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Endocrinology Vol. 140, No. 5 2232-2240
Copyright © 1999 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor (IGF) Binding Proteins Modulate the Glucocorticoid-Dependent Biological Effects of IGF-II in Cultured Fetal Rat Hepatocytes1

Pierrette Menuelle, Sylvie Babajko and Christiane Plas

Laboratoire de Biologie-Odontologie, Université Paris 7, Institut Biomédical des Cordeliers (P.M., C.P.), Paris, France; and Institut de la Santé et de la Recherche Médicale, Unité de Recherches sur la Régulation de la Croissance, Hôpital Saint Antoine (S.B.), Paris, France

Address all correspondence and requests for reprints to: Dr. P. Menuelle, Laboratoire de Biologie-Odontologie, U.F.R. Odontologie, Université Paris 7, Institut Biomédical des Cordeliers, 15 rue de l’Ecole de Médecine, 75270 Paris Cedex 06, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of insulin-like growth factor binding proteins (IGFBPs) in regulation by IGF-II of glycogenesis and DNA synthesis was investigated in hepatocytes isolated from fetal rat livers at days 15 and 18 of gestation and grown in the presence or absence of cortisol. IGFBP-1 was clearly revealed by Western ligand blot and immunoblot analysis of IGFBPs secreted into conditioned media. Its production and cellular messenger RNA (mRNA) were positively regulated by cortisol and increased in older cells. In the absence of IGFBP (fresh medium), glycogenesis, and DNA synthesis were stimulated by IGF-II and insulin. In each case, cortisol enhanced this stimulation. In the presence of IGFBPs (cell-conditioned media), IGF-II stimulation of DNA synthesis and to a lesser extent glycogenesis was inhibited. The degree of inhibition was directly related to IGFBP-1 production. IGFBPs had no effect on stimulation of glycogenesis and DNA synthesis by des(1–6)IGF-II, a structural analog of IGF-II that does not bind to IGFBPs. Insulin, whose biological effects were not modified by conditioned media, inhibited IGFBP-1 production. Comparison of the dose dependence of the two bioactivities showed that DNA synthesis was more sensitive to IGF-II than glycogenesis. Our results suggest that in the case of DNA synthesis the effects of IGF-II are mediated via the IGF-I receptor and those of insulin via the insulin receptor, whereas in the case of glycogenesis both are mediated via the insulin receptor. In conclusion, IGF-II and insulin stimulation of glycogenesis and DNA synthesis in cultured fetal hepatocytes depends on the presence of glucocorticoid and the stage of development. IGF-II action is negatively regulated by IGFBP-1 whose synthesis increases in the presence of glucocorticoids.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DESPITE extensive research, no specific endocrine mechanism has as yet been identified that plays the same role in fetal growth as GH does in childhood. Consequently, attention has been focused on the autocrine and paracrine action of growth factors, especially the insulin-like growth factors (IGFs) (reviewed in Ref. 1). IGF-I and IGF-II play essential roles in cell metabolism, proliferation and differentiation and to this extent have major effects on fetal and postnatal development and organogenesis in mammals (2, 3). Although circulating IGFs are produced principally in the liver, they are synthesized ubiquitously, which means they act on their target cells via endocrine and autocrine/paracrine mechanisms. In all biological fluids, IGFs are noncovalently bound to high affinity binding proteins (IGFBP-1 to -6) that modulate their action (4).

Cultured fetal hepatocytes are capable of synthesizing glycogen and actively proliferating (5, 6, 7), provided that they are cultured in the presence of cortisol. Glycogen synthesis in these cells is highly responsive to insulin and becomes more so with advancing stage of development (15–18 days of gestation) (8, 9, 10). IGF-II stimulates glycogenesis in 18-day-old fetal hepatocytes cultured in the presence of glucocorticoids and this stimulation is regulated by secreted IGFBPs, especially IGFBP-1, which is the predominant IGFBP secreted by these cells (11). Hepatic IGFBP-1 expression increases with stage of development, with a peak around birth (12), and is strongly enhanced in the presence of glucocorticoids (13). IGFBP-1 is expressed largely in the liver and endometrium, suggesting that it plays some specific role in each organ (14, 15).

The aim of this study was to investigate IGFBP-1 regulation of IGF-II-stimulated cell proliferation and glycogenesis in cultured fetal rat hepatocytes isolated at different stages of development and cultured with or without glucocorticoids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Uniformly labeled [14C]glucose was purchased from DuPont New England Nuclear (Boston, MA), [3H]thymidine from Amsersham S.A. (Les Ulis, France), recombinant human (rh) IGF-II from Boehringer Mannheim (Meylan, France) and porcine insulin was a gift from Eli Lilly Laboratories (Indianopolis, IN). Rabbit antihuman IGFBP-1 antibody was purified from amniotic fluid (16). Antihuman IGFBP-2 antibody was a gift from J. Schwander (Basel, Switzerland) and antihuman IGFBP-4 antibody a gift from T. Busby and D. Clemmons (Chapel Hill, NC). Goat antirabbit IgG polyclonal antibody coupled to horseradish peroxydase was purchased from the Sigma Chemical Co. (St. Louis, MO).

Cell culture
Primary cultures of hepatocytes from 15- and 18-day-old Sprague Dawley rat fetuses (IFFA CREDO, L’Arbresle, France) were prepared as previously described (6). Briefly, following mild trypsin treatment, isolated cells were plated on a collagen substrate, to which only hepatocytes adhere. After 6 h, the nonadhering hematopoietic cells were removed. NCTC 109 medium (ICN Biomedicals, Orsay, France) (17) with or without 10 µM cortisol and 10% FCS was used. More than 95% of the cell population was composed of morphologically typical hepatocytes and the remainder, of fibroblast-like cells (6). The number of cells per dish was 0.6 x 106, corresponding to 230 µg protein and 1.6 mg wet liver.

After either 12 or 24 h, the cultures were washed with serum-free NCTC 109 medium with or without cortisol and the medium renewed 10 min later. Tests of the effects of the glucocorticoid were then performed after 12 or 24 h (day 1) and after a further 24 h (day 2 of culture) when the glycogenic response to insulin is maximal (8). At the start of each experiment (Time 0), medium was replaced with either conditioned medium (in which fetal hepatocytes had been incubated for 24 h at 37 C) or control medium (maintained at 37 C for 24 h without cells). Because conditioning of the medium resulted in glucose consumption, the glucose concentration in test media was readjusted to 1 mg/ml (that in fresh NCTC 109 medium).

Western ligand blotting
The conditioned media were analyzed by Western ligand blotting according to Hossenlopp et al. (18). Briefly, lyophilized samples (500 µl equivalent of conditioned medium) and samples of fetal and adult rat serum (3 µl) were submitted to 12.5% SDS-PAGE under nonreducing conditions, followed by electrotransfer to nitrocellulose. The different IGFBP species were detected by incubation with a mixture of [125I]IGF-I and [125I]IGF-II and revealed by autoradiography.

Immunoblotting
After SDS-PAGE and transfer to nitrocellulose, the blots were probed as previously described (19), using anti-IGFBP-1, anti-IGFBP-2 and anti-IGFBP-4 antibodies at 1/1000 dilution. The antirabbit IgG antibody-horseradish peroxidase conjugate was added to bind the immunocomplexes, which were then visualized by chemiluminescence (ECL, Amersham, Les Ulis, France).

Isolation of RNA and Northern blotting
Total RNAs were isolated from frozen cultures using the standard CsCL/guanidine isothiocyanate method (20) and analyzed by Northern blotting as described by Babajko et al. (12). Forty micrograms of total RNA were loaded onto 1.2% agarose/8% formaldehyde gels, submitted to electrophoresis in MOPS 1x, transferred to Hybond-C membranes (Amersham) and covalently bound to the nylon by baking of the membranes at 80 C for 2 h. After prehybridization at 50 C in 50% formamide, 5 x SSC, 1% SDS, 5 x Denhardt’s, 50 mM phosphate buffer, pH 6.5, and 100 µg/ml sonicated salmon sperm DNA, the blots were hybridized to 3 x 106 cpm/ml hIGFBP-1, IGF-II, and type 1 IGF receptor (IGF-IR) complementary DNA (cDNA) probes for 20 h at 50 C in the same buffer plus 10% dextran sulfate. The blots were washed twice for 15 min in 2 x SSPE, 0.1% SDS at room temperature and once for 45 min in 0.5 x SSC, 0.1% SDS at 65 C, then autoradiographed at -80 C with intensifying screens.

Measurement of glycogenesis
Glycogen labeling was measured as previously described (6, 9). At Time 0 of the experiment on day 2, increasing dosages of insulin, IGF-II and des (1, 2, 3, 4, 5, 6)IGF-II and 1 µCi/mg [14C]glucose were added to the culture medium. [14C]glucose incorporation was then measured after 3 h of incubation.

DNA synthesis
DNA synthesis was assessed by [3H]thymidine incorporation and quantification of DNA content. Eighteen hours after plating (Time 0 of the experiment), medium was replaced with serum-free medium with or without either IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II or insulin at the concentrations indicated. Thereafter, medium was renewed at the beginning of each 4-h interval, up to 28 h. Cells were then labeled with 5 µCi/ml (0.5 µCi/mg) [3H]thymidine either for the seven successive 4-h periods up to 28 h, or for two successive 4-h periods between 20 h and 28 h. At the end of the 28-h incubation, cultures were washed 5 times with cold PBS without calcium and magnesium. Cells were lysed in 0.9 ml 0.3 N KOH, precipitated with 5% trichloroacetic acid, pelleted by centrifugation at 4 C, and the DNA solubilized in 0.5 N perchloric acid at 80 C (21). [3H]thymidine incorporation into DNA was measured and DNA content quantified according to Burton (22).

Definitions
For the responses to IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II and insulin, a "stimulation or inhibition index" was used, defined as the ratio of values obtained for treated cultures to the values obtained for controls. For each protocol considering glycogenesis or DNA synthesis measurements, at least three independent experiments were performed on different cell preparations. Data are expressed as means ± SD and the number of experiments given. For the representative experiments shown (see Figs. 3–6GoGoGoGo), each symbol on the graph represents the mean ± SD of triplicate culture measurements. Student’s t test for paired samples was used for statistical analyses of treated cultures and corresponding controls. For Northern blotting, at least two different samples were measured at least twice, representing a minimum of 4 points for each experimental condition. The cell population of each culture dish was approximately 0.6 x 106 hepatocytes, corresponding to 230 µg protein and 1.60 mg wet liver.



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  		Figure 3. Age-related effects of IGF-II, des (1 2 3 4 5 6 )IGF-II and insulin on glycogenesis in fresh medium. Fifteen- and 18-day-old fetal rat hepatocytes were grown in NCTC 109 medium containing 10% FCS and with or without 10 µM cortisol. After 24 h of culture, the medium was replaced with identical, but serum-free, medium and after a further 48 h, replaced again (Time 0). Different concentrations of IGF-II or des (1 2 3 4 5 6 )IGF-II, or 30 nM insulin were then added with labeled glucose and [14C]glucose incorporation into glycogen measured 3 h later. Stimulation indices are given for 15- (A) and 18-day-old cells (B) grown in the absence (open symbols) or presence of cortisol (solid symbols). Statistical significance is represented by * for P < 0.05, ** for P < 0.01, and *** for P < 0.001, as referred to corresponding triplicate cultures grown in the absence of hormone.

 


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  		Figure 4. Effects on IGF-II-stimulated glycogenesis of conditioning of the media by hepatocytes grown in the presence or absence of cortisol. Eighteen-day-old cells were grown in the presence of 10 µM cortisol as described in Fig. 3Go. On day 2, the medium was replaced with medium conditioned for 24 h as described in Fig. 1Go. Different concentrations of IGF-II or des (1 2 3 4 5 6 )IGF-II, or 30 nM insulin were then added with labeled glucose and [14C]glucose incorporation into glycogen measured 3 h later. Stimulation indices are given for medium conditioned by 15- (A, C) and 18-day-old cells (B, D) grown in the absence (A, B) or presence of cortisol (C, D). Open symbols correspond to responses obtained in cortisol-free medium, solid symbols correspond to those obtained in cortisol-containing medium. Statistical significance is represented by * for P < 0.05, ** for P < 0.01, and *** for P < 0.001, as referred to corresponding triplicate cultures grown in the absence of hormone.

 


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  		Figure 5. Stimulation of DNA synthesis by IGF-II and insulin in 15- and 18-day-old fetal hepatocytes grown in the presence and absence of cortisol. After 24 h of culture, medium was replaced with serum-free medium with or without increasing concentrations of IGF-II or insulin and 5 µCi/ml [3H]thymidine (0.5 µCi/mg). DNA content (A, B) and [3H]thymidine incorporation into DNA (C, D) were measured at the end of the 28-h incubation period in cells grown in the absence (open symbols) or presence (solid symbols) of cortisol. Statistical significance is represented by * for P < 0.05, ** for P < 0.01, and *** for P < 0.001, as referred to corresponding triplicate cultures grown in the absence of hormone.

 


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  		Figure 6. Dose dependence of the mitogenic and glycogenic responses to IGF-II, des (1 2 3 4 5 6 )IGF-II and insulin in 18-day-old fetal hepatocytes grown in the presence of cortisol. After 24 h of culture, medium was replaced with serum-free medium with or without increasing concentrations of peptide and renewed at the beginning of each 4-h interval up to 28 h. Cells were labeled with 5 µCi/ml [3H]thymidine (0.5 µCi/mg) for the two successive 4-h periods between Time 20 h and 28 h. [3H]thymidine incorporation into DNA (A) was measured at the end of each 4-h period. The same protocol as that described in Fig. 3Go was used to measure [14C]glucose incorporation into glycogen (B). Each response is given in terms of stimulation index. Statistical significance is represented by * for P < 0.05, ** for P < 0.01, and *** for P < 0.001, as referred to corresponding triplicate cultures grown in the absence of hormone.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of IGFBPs secreted into the culture media
The IGFBPs secreted into the culture media by 15- and 18-day-old fetal hepatocytes over a period of 24 h in the presence or absence of cortisol were analyzed by Western ligand blotting and compared with the IGFBPs in serum. In media conditioned by 15-day-old cells, whether in the presence or absence of cortisol, there was a major band corresponding to a 29–32 kDa protein and a minor one at 24 kDa. The bands were much more intense in cortisol-treated cells (Fig. 1AGo). In media conditioned by 18-day-old hepatocytes, the IGFBP profile was similar to that obtained for 15-day-old cells, although production of both peptides was increased, both in the presence and absence of cortisol. The conditioned media were then analyzed by immunoblotting to identify the bands detected by Western ligand blotting. The anti-IGFBP-1 antibody recognized a predominant peptide of 29 kDa within the 29–32-kDa doublet, the 32-kDa band within the doublet being recognized by the anti-IGFBP-2 antibody (Fig. 1BGo). The 24-kDa band corresponding to IGFBP-4 was poorly detectable by our antibody, because of its weak affinity. Immunoblotting confirmed the findings from Western ligand blotting, indicating that IGFBP-1 and IGFBP-2 production is positively regulated by glucocorticoids.



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  		Figure 1. Analysis of IGFBPs in media conditioned by fetal rat hepatocytes cultured in the presence and absence of cortisol. Samples of media conditioned by 24-h cultures in serum-free medium of 15- and 18-day-old cells with and without cortisol were submitted (0.5 ml eq/slot) to SDS-PAGE (12.5%) under nonreducing conditions and transferred to nitrocellulose. IGFBP-1 and IGFBP-2 were revealed by incubation either with 125I-labeled IGF (ligand blotting; A) or with specific anti-IGFBP polyclonal antibodies (immunoblotting; B). IGFBPs from adult and fetal rat serum (3 µl/slot) were analyzed simultaneously for comparison.

 
Effects of glucocorticoid and insulin on IGFBP-1, IGF-II, and IGF-IR gene expression
Expression of the IGFBP-1, IGF-II, and IGF-IR genes was analyzed by Northern blotting in 15- and 18-day-old hepatocytes cultured for 2 days in the presence or absence of cortisol and with or without 10 nM insulin during the last 6 h of culture (Fig. 2Go).



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  		Figure 2. Hybridization of IGFBP-1, IGF-II, and IGF-IR cDNA probes with 15- and 18-day-old fetal rat hepatocyte mRNAs. After 24 h of culture in the presence or absence of cortisol under the conditions described in Fig. 1Go, medium was replaced with serum-free medium with or without 10 nM insulin for a 6-h incubation. Forty micrograms of total RNA prepared from 15- and 18-day-old cells cultured in the presence (c, d, g, h) or absence (a, b, e, f) of cortisol and in the presence (b, d, f, h) or absence (a, c, e, g) of insulin were analyzed with the three cDNA probes. Ethidium bromide staining confirmed that equal amounts of RNA were present in each lane (A). Phospho-imaging was used to quantify the differences in IGF-II mRNA levels (B). Statistical significance is represented by * for P < 0.001, as referred to results for 15-day-old fetal rat hepatocytes grown in the absence of hormone taken as 100% (control).

 
In the presence of cortisol, IGFBP-1 messenger RNA (mRNA) levels increased, and more so in 15- than in 18-day-old cells. Similarly, IGF-II mRNA levels were increased (stimulation index : 1.82 ± 0.34, n = 6, P < 0.001), whichever the stage of development. Unlike IGFBP-1 mRNA, IGF-IR mRNA was barely detectable in 18-day-old hepatocytes. These findings confirm that glucocorticoids are involved in regulating IGF-II and IGFBP-1 gene expression.

Insulin depressed the cortisol-induced increases in IGFBP-1 mRNA. It had no significant effect on IGF-II mRNA in the absence of cortisol, but counteracted the response to cortisol, depressing these levels to the same extent independently of the stage at which the cells were isolated (e.g. inhibition index : 0.72 ± 0.08, n = 6, P < 0.001 in 18-day-old cells).

IGF-II-stimulated glycogenesis as related to stage of development and presence of IGFBPs
Before testing the modulation by IGFBPs of IGF-II-stimulated glycogenesis in fetal hepatocytes, it was necessary to examine IGF-II stimulation in the absence of IGFBPs (i.e. in fresh medium). Hepatocytes isolated at 15 and 18 days of gestation were grown in serum-free medium from day 1, then the medium renewed on day 2 at Time 0 of the experiment. [14C]glucose incorporation into glycogen over 3 h was measured after treatment with increasing concentrations of IGF-II or des (1, 2, 3, 4, 5, 6)IGF-II (which is incapable of binding IGFBPs) or with insulin at a concentration of 30 nM, which is known to exert maximal glycogenic stimulation.

In 15-day-old cells, IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II and insulin failed to stimulate glycogen labeling in the absence of cortisol, whichever the dosage of peptide used. In the presence of cortisol, glycogenesis was significantly increased by all three factors (Fig. 3AGo). In 18-day-old cells, by contrast, glycogen labeling in the absence of cortisol was stimulated by both IGF-II and des (1, 2, 3, 4, 5, 6)IGF-II (stimulation indices : 1.75 ± 0.18 and 1.82 ± 0.16 for 150 nM IGF-II and des (1, 2, 3, 4, 5, 6)IGF-II, respectively; n = 4, P < 0.001); these are similar to the 1.87 ± 0.18 stimulation by 30 nM insulin). Cortisol potentiated the action of all three peptides (stimulation indices at maximal concentrations : 3.38 ± 0.32, 3.41 ± 0.38 and 3.45 ± 0.30 for IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II and insulin, respectively; n = 3, P < 0.001) (Fig. 3BGo). These findings show that, as in the case of insulin, IGF-II- and des (1, 2, 3, 4, 5, 6)IGF-II-stimulated glycogenesis is dependent on the stage of development of the cell and the presence of glucocorticoid.

The next series of experiments was performed to investigate the response of peptide-induced glycogenesis to the presence of IGFBPs in the culture medium. The glycogenic effects of IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II and insulin were therefore tested in conditioned medium (i.e. serum-free medium incubated at 37 C for 24 h in the presence of cells cultured with and without cortisol). Stimulation of glycogenesis was measured in 18-day-old hepatocytes grown in the presence of cortisol because in these circumstances maximal stimulation could be attained. The same protocol as that described above (Fig. 3Go) was used, except that on day 2, the medium was replaced with conditioned medium (containing IGFBPs). As above, the effects of IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II and insulin were assessed by measuring [14C]glucose incorporation into glycogen over 3 h. Stimulation by 150 nM IGF-II decreased as follows: 15-day-old cells untreated with cortisol > 18-day-old cells untreated with cortisol > cortisol-treated 15-day-old cells > cortisol-treated 18-day-old cells (stimulation indices: 2.98 ± 0.23, 2.52 ± 0.21, 1.71 ± 0.19 and 1.12 ± 0.14, respectively; n = 3, P < 0.05) (Fig. 4Go, A–D). Des (1, 2, 3, 4, 5, 6)IGF-II was tested to determine whether or not binding of IGF-II to secreted IGFBPs was responsible for their inhibition of its glycogenic action. Whichever the conditioned medium used, des (1, 2, 3, 4, 5, 6)IGF-II stimulation remained constant and very close to that obtained either in fresh medium or with insulin, which fails to bind IGFBPs. It was therefore confirmed that IGF-II-stimulated glycogenesis was inhibited by IGFBP-1 and that the inhibition was correlated to cortisol-induced secretion of IGFBPs (see Fig. 1Go).

IGF-II-stimulated DNA synthesis as related to stage of development and presence of IGFBPs
Having demonstrated that the IGF/IGFBP system is involved in the regulation of glycogenesis in fetal hepatocytes, we considered it of interest to investigate its role in DNA synthesis. The effects of IGF-II and insulin over 28 h of culture were therefore studied in 15- and 18-day-old cells. At day 1 (24 h of culture), medium was replaced with serum-free medium with or without cortisol and containing [3H]thymidine and increasing concentrations of IGF-II or insulin. DNA content and [3H]thymidine incorporation measured 28 h later are shown in Fig. 5Go.

In the absence of added mitogen, cortisol provoked an increase in DNA content in both 15- and 18-day-old cells (from 7.42 ± 0.81 to 8.41 ± 0.91 µg/culture and from 7.10 ± 0.85 to 8.00 ± 0.95 µg/culture, respectively, n = 3, P < 0.05) (Fig. 5Go, A and B).

In the presence of cortisol, insulin increased DNA content and [3H]thymidine incorporation in both 15- (Fig. 5Go, A and C) and 18-day-old cells (Fig. 5Go, B and D). The effects were more pronounced in 18-day-old hepatocytes where enhanced [3H]thymidine incorporation was also evident in the absence of cortisol.

IGF-II stimulated [3H]thymidine incorporation in the absence of cortisol in 18-day-old hepatocytes. However, in contrast with the potentiation seen with insulin, cortisol inhibited the effect of IGF-II (stimulation indices for 70 nM IGF-II : 1.90 ± 0.23 in the absence, as opposed to 1.10 ± 0.12 in the presence of cortisol, n = 3, P < 0.01). Therefore, 28 h of treatment with insulin without renewal of medium stimulated DNA synthesis, its effect being potentiated by glucocorticoids, whereas stimulation by IGF-II was depressed by cortisol under these culture conditions.

Dose dependence of stimulation by IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II and insulin of DNA synthesis and glycogenesis
Because cortisol activated IGFBP production and suppressed stimulation of DNA synthesis by IGF-II, but not that by insulin, it seemed that the action of IGF-II may be modulated by IGFBPs. IGF-II-stimulated DNA synthesis over 28 h appeared to be more sensitive to the presence of secreted IGFBPs than did IGF-II-stimulated glycogenesis over 4 h. The effects of IGF-II were therefore tested under conditions where the amount of IGFBP would not exceed that secreted into the medium within 4 h of culture. Thus, culture media were replaced with fresh medium supplemented with IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II or insulin at the beginning of each 4-h interval up to 28 h. [3H]thymidine incorporation into DNA was measured in 18-day-old cells cultured in the presence of cortisol during the two successive 4-h periods between 20 h and 28 h of incubation in the presence of IGF-II, des (1, 2, 3, 4, 5, 6)IGF-II or insulin. These were the periods during which stimulation of DNA synthesis was the most effective (results not shown). At 70 nM, the three peptides stimulated [3H]thymidine incorporation to the same extent, but half-maximal responses were attained with 5–10 nM insulin or des (1, 2, 3, 4, 5, 6)IGF-II, and with 20 nm IGF-II (Fig. 6AGo). Under the same conditions, i.e. using 4-h conditioning of the medium, stimulation of DNA synthesis by IGF-II, as compared with that by des (1, 2, 3, 4, 5, 6)IGF-II, was weaker in the presence than in the absence of cortisol (results not shown).

[14C]glucose incorporation into glycogen was similarly enhanced by insulin, des (1, 2, 3, 4, 5, 6)IGF-II and IGF-II during the 4-h incubation period, half-maximal responses being attained with 3 nM insulin and 50 nM des (1, 2, 3, 4, 5, 6)IGF-II or IGF-II (Fig. 6BGo). Compared with des (1, 2, 3, 4, 5, 6)IGF-II, insulin was 15 times more potent in stimulating glycogenesis, whereas DNA synthesis was equally sensitive to the two factors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBPs and especially IGFBP-1 produced by 18-day-old fetal rat hepatocytes cultured in the presence of cortisol appear to play a role in regulating glycogenesis (10, 23). In our experiments, Western ligand- and immunoblot analyses of conditioned media of 15- and 18-day-old fetal cells and Northern blot analysis of IGFBP-1 mRNA showed that both IGFBP-1 protein production and its mRNA levels increased with stage of cell development and exposure to glucocorticoid. In vivo studies have revealed a dose-dependent increase in hepatic mRNA and serum IGFBP-1 concentration in chronically dexamethasone-treated rats (24, 25) and in rat H4IIE hepatoma cells, dexamethasone strongly stimulates IGFBP-1 gene expression (26, 27). During the perinatal period in the rat, between day 16 in utero and day 16 postnatally, IGFBP-1 gene transcription activity and circulating IGFBP-1 levels increase until birth, when serum glucocorticoid levels are high, and thereafter they decline (12). The dependence of IGFBP-1 expression on stage of development and presence of glucocorticoid observed in this study of fetal rat hepatocyte cultures therefore agrees with in vivo findings.

In this cell system, stimulation of glycogenesis by IGF-II in the absence of IGFBP was also dependent on stage of development and presence of glucocorticoid, similarly to stimulation by insulin (10, this paper). As previously observed (11), the action of secreted IGFBPs on the glycogenic effect of IGF-II was concurrent with their production. IGF-II stimulation was diminished in IGFBP-containing conditioned media and the inhibition was correlated to cortisol-induced secretion of IGFBP-1. The implication of IGFBPs in suppressing IGF-II-stimulated glycogenesis was corroborated by their lack of effect on basal glycogenesis, thus excluding the possibility of medium toxicity to the cells, and by the persistent action of insulin, which fails to bind IGFBPs. In addition, IGFBP secretion had no effect on glycogenic stimulation by des (1, 2, 3, 4, 5, 6)IGF-II, an analog of IGF-II that does not bind IGFBPs. Furthermore, the inhibitory effects of treatment with recombinant IGFBP-1 mimic those of conditioned medium (11). IGFBP-1 inhibition of IGF-II fits well with the suppression by IGFBP-1 of IGF action on glucose uptake and metabolism in rat skeletal muscle in vitro and on glucose consumption in mouse BALB/c3T3 fibroblasts (28, 29).

Given the effects on glycogenesis, it seemed appropriate to investigate the involvement of the IGF/IGFBP system in DNA synthesis. During late gestation, fetal hepatocytes not only differentiate, they also actively proliferate (30). Fetal rat hepatocytes in primary culture display a constitutively proliferative phenotype that is modified by activators and inhibitors of DNA synthesis, basal DNA synthesis being independent of added serum, mitogens or glucocorticoids (5, 6, 31, 32). Nevertheless, proliferation in cultured 15- and 18-day-old hepatocytes is enhanced in the presence of glucocorticoids, even when cells entering DNA synthesis are loaded with glycogen (31). In the present study, IGF-II stimulation of DNA synthesis was inhibited in the presence of cortisol, whereas IGFBP-1 secretion increased. This is consistent with the situation in vivo where a smaller fetal liver in growth-retarded rats coincides with elevated IGFBP-1 (25, 33). Strong IGFBP-1 expression has also been noted in association with intrauterine growth retardation in humans (34) and ovines (35). These in vivo data therefore support our observation that IGFBP-1 modulates IGF-II-stimulated DNA synthesis in developing fetal rat hepatocytes.

Insulin also stimulated DNA synthesis in these hepatocytes, but the effect was different from that of IGF-II, in that cortisol potentiated insulin action on both DNA synthesis and glycogenesis. This agrees with the mitogenic stimulation by insulin in 19-day-old fetal rat hepatocytes cultured in the presence of cortisol (7). For DNA synthesis, the concentrations of insulin and IGF-II required for a half-maximal response were very similar, but for glycogenesis, they were quite different. Considering the respective affinities of insulin and IGF-II for the heterologous receptors (23), it seems that insulin and IGF-II stimulate glycogenesis via the same receptor, but mitogenesis via distinct receptors. This would be in line with recent studies suggesting that the insulin and IGF-I receptors have distinguishing mechanisms of action, in part owing to different signaling pathways (36, 37). In man and other species, elevated insulin is associated with fetal growth and a shortage of insulin causes stunted fetal growth (reviewed in Ref. 38). Nevertheless, although they present drastic metabolic anomalies like hyperglycemia, insulin receptor-deficient mice are not growth-retarded (39, 40, 41), whereas IGF-I receptor-deficient animals are severely so (42). Apart from its metabolic action, insulin promotes growth in cultured cells and embryos in vitro (43). These findings would suggest that in fetal life the IGF-I receptor could replace the insulin receptor in mediating mitogenesis. Consistently with this hypothesis, mice lacking IRS-1 are growth-retarded and mildly insulin-resistant (44, 45), the insulin and IGF-I receptors mediating specific functions via the IRS-1 pathway. It is pertinent that cells overexpressing the insulin receptor are capable of proliferating in the presence of IGF-II, but not in the presence of IGF-I, which has weak affinity for the insulin receptor (46). In fetal hepatocytes, IGF-II binds to IGF-IR with affinity that is similar to that of IGF-I (23). Taken together, these observations corroborated with recent studies suggesting that insulin and IGF-I receptors have distinguishing mechanisms of action due in part to different signaling pathway (36, 37). Our results would therefore suggest that the action of insulin and IGF-II in glycogenesis is mediated via the insulin receptor (23, this paper), but in DNA synthesis, insulin acts via the insulin receptor, whereas IGF-II acts via the IGF-I receptor.

Changes in the IGF/IGFBP system may intercede in the effects of cortisol on cell differentiation and maturation (review in Ref. 24). In the present work, cortisol increased IGF-IR, IGF-II, and IGFBP-1 mRNAs and promoted IGFBP-1 protein production. However, older cells expressed less IGF-IR and IGF-II mRNA, but more IGFBP-1 mRNA than younger cells. At least in the latter, glucocorticoid stimulation of DNA synthesis could reflect a larger IGF-IR number and stronger IGF-II production. Also, IGF-II acting via autocrine/paracrine mechanisms in close proximity to the cell would be less exposed to sequestration by IGFBP-1 (whose synthesis increases with age). Nevertheless, as has been shown in the case of the insulin receptor (47), it is possible for the amounts of receptor mRNA to diverge from those of receptor protein. Augmented IGF-IR, IGF-II and IGFBP-1 mRNA levels have been observed in vivo in the livers of 20-day-old fetuses from rats treated with dexamethasone between days 15 and 19 of gestation (25). In our cultured fetal hepatocytes, insulin depressed cortisol-induced IGFBP-1 and IGF-II mRNA levels at both stages of cell development. In rat H4IIE hepatoma cells, insulin rapidly reduces IGFBP-1 mRNA and protein, either at transcriptional level or through mRNA processing (27). These results would suggest that, like glucocorticoids, insulin may promote IGF-II action in regulating the IGF/IGFBP system, but would do so in a different way. In our experiments, insulin also seemed capable of up-regulating itself via modulation of the IGF/IGFBP system.

Interactions between glucocorticoids, IGF-II and IGFBP-1 secreted by the cells may be of physiological significance. In the course of development in the rat, there is a marked rise in plasma glucocorticoid levels between days 15 and 19 of gestation, followed by a drop (48). Insulin levels are very high during late gestation with a peak at 20 days. Thereafter, they decrease until birth, remaining low during postnatal life (49). Serum levels of IGF-II are also elevated in fetal life (50, 51), in concert with the strong expression of IGF-II mRNA in the liver (52), and both decline rapidly at birth. Hepatic IGFBP-1 mRNA expression and serum IGFBP-1 levels in the rat increase during the last days of fetal life, reaching a maximum at birth and 1-day thereafter, and then decrease (12, 53). In the rat, it is during the final third of gestation that liver function matures, with active glycogen synthesis and cell proliferation (30, 54) and during which serum concentrations of glucocorticoids, insulin and IGF-II are high, whereas IGFBP-1 is only beginning to develop. Such conditions could be mimicked in fetal hepatocytes in culture, where insulin and IGF-II efficiently stimulate glycogenesis and cell proliferation. At birth, when these processes are decelerated and glycogen mobilization from the liver supplies the energy needs of the newborn, IGFBP-1 expression becomes maximal. In cultured hepatocytes, this would correspond to the inhibition of IGF-II-stimulated glycogen synthesis and cell growth. It can be hypothesized that during late gestation, the IGF/IGFBP system and insulin play complementary roles in regulating glucocorticoid-dependent fetal maturation. Our results show that in cultured fetal hepatocytes, cortisol-dependent DNA synthesis and glycogen storage are both modulated by insulin and IGF-II, and that the latter is negatively regulated by IGFBP-1.


   Acknowledgments
 
The authors are indebted to Michel Binoux for advice and helpful discussions. They also thank Emile Marie-Rose for skillful secretarial assistance.


   Footnotes
 
1 This work was supported by the Association de la Recherche contre le Cancer and by Université Paris 7. Back

Received July 31, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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