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Endocrinology Vol. 148, No. 10 4803-4811
Copyright © 2007 by The Endocrine Society

Activation of Endoproteolytic Processing of Insulin-Like Growth Factor-II in Fetal, Early Postnatal, and Pregnant Rats and Persistence of Circulating Levels in Postnatal Life

Qing Qiu, Jin-Yi Jiang, Michael Bell, Benjamin K. Tsang and Andrée Gruslin

Hormones, Growth, and Development Program (Q.Q., J.-Y.J., M.B., B.K.T., A.G.), Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9; and Departments of Obstetrics and Gynaecology (A.G.) and Cellular and Molecular Medicine (B.K.T.), University of Ottawa, The Ottawa Hospital, Ottawa, Ontario, Canada K1H 8L6

Address all correspondence and requests for reprints to: Andrée Gruslin, Department of Obstetrics and Gynecology, The Ottawa Hospital, Ottawa, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6. E-mail: agruslin{at}ottawahospital.on.ca; or Qing Qiu, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9. E-mail: qqiu{at}ohri.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The process of posttranslational modifications of IGF-II likely has important physiological consequences. In addition to mature IGF-II, glycosylated proIGF-II(156-amino acid peptide) and two glycosylated big IGF-II forms, IGF-II(1–104) and IGF-II(1–87), have been identified in the human circulation. Due to lack of an appropriate methodology, different IGF-II isoforms have not been demonstrated and characterized in the rat circulation, thus preventing a better understanding of the physiological and pathological roles of IGF-II. In the present study, we characterized each IGF-II form and assessed its content in the rat circulation throughout life time by using a highly sensitive Western blot analysis, which is void of the IGF binding protein interference and distinguished all IGF-II forms. For the first time, we demonstrated the presence of IGF-II variants, including proIGF-II, IGF-II(1–87), and mature IGF-II, in the rat circulation during postnatal life, challenging the current impression that IGF-II is absent from sera of adult rats. ProIGF-II is glycosylated and is the predominant form in the rat circulation. Endoproteolytic processing of proIGF-II was clearly activated in fetal, neonatal, and pregnant rats, likely reflecting its involvement in fetal development through the generation of specific forms of IGF-II (e.g. mature IGF-II) that are required for their distinct biological functions. Taken together, our data also suggest that serum IGF-II profiles may reflect underlying physiological conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-II IS A POTENT mitogen and is involved in the regulation of embryonic and placental growth (1) as well as tumorigenesis (2). It is activated by endoproteolytic processing (3). Human IGF-II is synthesized as proIGF-II peptide (156 amino acids) after the removal of a 24-amino acid signal peptide at the N terminal. ProIGF-II is then cleaved, generating IGF-II(1–104), a peptide with 104 amino acids (4). IGF-II(1–104) is subsequently endoproteolyzed, resulting in the formation of mature IGF-II, a 7.5-kDa peptide containing 67 amino acids (3). In addition to IGF-II(1–104), IGF-II(1–87) has also been identified in human and bovine serum (5, 6, 7, 8). IGF-II(1–104) and IGF-II(1–87), also referred to as "big" IGF-II, constitutes 10–15% of the total circulating IGF-II in human (9, 10, 11). Human pro and big IGF-II, but not mature IGF-II, is glycosylated (4, 8). To date, little information is available on the posttranslational modification of IGF-II in the rodent and on the presence of IGF-II variants in the circulation, even though this information would potentially be helpful in understanding the biological function of IGF-II and its variants. Human and rat IGF-II protein sequences share 85% homology and contain identical proprotein convertase cleavage sites, suggesting that the same endoproteolytic processing may occur in IGF-II maturation in both species and may generate similar IGF-II products (Fig. 1Go).


Figure 1
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  		FIG. 1. ProIGF-II peptide sequences in mouse, rat, and human as well as the schematic representation of the structures of IGF-II variants. Mouse and rat proIGF-II share 85% homology with human proIGF-II. They all contain the same cleavage motifs as indicated in the areas shaded and bold. The cleavage sites are indicated by arrowheads. The potential glycosylation sites of proIGF-II in rodent and the known glycosylation sites in human are indicated by bold and italic and are underlined. Glycosylated rodent proIGF-II, two big IGF-II isoforms including IGF-II(1–104) and IGF-II(1–87), and mature IGF-II are schematically represented. PCSK4, Proprotien convertase 4.

 
It is well established that the IGF system plays a critical role in the regulation of prenatal growth and development in the rodent and human (12). IGF-II has a similar prenatal expression pattern in both species (13). However, in contrast to human IGF-II, which continues to be expressed after birth, the expression of IGF-II mRNA in the rodent fetus gradually decreases and becomes extinguished 3 wk after birth in most tissues with the exception of the cerebral cortex, hypothalamus, and spinal cord (14, 15). Whereas IGF-II has a clearly established role in embryonic and fetal development, its role during postnatal life remains unclear (16). A number of studies using transgenic mice suggested that IGF-II may be involved postnatally in organ growth, carbohydrate and lipid metabolism, and tumorigenesis (17, 18). In that context, recent studies suggested that circulating IGF-II may prevent a wave of developmentally regulated apoptosis in the pancreatic islets during early postnatal life (19). In addition, during pregnancy, circulating IGF-II has been shown to regulate the structure and function of the placenta (20).

The paucity of information on the characteristics of circulating IGF-II and its potential postnatal function(s) is further compounded by the difficulties in adequately measuring IGF-II in the serum (16). Traditionally, immunoassays, including RIA and ELISA, have been used in the determination of circulating IGF-II, although the reliability of these assays is compromised by interference from IGF binding proteins (IGFBPs) present in the samples (21). To circumvent this problem, acid/ethanol extractions are commonly used in these assays. However, although mature IGF-II is soluble in acid/ethanol (22), big IGF-II is not completely extracted with this method (23). Furthermore, all IGF-II peptide variants (pro-, big, and mature IGF-II) display similar immunoreactivity to antibodies raised against mature IGF-II, thus rendering these immunoassays incapable of distinguishing different IGF-II peptides. Serum IGF-II levels measured by RIA are remarkably stable, not changing with altered hormonal or metabolic status or under various pathological conditions (16). Even in nonislet cell tumor-induced hypoglycemia (NICTH), in which big IGF-II is dramatically increased and believed to play a pathological role in the development of hypoglycemia, deviations from normal levels in the circulating concentrations of IGF-II have not been detected (9, 24, 25).

In the present study, we assessed the peptide contents of all IGF-II isoforms in the circulation of fetal, postnatal, and pregnant rats with a highly sensitive assay that is not complicated by the presence of IGFBPs in serum samples. We characterized all IGF-II isoforms in the rat circulation and demonstrated for the first time that, in contrast to reports showing extinct expression 3 wk of postnatal life (26), IGF-IIs are persistently present in the postnatal circulation. We also demonstrated that the conversion of proIGF-II to big and mature IGF-II is activated in the fetal, neonatal, and pregnant rats, indicating that IGF-II processing is likely of physiological importance during these critical stages of development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and blood collection
All animal studies were carried out in compliance with the Guidelines of the Canadian Council on Animal Care and approved by the Animal Care and Ethics Committee of the Ottawa Health Research Institute. Eight 4-month-old healthy (two males and six females) and eight pregnant Sprague Dawley rats were provided by Charles River Laboratories (Wilmington, MA). Blood samples from eight adult rats and four pregnant rats at gestational age embryonic d 18.5 were collected by direct cardiac puncture under isoflurane anesthesia. After collecting the maternal blood samples, the pregnant rats were killed and fetal blood samples were also collected by decapitation. The other four pregnant rats were maintained for delivering and carrying pups. Four neonatal rats from four different litters were killed at 1, 5, 10, 15, 20, 25, and 50 d of age by decapitation (1–10 d old) or heart puncture (15–50 d old) for blood collection. In addition, blood samples (50 µl) from the maternal rats were also collected from the dorsal pedal vein at postnatal d 5. Sera were separated and stored at –20 C for subsequent analysis.

Determination of IGF-II profile by Western blot analysis
Aliquots of 1 µl of sera were diluted with water to 10 µl and subjected to electrophoresis with 15% tricine SDS-PAGE. To quantify the IGF-II concentrations in the serum samples, serially diluted recombinant mouse mature (rm) IGF-II (100, 50, 25, 12.5, and 6.25 pg) were loaded (as reference) onto the same gel along with the samples under nonreducing condition. To obtain the best separation of IGF-II variants, electrophoresis was run at constant voltage of 100 V until the 7-kDa prestained protein molecular mass marker migrated to the bottom of the gel. The separated proteins were blotted onto a nitrocellulose membrane, which was treated with antibody extender solution (Pierce, Rockford, IL), blocked with ECL Advance blocking agent (GE Healthcare, Buckinghamshire, UK), and subsequently probed with mouse antirat IGF-II monoclonal antibody (clone S1F2; Upstate, Lake Placid, NY). Bands of IGF-II variants were visualized with ECL or advanced ECL reagents and their relative contents were densitometrically quantified with {alpha} Ease FCTM software (Alpha Innotech, San Leandro, CA). The assay detected a quantity of rmIGF-II (see Fig. 4AGo) and recombinant human IGF-II (data not shown) as low as 6.25 pg, which is 500 times more sensitive than the Western blot reported by others [detection limit for recombinant human IGF-II: 3 ng (27)]. To confirm the specificity of IGF-II variants detected by S1F2 IGF-II monoclonal antibody, samples were treated with reducing reagent and the Western blot analysis was performed using goat antimouse IGF-II polyclonal antibody (no. I 2282; Sigma, St. Louis, MO) as primary antibody. To compare the relative contents of IGF-II variants determined in different blots, an aliquot of 50 pg rmIGF-II was loaded to each gel as an internal control. The density of IGF-II variants were normalized by the density of its internal control.


Figure 4
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  		FIG. 4. ProIGF-II predominantly exists in the circulation of adult rats and fails to be extracted by acid/ethanol treatment. A, Western blots showing density of proIGF-II in rat sera and that of serially diluted rmIGF-II. Aliquots of 1 µl sera from four adult rats (two male and two female) were applied to Western blot analysis, respectively, along with a serial dilution of rmIGF-II (6.25, 12.5, 25, 50, 100 pg) as references. The density of bands on the blot was obtained after a 1-min exposure. B, Western blot in the boxed area of the above panel showing bands representing big and mature IGF-II with 10 min of exposure. C, Graphic representation of the concentrations of pro-, big, and mature IGF-II in the rat circulation. The densities of bands representing pro-, big, and mature IGF-II in the serum samples were densitometrically measured and calculated based on the standard curve of rmIGF-II. The numbers on the top of the bars represent the percentages (mean ± SE) of each variant of IGF-II content in total IGF-II content. D, Failure of proIGF-II extraction by acid/ethanol treatment, a common procedure used in RIA. Aliquots of 20 µl of adult rat sera and newborn rat sera were extracted with acid/ethanol solution and neutralized with Tris. The 1 µl of serum and 20 µl extracted solutions equivalent to 1 µl serum were subjected to Western blot analysis.

 
Antibody preabsorption
Mouse recombinant IGF-II was added to goat antimouse IGF-II antibody solution (100 ng/ml) to make a final concentration of mouse recombinant IGF-II at 5 µg/ml and incubated at least 1 h at room temperature. Duplicates of mouse serum samples were subjected to electrophoresis and transferred to nitrocellulose membrane. The membrane was split into two: one was probed with antibody without preabsorption and the other incubated with antibody preincubated with recombinant IGF-II. Other procedures were followed as described above in IGF-II profile determination.

Furin-mediated proIGF-II endoproteolytic processing
Aliquots of recombinant proIGF-II (10 ng; GroPep Ltd., Adelaide, Australia) were treated with recombinant furin (0.25 U; ABR Affinity BioReagents, Golden, CO) at 37 C for different durations (0, 0.5, 1, 2, 4, 8 h) in Tris-HCl buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl2] at a final volume of 30 µl. The molecules of processed proIGF-II and generated IGF-II(1–104) were assessed by Western blot analysis with mouse antirat IGF-II antibody. The reduced densities of proIGF-II signals and increased densities of IGF-II (1–104) signals, as furin incubation periods increased, were quantitatively analyzed.

Deglycosylation treatment
In the initial experiments, treatment with either enzymatic or chemical deglycosylating reagent failed to completely remove the sugar moieties. In this study, the rat sera were treated with sialidase to enzymatically remove sialic acids, followed by trifluoromethanesulfonic acid (TFMS) hydrolysis. The chemical deglycosylation treatment was performed with chemical deglycosylation kit (Sigma). In brief, aliquots of 10 µl sera were treated overnight at 37 C in glass reaction vials with 1 µl sialidase (Roche Diagnostics, Laval, Québec, Canada) in 10 mM phosphate buffer (pH 6.0) at a final volume of 20 µl and lyophilized. An aliquot of 60 µl precooled TFMS was added to the precooled sample vial at –20 C and the reaction vial was incubated for 2 h at –20 C with occasional shaking. Excess TFMS was neutralized with 120 µl of 60% pyridine precooled at –20 C. The 100 µl of reaction mixture was dialyzed overnight against 10 mM Tris-HCl buffer with 50 mM NaCl (pH 7.5), using a dialysis cassette with the cut-off size of 3500 Da. The dialyzed samples were concentrated to 100 µl with 3 kDa cut-off microcentricon. The concentrated sample (10 µl) was subjected to Western blot analysis to determine the IGF-II variants as described above. As a control, human serum obtained from a healthy subject was treated with the same deglycosylation procedure.

Acid-ethanol extraction
An aliquot (20 µl) of sera was mixed with 250 µl of acid/ethanol extract solution (12% 2 N HCl and 87.5% ethanol) by vortex. The mixture was left at room temperature for 30 min and then centrifuged (14,000 rpm, 5 min). The acidic supernatant was neutralized with 125 µl of 855 mM Tris, vacuum dried to approximately 20 µl, and reconstituted to 200 µl with H2O. An aliquot (20 µl) of the extract and 1 µl of original serum were subjected to Western blot analysis.

Statistics
Results are expressed as mean ± SEM. Statistical comparisons of IGF-II variants and ratio of proIGF-II content to the content of processed IGF-II isoforms within nonpregnant, pregnant, and lactating groups were made by ANOVA, followed by Bonferroni test, using Sigmastat software (Systat Software, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pro-, big, and mature IGF-II exists in the rodent circulation
Using Western blot analysis, multiple bands ranging from 7.5 to 34 kDa were detected in newborn rat sera with mouse antirat IGF-II(1–67) monoclonal antibody, one known to cross-react with big and proIGF-II (Fig. 2AGo). Multiple bands ranging from 22 to 34 kDa represent proIGF-II, whereas two bands between 11 and 17 kDa were big IGF-IIs. Mature IGF-II was observed in the lower portion of the gel. Similar results were noted using goat antimouse IGF-II polyclonal antibody in the assay. To further confirm the specificity of the assay, the goat antimouse IGF-II antibody was preabsorbed with or without recombinant mouse IGF-II, and all bands disappeared in the blot probed with the preabsorbed antibody (Fig. 2BGo). Similar results were observed in our previous study (3) in confirming specificity of mouse antirat IGF-II antibody. This information suggests that all bands detected were specific and represented different IGF-II forms. However, it is also questionable whether the antibody has different affinities toward IGF-II molecular forms, which may lead to a misinterpretation of the peptide content when comparing the signals for each IGF-II form. It has been shown that proIGF-II is endoproteolytically processed to generate IGF-II(1–104) by furin (4). The change of signal densities between IGF-II forms in furin-mediated proIGF-II processing was monitored by Western blot analysis. This revealed that the reduction in density of proIGF-II is proportional to the increased density of IGF-II(1–104) (Fig. 2CGo). These data suggest that the quantification and comparison of the various IGF-II forms is valid using this Western blot analysis.


Figure 2
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  		FIG. 2. Presence of pro-, big, and mature IGF-II in the rat circulation as detected by Western blot analysis. A, Western blots, using two different antibodies, respectively, showing IGF-II variants exist in the newborn rat circulation. An aliquot of 1 µl of newborn rat serum was subjected to Western blot analysis using mouse antirat IGF-II monoclonal antibody. The experiment was repeated using goat-antimouse polyclonal antibody and generated similar results. B, Confirmation of the specificity of the Western blot analysis by antibody absorption test. A duplicate of mouse serum sample was subjected to Western blot analysis using antibody with or without IGF-II preabsorption, respectively. Disappearance of the bands using antibody preabsorbed with recombinant mouse IGF-II confirmed the specificity of the antibody used in the assay. C, The signal densities shown on Western blot between IGF-II forms are equivalent to their relative molar amount of peptides. Aliquots of recombinant proIGF-II were treated with recombinant furin for different durations (0, 0.5, 1, 2, 4, 8 h). The processed proIGF-II and generated IGF-II(1–104), resulting from furin-mediated proIGF-II endoproteolysis, were monitored by Western blot analysis with mouse antirat IGF-II antibody (upper panel). Quantitative data revealed that reduced density of proIGF-II signal and increased density of IGF-II(1–104) signal are equivalent (lower panel), suggesting that the antibody has similar affinity for both molecular forms and the signals are comparable in reflecting relative amounts of peptides in molarities between IGF-II forms.

 
Pro- and big IGF-II, but not mature IGF-II, is glycosylated in the rodent circulation
The migratory differences of IGF-II proteins detected by electrophoresis could be due to either different sizes of IGF-II peptides (i.e. different isoforms) or the same isoform but with various carbohydrate residues. In the human, pro-, and big IGF-II is glycosylated (4). To identify whether those peptides in the rodent model are glycosylated, rat fetal sera were treated with sialidase, followed by chemical deglycosylation treatment with TFMS. These treatments resulted in the disappearance of the bands between 22 and 34 kDa and the appearance of one at 18.5 kDa (Fig. 3Go), suggesting that they were proIGF-II with various glycosylations and that the 18.5-kDa band is a deglycosylated proIGF-II. Unlike the appearances of two deglycosylated forms of big IGF-IIs [IGF-II(1–104) and IGF-II(1–87)] in human serum, only one deglycosylated big IGF-II form appearing in size similar to human IGF-II(1–87) was observed in rat serum (Fig. 3Go). The data suggest that big IGF-II form is glycosylated IGF-II(1–87). IGF-II(1–104) was not detected in the rat serum (Fig. 3Go). As expected, the 7.5-kDa mature IGF-II was not glycosylated as indicated by the lack of change in its migration after deglycosylation reaction (Fig. 3Go).


Figure 3
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  		FIG. 3. Pro- and big IGF-IIs, but not mature IGF-II, are glycosylated in the rat circulation. The newborn rat serum sample was treated with sialidase and followed by TFMS hydrolysis to remove the sugar moieties attached to IGF-II. The serum samples, with or without deglycosylation treatment, were subjected to Western blot analysis to detect IGF-II variants. A normal human serum sample was analyzed with the same treatment as the control.

 
ProIGF-II predominantly exists in the circulation of adult rats
Because serum levels of IGF-II are undetectable with conventional immunoassays in adult rats, it has been accepted that IGF-II is absent from the circulation of those adult rodents. Surprisingly, we observed that proIGF-II was predominantly present in the circulation of four adult rats (two males and two females, Fig. 4AGo). The big and mature IGF-IIs were also clearly detected as shown in the blot with a longer exposure time (Fig. 4BGo). Quantified data obtained by using rmIGF-II as references, revealed that proIGF-II was 33.3 ± 5.1 nM and constituted 95.2 ± 1.2% of the total circulating IGF-II, whereas concentrations of mature and big IGF-II were 0.7 ± 0.2 and 0.9 ± 0.5 nM, respectively (Fig. 4CGo). To determine the reason(s) for the failure of the conventional immunoassays to detect proIGF-II in the adult rat circulation, sera were treated with acid/ethanol (to liberate IGF-II from IGFBPs) according to a common procedure used in the immunoassay to avoid interference from those binding proteins. IGF-II isoforms in the extracts were then examined by Western blotting. Although the density of mature IGF-II in the extracted solution in newborn rat was not different, compared with that in the serum, the density of proIGF-II in the extracted solution was much weaker in newborn rat, and it was not recovered at all in the adult sera (Fig. 4DGo).

Endoproteolytic processing of proIGF-II is activated during fetal and early postnatal life in the rat model
Serum concentration of IGF-II as determined by RIA has been shown to be highest in rat fetuses and to gradually decrease after birth (26). In this study, we investigated the changes in serum level of each IGF-II variants during this period. Serum levels of mature and big IGF-II gradually decreased after birth, following a similar trend as previously described by RIA (26). However, as shown in Fig. 5Go, the changes in proIGF-II levels were biphasic, with the content of proIGF-II increased to a peak at d 15 after birth and decreased thereafter. During early postnatal life (before 15 d after birth), a decrease in the processed forms of IGF-II (big and mature IGF-II) was accompanied by an increase in proIGF-II, reflecting a reduction in endoproteolytic processing of proIGF-II. In contrast, the levels of all IGF-II isoforms decreased after 15 d postnatal age, suggesting a reduction of IGF-II expression. In proIGF-II, a less glycosylated form of 22 kDa was dominant in the fetal and neonatal rat circulation, whereas proIGF-II of 26 kDa, with a higher degree of glycosylation, was principal component in postnatal d 5 and after (Fig. 5AGo). The relative abundance of proIGF-II in relation to the total circulating IGF-II increased beyond 95% 25 d after birth and thereafter (Fig. 5BGo), suggesting the proIGF-II endoproteolysis is not activated in later postnatal life.


Figure 5
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  		FIG. 5. Endoproteolytic processing is activated in fetal and neonatal rats. A, A representative image of Western blot detecting IGF-II variants from fetal and early postnatal rat sera. Blood samples were collected from fetal rats at gestational day (GD) 18.5 and young rats at postnatal d 1, 5, 10, 15, 20, 25, and 50. Aliquots (1 µl) of sera were subjected to Western blot analysis. B, The content of each IGF-II variant was quantified by densitometric determination in four replicates and graphically represented (upper panel). The percentages of each IGF-II variant content present in the total circulating IGF-II are also presented (lower panel). Values are expressed as means ± SE (n = 4).

 
Endoproteolysis of proIGF-II is reactivated during pregnancy in the rat
We then investigated whether pregnancy influences the profile of IGF-II isoforms. Consistent with results shown in normal adult rats, the majority of IGF-II present in the circulation was highly glycosylated proIGF-II (≥26 kDa) in nonpregnant rats (Fig. 6AGo). The content of proIGF-II decreased during pregnancy, compared with that of nonpregnant rats (P < 0.01), but increased again after parturition (P < 0.05), whereas the contents of mature IGF-II significantly increased during pregnancy, compared with that found in nonpregnant rats (P < 0.05) (Fig. 6Go, A and B). The ratio of proIGF-II to its processed forms (big + mature IGF-II) was significantly lower in pregnant than that in nonpregnant rats (P < 0.001) and rats after parturition (P < 0.05) (Fig. 6CGo), clearly suggesting that pro-IGF-II processing is activated during pregnancy.


Figure 6
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  		FIG. 6. Endoproteolysis of proIGF-II is activated during pregnancy in the rat. Aliquots of 1 ml sera from nonpregnant and pregnant (gestation age d 18.5) rats as well as lactating rats at d 5 after parturition was applied to Western blot analysis to determine the contents of IGF-II variants. An aliquot of 50 pg rmIGF-II was loaded to each gel as a reference of internal control to confirm the densities of IGF-II are comparable between blots. A, Western blots showing the relative density of rat serum IGF-II variants. B, Graphic representation of pro-, big, and mature IGF-II contents in each group. C, The bar graph represents the ratio of the contents of proIGF-II to processed IGF-II forms (big and mature IGF-II). Values are expressed as means ± SE (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have, for the first time, demonstrated that pro-, big, and mature IGF-IIs are persistently present in rat circulation. We have shown that proIGF-II is glycosylated and is the predominant form of IGF-II in the circulation of adult rats. In contrast to both glycosylated IGF-II(1–87) and IGF-II(1–104) found in human sera, only glycosylated IGF-II(1–87) in rats was detected. Furthermore, we observed in this model that IGF-II endoproteolytic processing is active during fetal and early neonatal periods and is reactivated during pregnancy, suggesting that this process is of physiological importance at these critical developmental stages. We suggest that this is related to the growth-promoting function of mature IGF-II.

We have previously shown that posttranslational modification of IGF-II alters its biological functions (3). In the human, proIGF-II is endoproteolyzed by furin and other proprotein convertases at the cleavage motif RLRR104{downarrow} to generate IGF-II(1–104) (4). IGF-II(1–104) is further processed by proprotein convertase 4 at KSER68 {downarrow} to produce mature IGF-II (N terminal fragment) and IGF-II(69–104) (C-terminal fragment) (3). These cleavage sites are identical in the human and rodent proIGF-II (Fig. 1Go), suggesting that the same endoproteolysis may occur in the rodent proIGF-II. This notion is supported by the fact that the IGF-II(69–102) peptide named peptin is secreted from rat pancreatic islet ß-cells (28). The Arg(104) and Arg(103) of the C terminal of IGF-II(69–104) peptide could be removed subsequently by carboxypeptidase (29), thus producing the peptide fragment of IGF-II(69–102). Failure of the detection of IGF-II(1–104) in rat sera could be explained by the results of quick turnover of IGF-II(1–104) into mature IGF-II. In addition to endoproteolysis, glycosylation also occurs as part of the posttranslational modifications of proIGF-II. The degree of glycosylation of proIGF-II was also development dependent, as evident by proIGF-II with smaller size (22 kDa) and larger size (≥26 kDa) being dominant before and after postnatal d 5, respectively. The inverse association of contents of highly glycosylated proIGF-II [(≥26 kDa) and processed forms (big and mature IGF-II) suggests that it is possible the different degree of glycosylation status could negatively influence the IGF-II processing. Our study provides information of these modifications, which will help in identification of isoforms of IGF-II and in the clarification of their biological properties.

IGF-II profiles in the circulation may be reflective of the physiological and pathological importance of IGF-II under various conditions. A higher rate of conversion of proIGF-II to big and mature IGF-II in fetuses was observed in this study. This supports the concept that big and mature IGF-IIs are biologically active and is consistent with the physiological role of IGF-II during fetal development. Whereas there is information on the growth-promoting role of IGF-II during embryonic and fetal development, its physiological function during postnatal/adult life remains unknown (16). The profile of proIGF-II to big and mature IGF-II in early postnatal rats (before d 15) (Fig. 5Go) is consistent with our notion that bioactive forms of IGF-IIs are present in the circulation and are important for early postnatal development in rats. For example, it has been shown that the persistent presence of circulating IGF-II postnatally in transgenic mice prevents the wave of developmentally regulated apoptosis in pancreatic islets in the second week after birth (19). During pregnancy, the reactivation of endoproteolytic processing of proIGF-II that we observed may enhance its endocrine effects on placental structure and function. This hypothesis is supported by the observation that increased maternal circulating mature IGF-II resulting from a direct IGF-II infusion resulted in an increased volume and surface area of the exchange region of the placenta near term in the guinea pig (20). To date, there is very limited information regarding changes in IGF-II profiles under pathological conditions, although these have been documented in at least two conditions, namely NICTH (9) and fetal growth restriction (3). A very significant increase in serum proIGF-II has been shown in NICTH with this variant constituting 60–80% of the total circulating IGF-II, compared with 10–20% under normal conditions (9). We also previously reported a significant increase in the ratio of pro-IGF-II to mature IGF-II in mothers carrying growth-restricted fetuses, reflecting a decrease in processing and likely a decline in biological function of this growth factor (3).

Because IGF-II mRNA expression is extinguished in most tissues postnatally except for the nervous system (14) and because proIGF-II is the predominant form present in the circulation of nonpregnant adult rats (present study), it is likely that the major source of proIGF-II originates from the nervous system and is not endoproteolytically processed. However, the placenta, an organ in which IGF-II mRNA is highly expressed (13), may be the source of circulating IGF-IIs during pregnancy. Due to the presence of proprotein convertases, such as proprotien convertase 4 (3), which are involved in the endoproteolytic processing of IGF-II in the placenta, proIGF-II is converted to big and mature IGF-II intracellularly before these forms are secreted. This may explain the change of IGF-II profiles seen during pregnancy.

Immunoassays are the most commonly used method in the determination of circulating IGF-II concentration. In addition, a number of methods have been described for the measurement of big IGF-II. In earlier assays, big IGF-II and mature IGF-II were separated from different fractions using size-exclusion gel chromatography and followed by determination with RIA (9, 30). This tedious and time-consuming protocol limited the application of the assay. Recently a sandwich assay, with a coating antibody specific for E peptide (amino acid 78–88) and a detection antibody against mature IGF-II has been developed for the measurement of proIGF-II (23, 31). However, this assay has limited value because both the E peptide and the mature IGF-II peptide are present in proIGF-II and big isoforms, IGF-II(1–87) and IGF-II(1–104), thus preventing differentiation between these variants. In contrast, through separation by tricine-SDS-PAGE, the assay used in the present study effectively distinguished every IGF-II variant. An assay using a similar strategy has been reported in the detection of human proIGF-II in NICTH patients (32), although it required 5 µl sera and may not be sensitive enough to detect IGF-II in rodents. In this study, the requirement of a small amount (1 µl) of serum sample makes it possible to assess the IGF-II profile changes between certain developmental periods in rats by repeated determination of IGF-II levels without killing the animals. Furthermore, IGF-IIs are dissociated from binding proteins by denaturation before the gel electrophoresis to eliminate interference by IGFBPs, a previous limitation encountered in the conventional immunoassays (19, 21, 23, 26, 33). In addition, the data from furin-mediated proIGF-II processing in this study and proprotien convertase 4-mediated IGF-II(1–104) processing in our previous study (3) suggest that the signal densities of all IGF-II forms shown on Western blot analysis are equivalent to their absolute amounts (molar mass) of peptides. The Western blot signals are comparable between each form in the quantification of IGF-II content.

In conclusion, we have demonstrated for the first time, by using a highly sensitive assay that allows the determination of the content of IGF-II variants, that pro-, big, and mature IGF-IIs are persistently present in the circulation of adult rats. Endoproteolytic processing of proIGF-II was clearly activated in fetal, neonatal, and pregnant rats, likely reflecting its involvement in fetal development through the generation of specific and active isoforms of IGF-II (e.g. mature IGF-II) that are required for their distinct biological functions. Taken together, our data also suggests that serum IGF-II profiles may reflect underlying physiological conditions.


   Footnotes
 
This work was supported by the Physicians’ Service Incorporated Foundation (to A.G. and Q.Q.) and Canadian Institutes of Health Research (to A.G. and Q.Q.).

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online July 12, 2007

Abbreviations: IGFBP, IGF binding protein; NICTH, nonislet cell tumor-induced hypoglycemia; rm, recombinant mouse mature; TFMS, trifluoromethanesulfonic acid.

Received April 24, 2007.

Accepted for publication July 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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