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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 8 2875-2877
Copyright © 1998 by The Endocrine Society

Original Studies

Serum High Molecular Weight Form of Insulin-Like Growth Factor II from Patients with Non-Islet Cell Tumor Hypoglycemia Is O-Glycosylated1

Naomi Hizuka, Izumi Fukuda, Kazue Takano, Kumiko Asakawa-Yasumoto, Yumiko Okubo and Hiroshi Demura

Department of Medicine II, Tokyo Women’s Medical University, Tokyo, 162-8666, Japan

Address all correspondence and requests for reprints to: Naomi Hizuka, Department of Medicine II, Tokyo Women’s Medical University, 8–1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: naomi-hi{at}hi-ho.ne.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Non-islet cell tumor hypoglycemia (NICTH) is one of major causes of fasting hypoglycemia. In some patients with NICTH, insulin-like growth factor II (IGF-II) produced by and secreted from the tumors is thought to be a hypoglycemic agent. In patients with NICTH, the major form of IGF-II is high molecular weight form of IGF-II, designated as big IGF-II. The generation of big IGF-II in the NICTH syndrome is unclear. It has been reported that in the patients with NICTH big IGF-II lacks normal E-domain O-linked glycosylation, suggesting that the patient’s big IGF-II might be generated by abnormal processing of pro-IGF-II. However, we have found that the apparent size of big IGF-II varies in sera from the patients with NICTH, and that there is a possibility that slower migration pattern of IGF-II might be because of a different size of sugar moiety attached to pro-IGF-II. In the present study using the sera from 10 patients with NICTH, we investigated the effect of O-glycosidase digestion on migration of IGF-II and analyzed the results by Western immunoblot. By Western immunoblot analysis the big IGF-II was reduced in size to 9.5 kDa in the enzyme-treated sera of the 10 patients with NICTH. The migration pattern is similar to that observed in sera of normal subjects after O-glycosidase digestion. These data indicate that big IGF-II from patients with NICTH is O-glycosylated, and the sizes of the sugar moiety are larger than those from normal subjects suggesting abnormal glycosylation in NICTH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NON-ISLET cell tumor hypoglycemia (NICTH) is one of the major causes of fasting hypoglycemia (1). In some patients with NICTH, insulin-like growth factor II (IGF-II) produced by and secreted from the tumors is thought to be a hypoglycemic agent (2, 3). However, the mechanisms of hypoglycemia are still unknown, because it has been reported that serum IGF-II levels are not always elevated (4, 5, 6). It has been reported that the high molecular weight form of IGF-II (big IGF-II) increases in sera and tumors (3, 6, 7, 8), and that the big IGF-II circulates with IGF binding protein 3 (IGFBP-3) as a binary complex but not as the ternary complex (150 kDa) of big IGF-II-IGFBP-3-acid-labile subunit (ALS) in this syndrome (7, 9, 10). After successful treatments, the hypoglycemia disappears, the circulating big IGF-II significantly decreases (6, 11), and the 150-kDa complex increases (10, 12). Therefore, it has been suggested that the impaired formation of 150-kDa complex of big IGF-II could be one of the major causes of hypoglycemia.

The generation of big IGF-II in the NICTH is less clear. Initially IGF-II is synthesized as pro-IGF-II (Fig. 1Go) that consists of 67 amino acids of IGF-II with a carboxyl 89 amino acid extension (E domain), and the mature form is produced by cleavage of the E domain (13). A relatively small amount of big IGF-II is yielded in the processing of pro-IGF-II. Big IGF-II has been isolated from normal sera. The big form, designated as pro-IGF-II-(E1–21), is produced by cleavage after the single lysine at position 21 of the E domain in the processing of pro-IGF-II (14). The pro-IGF-II-(E1–21) is O-linked glycosylated on threonine at position 8 of the E domain (15). It has been reported that big IGF-II in NICTH lacks normal E-domain O-linked glycosylation, suggesting that big IGF-II in NICTH might be generated by an abnormal processing of pro-IGF-II (16). However, we found that the size of big IGF-II in sera from the patients with NICTH is varied (6, 8), and there is a possibility that the size difference of IGF-II might be because of a difference in the size of the sugar moiety attached to IGF-II. Therefore, in the present study, we investigated whether big IGF-II from patients with NICTH is O-glycosylated, and if so, whether the size of the sugar moiety is different among the patients.



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  		Figure 1. Amino acid sequence of human pro-IGF-II. Amino acids enclosed by boxes indicate region of pro-IGF-II-(E1–21). *, O-glycosylation site.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Serum samples

Serum samples were obtained from 10 patients with NICTH. The clinical findings of the patients are shown in Table 1Go. The characterization of IGF-II in sera from these 10 patients has been previously reported (6, 8, 11). Serum samples were also obtained from 5 normal women (age 26–45 yr). The samples were kept at -20 C until they were assayed.


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  		Table 1. Clinical findings in 10 patients with NICTH

 
Enzyme

Neuramidase was purchased from Seikagaku Kogyo Co Ltd. (Tokyo, Japan), and Endo-a-N acetylgalactosaminidase (O-glycosidase: O-Glycanase) from Genzyme Corp. (Cambridge, MA).

Removal of O-linked sugars

Serum IGF-II was extracted with acid-ethanol (12.5% 2N HCl/87.5% ethanol) from 0.2 mL serum, neutralized with 2 M NH4HCO3, and dried in a Speed Vac concentrator (Savant Instruments, Hickville, NY). The dried samples were reconstituted with 0.2 mL 20 mM phosphate buffer (pH 6.0). Each sample (90 µL) was incubated with 5 mU neuramidase for 3 h at 37 C in a total volume of 0.1 mL and then incubated with 1 mU O-glycosidase overnight at 37 C. An aliquot (80 µL) of each sample was diluted in sample buffer (80 µL) to a total volume of 160 µL before separation by electrophoresis on SDS polyacrylamide gel.

Western immunoblot analysis of IGF-II

The samples (NICTH patient, 40 µL; normal subject, 60 µL) treated with or without neuramidase and O-glycosidase, were analyzed by Western immunoblotting (6, 8). Briefly, the samples were electrophoresed on 16% SDS-polyacrylamide gel under nonreducing conditions. The size-fractionated proteins were electroblotted onto nitrocellulose sheet. The sheet was blocked with 5% (wt/vol) skim milk, and then incubated with anti-IGF-II antibody (Amano Pharmaceutical Co., Nagoya, Japan). After extensive washing, the sheet was incubated with horseradish peroxidase-conjugated antimouse IgG, and then IGF-II-anti-IGF-II antibody complexes were detected with enhanced chemiluminescence (ECL) system (Amersham Co., Buckinghamshire, England).

RIA for IGFs

Serum IGF-I and IGF-II were measured by RIA using acid-ethanol extracted samples as reported previously (17, 18). In these RIAs, the normal values for serum IGF-I and IGF-II in adults ranged from 88–240 ng/mL and from 374–804 ng/mL, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Using Western immunoblot analysis, we observed that in normal subjects, the majority of serum IGF-II was detected at 7.5 kDa, the expected size for IGF-II, and a minor amount at 11 kDa. When the sera from normal subjects were digested with neuramidase and O-glycosidase, the 11-kDa form was reduced in size to approximately 9.5 kDa (Fig. 2Go). In contrast, we found that, in the sera from 10 patients with NICTH, most of the circulating IGF-II migrated between 11 and 18 kDa, the fragment size predicted for big IGF-II, and a lesser amount at 7.5 kDa, as expected for IGF-II (Fig. 2Go). The 11- to 18-kDa forms of IGF-II in sera from the patients with NICTH was reduced in size to approximately 9.5 kDa after neuramidase and O-glycosidase digestion comparable with that observed in the enzyme digestions of sera from normal subjects (Fig. 2Go).



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  		Figure 2. Effect of neuramidase and O-glycosidase treatment on size of big IGF-II in sera from patients with NICTH (nos. 1–10) and normal subjects (N1-N5). Extracted IGF-II from sera were treated either without (-) or with (+) neuramidase and O-glycosidase, and then were analyzed by Western immunoblot. Molecular weight markers are indicated on left. *, Recombinant hIGF-II.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the patients with NICTH, the major circulating form of IGF-II is big IGF-II. The presence of big IGF-II could be related to hypoglycemia in the NICTH syndrome, because serum big IGF-II is decreased significantly, and the hypoglycemia disappears after successful removal of the tumor (6, 11). However, the generation of big IGF-II in NICTH is unclear. In normal sera, a relatively small amount of big IGF-II is detected. The big form of IGF-II, designated as pro-IGF-II-(E1–21), is produced by cleavage after the single lysine at position 21 of the E domain in processing of pro-IGF-II (14). The pro-IGF-II-(E1–21) has O-linked glycosylation on threonine at position 8 of the E domain (15).

It would be of interest to know the sugar moiety of big IGF-II in NICTH. Recently, Perdue et al. (19) reported that recombinant unglycosylated pro-IGF-II was about 10 times more potent than glycosylated pro-IGF-II or IGF-II in stimulating thymidine incorporation, suggesting that the sugar moiety modulates the biological activity. Previously, Daughaday et al. (16) reported that big IGF-II in two patients with NICTH lacked normal E-domain O-linked glycosylation, suggesting that big IGF-II in NICTH might be generated by abnormal processing of pro-IGF-II. In contrast to these reports, we found by Western immunoblot analysis, that after neuramidase and O-glycosidase digestion of sera from 10 patients with NICTH, the big IGF-II was reduced in size to 9.5 kDa, similar to that observed in enzyme-treated sera from normal subjects. The differences in the results might be because of the techniques that were used to assess the size of IGF-II. The Western immunoblot could be a more sensitive method than gel-filtration. Our data suggest that the big IGF-II from patients with NICTH is O-glycosylated pro-IGF-II-(E1–21), but, the sizes of sugar moiety are larger than those from normal subjects, suggesting abnormal glycosylation in NICTH. Furthermore, the data do not support an increased bioactivity from the production of a nonglycosylated form of IGF-II.

The mechanism by which big IGF-II produces hypoglycemia remains unclear. Zapf et al. (7) reported that the insulin-like bioactivity of big IGF-II was similar to that of normal IGF-II, suggesting that the intrinsic bioactivity of big IGF-II does not have a major impact on the generation of the hypoglycemia. It has been suggested that the increased bioavailability of big IGF-II, because of an impaired formation of 150 kDa complex, could be a primary factor in hypoglycemia (12). Because the serum ALS levels were low in the patients with NICTH (10), the decreased ALS in sera might be related to the impaired formation of the 150-kDa complex. However, Baxter et al. (12) reported that in one patient with NICTH the big IGF-II inhibited ALS binding to IGFBP-3 in vitro. Therefore, the intrinsic inability of big IGF-II to form the complex could be a major cause of the impaired formation of the 150-kDa complex. Further study of inhibition of ALS binding to IGFBP-3 by abnormal glycosylated big IGF-II is required.


   Acknowledgments
 
We are greatly indebted to Dr. Tomioka, Takamatsu Red Cross Hospital; Dr. Hara, Ohme City Hospital; Dr. Katoh, Omiya Red Cross Hospital; Dr. Hara, Hiroshima University; Dr. Satoh, Showa Hospital; Dr. Kotani, Itami City Hospital; Dr. Shimizu, Toranomon Hospital; Dr. Kuzuya, Omiya Medical Center, Jichi Medical School; and Dr. Horiuchi, Teikyo University for providing serum samples from patients with NICTH, subjects 1–10, respectively. We also greatly thank Dr. M. A. Lesniak for helpful suggestions for our manuscript.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for General Scientific Research (08671184) and a Grant-in Aid for Encouragement of Young Scientists (08770833) from The Ministry of Education, Science and Culture, and a research grant from the Foundation for Growth Science, Japan. Back

Received February 5, 1998.

Revised April 8, 1998.

Accepted May 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Takayama-Hasumi S, Eguchi Y, Satoh A, Morita C, Hirata Y. 1990 Insulin autoimmune syndrome is the third leading cause of spontaneous hypoglycemic attacks in Japan. Diabetes Res Clin Pract. 10:211–214.[CrossRef][Medline]
  2. Daughaday WH. 1991 Autocrine, paracrine and endocrine manifestation of insulin-like growth factor secretion by tumors. In: Spencer EM, ed. Modern concepts of insulin-like growth Factors. New York: Elsevier Science Publishing Co.; 557–565.
  3. Daughaday WH, Emanuele MA, Brooks MH, Barbato AL, Kapadia M, Rotwein P. 1988 Synthesis and secretion of insulin-like growth factor II by a leiomyosarcoma with associated hypoglycemia. N Engl J Med. 319:1434–1440.[Abstract]
  4. Zapf J, Walter H, Froesch ER. 1981 Radioimmunological determination of insulin-like growth factors I and II in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia. J Clin Invest. 68:1321–1330.
  5. Widmer U, Zapf J, Froesch ER. 1982 Is extrapancreatic tumor hypoglycemia associated with elevated levels of insulin-like growth factor II? J Clin Endocrinol Metab. 55:833–839.[Abstract]
  6. Hizuka N, Fukuda I, Takano K, Okubo Y, Asakawa-Yasumoto K, Demura H. 1998 Serum insulin-like growth factor II in 44 patients with non-islet cell tumor hypoglycemia. Endocr J. 45(Suppl):S61–S65.
  7. Zapf J, Futo E, Peter M, Froesch ER. 1992 Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia? J Clin Invest. 90:2574–2584.
  8. Enjoh T, Hizuka N, Perdue JF, et al. 1993 Characterization of new monoclonal antibodies to human insulin-like growth factor-II and their application in Western immunoblot analysis. J Clin Endocrinol Metab. 77:510–517.[Abstract]
  9. Fukuda I, Hizuka N, Takano K, et al. 1994 Circulating forms of insulin-like growth factor II (IGF-II) in patients with non-islet cell tumor hypoglycemia. Endocrinol Metab. 1:89–95.
  10. Baxter RC, Daughaday WH. 1991 Impaired formation of the ternary insulin-like growth factor-binding protein complex in patients with hypoglycemia due to nonislet cell tumors. J Clin Endocrinol Metab. 73:696–702.[Abstract]
  11. Fukuda I, Hizuka N, Takano K, Asakawa-Yasumoto K, Shizume K, Demura H. 1993 Characterization of insulin-like growth factor II (IGF-II) and IGF binding proteins in patients with non-islet-cell tumor hypoglycemia. Endocr J. 40:111–119.[Medline]
  12. Baxter RC, Holman SR, Corbould A, Stranks S, Ho JP, Braund W. 1995 Regulation of the insulin-like growth factors and their binding proteins by glucocorticoid and growth hormone in nonislet cell tumor hypoglycemia. J Clin Endocrinol Metab. 80:2700–2708.[Abstract]
  13. Daughaday WH, Rotwein P. 1989 Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev. 10:68–91.[CrossRef][Medline]
  14. Zumstein PP, Luthi C, Humbel RE. 1985 Amino acid sequence of a variant pro-form of insulin-like growth factor II. Proc Natl Acad Sci USA. 82:3169–3172.[Abstract/Free Full Text]
  15. Hudgins WR, Hampton B, Burgess WH, Perdue JF. 1992 The identification of O-glycosylated precursors of insulin-like growth factor II. J Biol Chem. 25:267:8153–8160.
  16. Daughaday WH, Trivedi B, Baxter RC. 1993 Serum "big insulin-like growth factor II" from patients with tumor hypoglycemia lacks normal E-domain O-linked glycosylation, a possible determinant of normal propeptide processing. Proc Natl Acad Sci USA. 90:5823–5827.[Abstract/Free Full Text]
  17. Miyakawa M, Hizuka N, Takano K, et al. 1986 Radioimmunoassay for insulin-like growth factor I using biosynthetic IGF-I. Endocrinol Jpn. 33:795–801.[Medline]
  18. Asakawa K, Hizuka N, Takano K, et al. 1990 Radioimmunoassay for insulin-like growth factor II. Endocrinol Jpn. 37:607–614.[Medline]
  19. Yang CQ, Zhan X, Hu X, Kondepudi A, Perdue JF. 1996 The expression and characterization of human recombinant proinsulin-like growth factor II and a mutant that is defective in the O-glycosylation of its E domain. Endocrinology. 137:2766–2773.[Abstract]



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