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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 4 1795-1800
Copyright © 2001 by The Endocrine Society

Original Studies

Insulin Production in a Neuroectodermal Tumor that Expresses Islet Factor-1, But Not Pancreatic-Duodenal Homeobox 1

Takaaki Nakamura, Akio Kishi, Yoshihiko Nishio, Hiroshi Maegawa, Katsuya Egawa, Norman C. W. Wong, Hideto Kojima, Mineko Fujimiya, Ryohachi Arai, Atsunori Kashiwagi and Ryuichi Kikkawa

Department of Anatomy (T.N., M.F., S.A.) and Third Department of Medicine (A.K., H.K., Y.N., H.M., K.E., A.K., R.K.), Shiga University of Medical Science, Otsu, Shiga 520-2129, Japan; and Departments of Medicine and Biochemistry and Molecular Biology, University of Calgary Health Sciences Center (N.W.), Calgary, Alberta, Canada T2N 4N1

Address all correspondence and requests for reprints to: Dr. Atsunori Kashiwagi, Shiga University of Medical Science, Otsu, Shiga 520-2129, Japan. E-mail: kasiwagi{at}belle.shiga-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied a 60-yr-old female with a brain tumor who showed severe symptoms of hypoglycemia (plasma glucose, 2.2 mmol/L) and hyperinsulinemia (1.28 nmol/L) after radiotherapy. The cystic brain tumor contained proinsulin and insulin at concentrations of 13.6 and 1.22 nmol/L, respectively. Immunohistochemical studies showed the tumor cells were ectodermal in origin but not endodermal, based on three diagnostic features of neuroectodermal tumors 1) pseudorosette formation noted under light microscopy, 2) finding of a small number of dense core neurosecretory granules on electron microscopy, and 3) positive immunostaining for both neuronal specific enolase and protein gene product 9.5. These cells also expressed the transcription factor, neurogenin-3, NeuroD/ß2, and islet factor I, which are believed to be transcription factors in neuroectoderm as well as in pancreatic islet cells, but not pancreatic-duodenal homeobox 1, Pax4, or Nkx2.2. In addition, they did not express glucagon, somatostatin, or glucagon-like peptide-1. Our results show the presence of proinsulin in an ectoderm cell brain tumor that does not express the homeobox gene, pancreatic-duodenal homeobox 1, but expresses other transcription factors, i.e. neurogenin3, NeuroD/ß2, and islet factor-1, which are related to insulin gene expression in the brain tumor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPOGLYCEMIA IS AN uncommon manifestation associated with tumor. The cell origin of these tumors can be simply divided into two broad categories, either pancreatic islet or extrapancreatic nonislet cell origins. Whereas excessive expression of the insulin gene in the pancreatic islet cell-derived tumor, insulinoma, is the underlying cause of hypoglycemia (1, 2, 3), that associated with nonislet cell tumors has a variety of causes. Possible causes include expression of insulin-like growth factor II (IGF-II) (4, 5, 6), which binds the insulin receptor; metastasis of the tumor to the liver, leading to impaired hepatic production of glucose (7); and, rarely, expression of autoantibodies against insulin or its receptor (8, 9). In nonislet cell tumors, there is considerable debate over whether they can secrete insulin. We can refer only one case report showing that a poorly differentiated small cell carcinoma of the cervix produces insulin in addition to other pancreatic peptides including glucagon, glucagon-like peptide-2 (GLP-2), and pancreatic polypeptide (10). This rare finding will indicate whether our nonislet cell tumors may also produce insulin and the potential mechanism(s) underlying this expression.

Immunohistochemical studies provide an easy way to distinguish islet from nonislet cell tumors. Insulinomas arising from endoderm, express epithelial markers but normal neurons of neural tube or crest origin do not express the epithelial markers once they enter the process of differentiation (11, 12). Neuroendocrine carcinomas, such as small cell carcinoma of the lung, express epithelial and neuronal or neuroendocrine markers (13). The characteristics of these tumors resemble those of endocrine origin, such as ß-cells but not neurons. In fact, the finding of epithelial marker, such as Ep-CAM (epithelial glycoprotein 40) is used to rule out tumors of neuroectoderm origin. As Ep-CAM is expressed on the basolateral surface of epithelium and is normally found in almost all types of neuroendocrine carcinomas (14) and pancreatic ß-cells (15). On the other hand, neurons express neuronal specific enolase (NSE) or protein gene product (PGP9.5) in cytoplasm. Therefore, this panel of markers is useful in distinguishing neuroendocrine cells from those of neuroectodermal origin.

Added to the separation of islet from nonislet cell tumors using specific markers, it is possible to examine what potential mechanism(s) in the tumor cells is underlying the expression of this gene using our current understanding of insulin gene regulation. For example, we can search for transcription factors that play important roles in pancreatic islet ß-cell differentiation and insulin gene expression. The transcription factor, pancreatic- duodenal homeobox 1 (Pdx1) is essential for both developmental and regulated expression of the insulin gene (16, 17). Tumors of islet ß-cell origin that express this transcription factor can produce insulin. Therefore, the finding of Pdx1 is a clue that tells us whether a tumor arises from islet or nonislet ß-cells. In contrast, other transcription factors; islet factor-1 (ISL-1), neurogenin-3 (ngn3), NeuroD/ß2, Pax4, and Nkx2.2, are also expressed in pancreatic islet ß-cells and nonislet ß-cells, but their roles in insulin gene expression are not well known in nonislet ß-cells. According to previous studies, enteroendocrine and neuroectoderm cell differentiation requires these transcription factors, including the basic helix-loop-helix factor, NeuroD/ß2, ngn3, Pax4, and Pax6 (18, 19). Indeed, mice homozygous for a null mutation in some of these transcription factors showed marked morphological changes, including defects in pancreatic islet cell function (20, 21, 22). Therefore, to understand insulin production in nonislet cell tumor, we need not only immunohistochemical detection of insulin, but also the expression of various transcription factors in the tumor cells.

In the present study we studied a patient with a brain tumor that produced insulin, and we found that NeuroD/ß2, ngn3, and ISL-1, rather than Pdx1, were expressed in this brain tumor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Characteristics of the brain tumor

A 60-yr-old female was referred to our hospital for assessment of dementia. Magnetic resonance imaging, studies showed a brain tumor with cystic formation. Chemotherapy consisting of fluorouracil (500 mg/day) and cyclophosphamide (50 mg/day) was given, but there was no significant response. Next, she received radiotherapy to the tumor (first focal exposure with 5 x 5-cm field; 3 Gy). Within 30 min following this treatment she suffered severe symptoms of hypoglycemia that included sweating, palpitations, tremor, and mental confusion. All symptoms were relieved by iv administration of glucose. Although blood cell count, liver and kidney function tests were all normal after the hypoglycemic attacks, plasma glucose and immunoreactive insulin concentrations were 2.2 mmol/L and 1.28 nmol/L, respectively. Mild increases in serum cortisol (0.84 µmol/L), glucagon (613 ng/L), somatostatin (25 ng/L), ACTH (24 ng/L), and TSH (3.5 mIU/L) were noted after the episodes. These changes are probably due to normal physiological responses to hypoglycemia. Before radiotherapy, the fasting blood glucose (5.4 mmol/L) and immunoreactive insulin (49.4 pmol/L) concentrations were both within normal range.

The onset of hypoglycemia immediately following radiotherapy suggests that it may have disrupted tissue surrounding the tumor, thus allowing the cystic fluid to be released into the systemic circulation, leading to severe hypoglycemia. This reasoning led to surgical removal of the tumor. Histological studies of the tumor revealed a poorly differentiated neuroendocrine carcinoma. Analysis of fluid in the cystic portion of the tumor showed it to contain proinsulin and insulin at concentrations of 13.6 and 1.22 nmol/L, respectively. The hypothesis that the tumor fluid underlies the etiology of the hypoglycemia is supported by additional studies using computed tomography, which showed no abnormality in the pancreas or liver. Similarly, negative results from magnetic resonance imaging and the calcium infusion test during angiographic study of liver and pancreas were also noted.

Hormone assays

The insulin concentration was measured by double antibody RIA with human insulin as a standard (23). The cross-reactivity with proinsulin using this assay is less than 1%. The proinsulin concentration was measured using human proinsulin-specific antibody (SRL, Tokyo, Japan).

Immunohistochemical studies

Tissue sections 20 µm thick were fixed for 24 h with 4% paraformaldehyde, 0.2% picric acid, and 0.5% glutaraldehyde in 0.2 mol/L phosphate buffer (pH 7.4) at 4 C. After washing for 24 h with 0.1 mol/L phosphate-buffered saline (PBS) at 4 C, the sections were incubated for 48 h with antibodies of interest diluted to 1:5000 in PBS containing 0.3% Triton X-100 (PBST) at 4 C. The antibodies used were rabbit antiserum to a synthetic peptide of Pdx1 (24), ISL-1 (25), and guinea pig antiserum to human recombinant insulin (Austral Biological, Inc., La Brea, CA). After washing for 10 min with PBST, the sections were incubated for 2 h with biotinylated IgG (Vector Laboratories, Inc., Burlingame, CA) diluted to 1:1000 in PBST at room temperature and then reacted for 1.5 h with avidin-biotin peroxidase complex (Vector Laboratories, Inc.) diluted to 1:1000 in PBST at room temperature. The immunoreaction was then visualized by developing with 0.05 mol/L Tris-HCl buffer (pH 7.6) containing 0.01% 3,3'-diaminobenzidine, 1% ammonium nickel sulfate, and 0.0003% H2O2 for 30 min at room temperature. The sections were mounted on the gelatin-coated glass slides, dehydrated by graded ethanol, coverslipped with Entellan (Merck & Co., Darmstadt, Germany), and observed under light microscopy.

For immunofluorescence double staining, fixed sections processed as described above were incubated for 48 h with either the mixture of primary antibodies against insulin and Pdx1 or ISL-1 diluted to 1:2000 in PBST at 4 C. After washing with PBST, the sections were incubated for 2 h at room temperature with either the mixture of fluorescein isothiocyanate-labeled antirabbit IgG (Vector Laboratories, Inc.) and Texas Red-labeled antiguinea pig IgG (Vector Laboratories, Inc.) for Pdx1 or ISL-1/insulin double staining. After rinsing with PBST, the sections were mounted on the glass slides, dried, coverslipped with liquid paraffin, and observed under a confocal laser scanning image system (MRC-600, Bio-Rad Laboratories, Inc., Hercules, CA). The specificity of the positive staining was examined by the immunocytochemical absorption study. The primary antibodies were replaced with an antigen-antibody mixture, where the antigens used were recombinant human insulin, Pdx1, and ISL-1 at the concentrations of 10 µmol/L, respectively.

Electron microscopy

The sections were fixed with PBS and incubated for 1 h with 1% OsO4 in 0.1 mol/L PBS at 4 C, followed by dehydration in a graded series of ethanol and propylene oxide and then were embedded in epoxy resin. Ultrathin sections were cut with an ultramicrotome (Ultracut E, Reichert-Jung, Vienna, Austria) vertically or horizontally and mounted on 200-mesh copper grids. Sections were stained for 20 min with 2% uranyl acetate, followed by an additional 5 min with Reynolds’ solution, and then were observed under electron microscopy (H-7100, Hitachi, Tokyo, Japan).

RNA extraction and RT-PCR analysis

After surgical removal, the brain tumor was dissected, washed with PBS, snap-frozen, and then stored at –70 C. Total RNA was extracted using acid guanidinium isothiocyanate-phenol-chloroform as described previously (26). For RT-PCR studies, we used 5 µg total RNA as a template to synthesize complementary DNA using RNA reverse transcriptase (Superscript II, Life Technologies, Tokyo, Japan). For the subsequent PCR, 1 µL of the reaction mixture was used each time. Oligonucleotide primers (5' and 3') for amplification of the complementary DNA of interest were used. The PCR product was identified by agarose gel electrophoresis, and finding the band of expected size visualized with ethidium bromide was used to assign identity, which was further confirmed by sequence analysis (PE Applied Biosystems, Foster City, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin-expressing tumor cells originate from ectoderm, not endoderm. Histological examination of the extracted brain tumor was performed using hematoxylin-eosin stain (Fig. 1AGo) and revealed that tumor cells were small with large nuclei, a feature suggestive of neuroendocrine cell carcinomas. We further searched for the presence of human insulin using immunohistochemical analysis. Results showed that the tumor cells stained positively for insulin (Fig. 1BGo). Therefore, to determine whether the cells had storage granules for insulin, we examined the cells using electron microscopy. The tumor cells had abundant granules of pleiomorphic shape throughout their cytoplasm. Most of these granules were small, and they were sometimes found outside the cell membrane (Fig. 1CGo). These data show clearly that the cells produce and possibly secrete insulin.



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  		Figure 1. A, Hematoxylin-eosin stain. Tissue stained with this dye reveals small cells with large nucleus, a feature suggestive of a neuroendocrine cell carcinoma. Bar, 25 µm. B, Immunohistochemical staining for insulin. The insulin signal is strong, and pseudorosette formation is observed in the tumor cells. Bar, 50 µm. C, Electron micrograph. The tumor cells have abundant granules (arrow) of pleiomorphic shape throughout the cytoplasm. Most of these granules are small, and they are sometimes found outside the cell membrane. Bar, 4 µm.

 
As neurons stain with anti-PGP.9.5 antibodies, we used confocal laserscan microscopy to search for colocalization (Fig. 2Go). In the staining for insulin (Fig. 2AGo-1) and PGP9.5 (Fig. 2AGo-2) in tumor cells, those immunoreactivities overlapped (Fig. 2AGo-3). To support this result, we stained the cells looking for Ep-CAM and NSE, which are markers for epithelium and neuron, respectively. Although the tumor stained positively for NSE, suggesting that it was neuronal in origin, the cells were Ep-CAM negative (data not shown), suggesting that they were not derived from epithelial origin.



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  		Figure 2. Double staining for insulin/PGP9.5 in brain tumor cells. Insulin (A-1) and PGP9.5 (A-2) show that immunoreactivities overlap (A-3) in the tumor cells. Bar, 50 µm. Double staining for insulin/Pdx1 in the tumor cells. A strong signal is obtained (B-1; bar, 25 µm) with human antisera against insulin, but antisera against Pdx1 (B-2) shows very little. The overlapped image for insulin (red)/Pdx1 (green) from human pancreas served as the positive control (B-3). Bar, 25 µm. Double staining for insulin/ISL-1. Insulin (C-1) and ISL-1 (C-2) stainings show that immunoreactivities overlap (C-3) in the cells. Bar, 50 µm.

 
Expression of ISL-1, but not Pdx1, in insulin-producing brain cells

To identify features that enabled these cells to express insulin, we stained the tumor with antibodies against Pdx1 and ISL-1, two transcription factors believed to be important for insulin gene expression. Additionally, we stained the same cells for insulin. Although the cells stained positively for insulin (Fig. 2BGo-1), they did not appear to contain Pdx1 (Fig. 2BGo-2). On the other hand, double staining with antibodies against insulin and Pdx1 (Fig. 2BGo-3) showed colocalization of both proteins in human pancreatic islet cells.

Whereas tumor cells stained for insulin yielded red in the cytoplasm (Fig. 2CGo-1), those stained with the ISL-1 antibody showed the presence of green (Fig. 2CGo-2) located predominantly in the cytoplasm. Double staining of cells with both insulin and ISL-1 showed colocalization of the two proteins in the cytoplasm, as reflected by an identical pattern outlined in yellow (Fig. 2CGo-3). These data suggest that insulin expression in tumor cells correlates with the finding of cytoplasmic ISL-1, but not Pdx1. Together these findings indicate that Pdx1 is not always essential for insulin gene expression in nonislet tumor cells.

To be certain that the cells did not express Pdx1, we measured the expression of this and other nuclear transcription factors in the tumor and compared it with that in human pancreas cells (Fig. 3Go) using RT-PCR. The results showed that the tumor cells expressed ISL-1, ngn3, and NeuroD/ß2, but not Pdx1, Pax4, or Nkx2.2. As expected, five transcription factors, but not ngn3, were expressed in human pancreatic cells. Additionally, the messenger RNA (mRNA) encoding insulin was present in the tumor cells. The RT-PCR product in tumor cells reflecting insulin expression was verified by nucleotide sequencing of the 187-bp product and was shown to be identical to that of human insulin mRNA.



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  		Figure 3. The expression of nuclear transcription factors (Pdx1, ISL-1, Nkx2.2, Pax4, ngn3, and NeuroD/ß2) in tumor cells. RT-PCR analyses were performed using total RNAs isolated from tumor cells (1 ) and human pancreas (2 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hypoglycemia arising from nonislet cell tumors is rare, and the underlying etiology is usually associated with high levels of IGF-II. The unique feature of the presented case is that hypoglycemia occurred immediately after radiation therapy, and it is probably due to extremely high levels of plasma insulin (1.28 nmol/L) in the absence of liver dysfunction. We found only one previous report of a poorly differentiated small cell carcinoma of the cervix that produced insulin (10). Surgical removal of the tumor and analysis of the tissue indicated that we could exclude small cell carcinoma because the tumor cells did not stain for the epithelial antigen, Ep-CAM. Additional evidence showed that the tumor cells originating from neuroectoderm revealed positive immunostaining for both NSE and PGP9.5, which are neuron-specific proteins, in the morphological studies. Other features of the tumor were pseudorosette formation noted under light microscopy and/or demonstration of small numbers of dense core neurosecretory granules on electron microscopic analysis. These features are characteristics of neuroectodermal tumor (27). Thus, insulin expression in a tumor of ectoderm origin provides a unique opportunity to examine the expression of this hormone in nonislet cells. We further probed the tumor with antibodies against human chromogranin A, somatostatin, serotonin, pancreatic polypeptide, glucagon, and glucagon-like peptide-1 (GLP-1) to determine whether the tumor cells had other endocrine characteristics. Subsequent studies showed that these neuroectoderm cells expressed only insulin and chromogranin A, but not other endocrine peptides (i.e. glucagon, pancreatic polypeptide, somatostatin, or GLP-1; data not shown). These results also strongly support that the brain tumor cells are neuroectodermal in origin, but not derived from neuroendocrine. In addition to the results obtained by immunostaining, the expression of specific transcription factors in the tumor cells was further explored using RT-PCR. The patient’s brain tumor cells expressed only ISL-1, ngn3, NeuroD/ß2, and insulin, not Pdx1, Nkx2.2, or Pax4, which are believed to play important roles in pancreatic endocrine cell differentiation (16, 17). Furthermore, the transcription factors ISL-1, NeuroD/ß2, and ngn3 also participate not only in insulin gene expression of pancreatic islet cells, but also in neuronal differentiation (18, 19). These results raise the possibility that ISL-1, NeuroD/ß2, and/or ngn3 may be more important for insulin expression than Pdx1 in nonislet cell tumors.

Pdx1, a homeotic selector, plays an important role in the formation of pancreatic ß-cells (17) and trans-activates the insulin, amylin (28), and somatostatin genes in both pancreas and duodenum (29). One potential reason why the tumor cells do not express Pdx1 is because they are neuroectodermal in origin. Additionally, ISL-1 is an important transcription factor that not only binds to the enhancer region of the insulin gene, but also regulates amylin (30), proglucagon (31) and somatostatin (32) genes. Initial reports localize ISL-1 in the pancreatic islet cells, suggesting a possible role for this protein in the regulation of insulin gene expression and/or islet cell development (18). In the patient’s tumor cells, immunoreactive ISL-1 was mainly observed in cytoplasm compared with the nuclear localization in pancreatic islet cells. However, it is still uncertain whether ISL-1 expression in cytoplasm related to the expression of insulin gene in nonislet cells. Our immunohistochemical study indicates that a small amount of ISL-1 in the nucleus may be enough to regulate insulin gene expression in the tumor cells. Moreover, we show herein the positive gene expression of ngn3 and NeuroD/ß2, which are members of a family of basic helix-loop-helix transcription factors and are involved in the determination of neural precursor cells in the neuroectoderm (33). ngn3 is believed to regulate the gene expression of NeuroD/ß2 in pancreas and neuron (34). Therefore, this gene may induce the gene expression of insulin through the action of NeuroD/ß2 in the tumor cells. Indeed, in mice lacking a functional NeuroD/ß2 gene, the nervous system appeared to develop normally despite the existence of defects in pancreatic islet cells in differentiating pancreas (35). Thus, NeuroD/ß2 might be more critical for insulin gene expression than neuronal differentiation in the tumor cells. Our data concerned with gene expression of these transcription factors strongly indicate that ngn3, NeuroD/ß2, and ISL-1 need to produce insulin in nonislet cells.

In summary, our studies show that the tumor expressed proinsulin as well as ISL-1 mRNAs, but not PdxX1, Pax4, or Nkx2.2, suggesting that tumor cells of ectoderm origin can produce proinsulin without expressing the homeobox gene, Pdx1. These findings raise the question of differential regulation of insulin gene expression in nonislet cells.


   Acknowledgments
 
Our most sincere thanks to Dr. Yoshitaka Kajimoto, Osaka University Graduate School of Medicine, for the generous gift of the mouse antisera against Pdx1. We also thank Hirotsugu Imaeda, Shiga University of Medical Science, for the excellent technique of immunohistochemistry.

Received September 28, 2000.

Revised December 22, 2000.

Accepted January 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Protein-Tyrosine Phosphatase 1B Associates with Insulin Receptor and Negatively Regulates Insulin Signaling without Receptor Internalization
J. Biochem., July 1, 2004; 136(1): 89 - 96.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Endocrinol. Metab.Home page
A. Kishi, T. Nakamura, Y. Nishio, H. Maegawa, and A. Kashiwagi
Sumoylation of Pdx1 is associated with its nuclear localization and insulin gene activation
Am J Physiol Endocrinol Metab, April 1, 2003; 284(4): E830 - E840.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
K. Egawa, H. Maegawa, K. Shi, T. Nakamura, T. Obata, T. Yoshizaki, K. Morino, S. Shimizu, Y. Nishio, E. Suzuki, et al.
Membrane Localization of 3-Phosphoinositide-dependent Protein Kinase-1 Stimulates Activities of Akt and Atypical Protein Kinase C but Does Not Stimulate Glucose Transport and Glycogen Synthesis in 3T3-L1 Adipocytes
J. Biol. Chem., October 4, 2002; 277(41): 38863 - 38869.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
O. Sekine, Y. Nishio, K. Egawa, T. Nakamura, H. Maegawa, and A. Kashiwagi
Insulin Activates CCAAT/Enhancer Binding Proteins and Proinflammatory Gene Expression through the Phosphatidylinositol 3-Kinase Pathway in Vascular Smooth Muscle Cells
J. Biol. Chem., September 20, 2002; 277(39): 36631 - 36639.
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 DiabetesHome page
H. Kojima, T. Nakamura, Y. Fujita, A. Kishi, M. Fujimiya, S. Yamada, M. Kudo, Y. Nishio, H. Maegawa, M. Haneda, et al.
Combined Expression of Pancreatic Duodenal Homeobox 1 and Islet Factor 1 Induces Immature Enterocytes to Produce Insulin
Diabetes, May 1, 2002; 51(5): 1398 - 1408.
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