochemical study of 18 cases.
Mod Pathol
11
:
444
–449,
1998
39.
Maake C, Reinecke M: Immunohistochemical localization of insulin-like growth factor 1 and 2 in the endocrine pancreas of rat, dog, and man, and their coexistence with classical islet hormones.
Cell Tissue Res
273
:
249
–259,
1993
40.
Fournet JC, Mayaud C, de Lonlay P, Gross-Morand MS, Verkarre V, Castanet M, Devillers M, Rahier J, Brunelle F, Robert JJ, Nihoul-Fekete C, Saudubray JM, Junien C: Unbalanced expression of 11p15 imprinted genes in focal forms of congenital hyperinsulinism: association with a reduction to homozygosity of a mutation in ABCC8 or KCNJ11.
Am J Pathol
158
:
2177
–2184,
2001

Address correspondence and reprint requests to Benjamin Glaser, Director, Endocrinology and Metabolism Service, Hebrew University, Hadassah Medical Center, POB 12000, 91120 Jerusalem, Israel. E-mail: beng@cc.huji.ac.il.

Received for publication 11 May 2001 and accepted in revised form 17 September 2001.

Cdk, cyclin-dependent kinase; HI, hyperinsulinism of infancy; IOD, integrated optical density; KATP, ATP-sensitive K+ channel; LOH, loss of heterozygosity; SUR1, sulfonylurea receptor-1.