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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad Complutense de Madrid), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain
Address all correspondence and requests for reprints to: Luis Goya, Instituto del Frío (Consejo Superior de Investigaciones Científicas), Ciudad Universitaria, 28040 Madrid, Spain. E-mail: luisgoya{at}if.csic.es
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Abstract |
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Introduction |
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However, in experiments in vivo, the simultaneous fluctuations of fuels and hormones that occur in diabetic and undernourished animals render it difficult to demonstrate specific regulation and the molecular mechanisms involved. It seemed appropriate, therefore, to investigate in vitro the underlying mechanisms for IGFs regulation. The in vitro system that more closely resembles normal developing liver is the primary culture of fetal hepatocytes (5, 6, 7). Because IGFs expression in primary fetal cultures is limited and subject to plastic substratum-induced changes in the differentiation state of the liver cells, very scant data are available in the literature about the IGF response of fetal hepatocytes in culture to different conditions (8, 9, 10, 11). To overcome these difficulties, a selective primary culture of fetal rat hepatocytes from fetuses on d 21 gestation has been established in our laboratory (5). The main goal of this primary culture of fetal hepatocytes was to confirm the results obtained in vivo in our laboratory (e.g. that nutrients and insulin are the main regulatory factors of IGF synthesis and secretion during the late fetal stages). In this model, we have previously reported a regulatory role for glucose on IGF-I and -II synthesis and secretion (5). Therefore, the aim of this work was to demonstrate a role for insulin in the control of the IGF system and compare the molecular pathways involved with those reported for glucose.
Insulin has been widely reported to regulate IGF-I transcription in vivo (1) as well as in adult hepatocytes in culture (12, 13, 14, 15), but this article describes the specific regulatory role for insulin on IGF-I and -II gene expression in cultures of fetal hepatocytes. Doses of insulin higher than those required in primary cultures of adult hepatocytes were necessary to evoke a response of the IGF system in fetal cell cultures. Gene expression, by RNase protection assay, and peptide secretion of IGF-I and -II, by RIA and RRA, respectively, have been tested in cultures of late fetal rat hepatocytes in the presence of different doses of insulin. In this article we report, for the first time, the posttranscriptional effect of insulin on IGF-I and -II synthesis during late fetal stages of perinatal development. Therefore, although both glucose and insulin specifically regulate the IGF system, clear differences in the molecular mechanisms can be observed and will be discussed. This system should be a useful tool for further studies of molecular mechanisms of IGF-I and -II regulation.
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Materials and Methods |
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Experimental models
Wistar rats bred in our laboratory with controlled temperature
and artificial dark-light cycle (0600–1800 h) were used throughout the
study. Females were caged with males and mating was confirmed by the
presence of spermatozoa in a vaginal smear. Each dam was housed
individually from the 14th d of pregnancy. Animals were fed a standard
laboratory diet ad libitum (19 g protein-coupled, 56 g
carbohydrate, 3.5 g lipid, 4.5 g cellulose per 100 g,
plus salt and vitamin mixtures). Water was given ad libitum.
Dams were killed and fetuses were exposed after abdominal incision. All
experiments were conducted in accordance with the principles and
procedures outlined in the NIH guide for care and use of experimental
animals (Bethesda, MD).
Cell extraction
Fetal hepatocytes. Primary cultures of hepatocytes from
21-d-old Wistar rat fetuses were prepared by a nonperfusion collagenase
dispersion method (5, 6). The protocol involves incubation
of the minced tissue with Ca2+-free Krebs
bicarbonate buffer containing 0.5 mM EGTA in a 150-ml
conical flask for 30 min at 37 C in a shaking water bath (100
cycles/min) under continuous gassing
(O2/CO2, 19:1). The cell
suspension was centrifuged at 50 g for 5 min and the supernatant
was discarded. Cells were then resuspended in Krebs bicarbonate buffer
containing 2.55 mM Ca2+ and 0.5 mg
collagenase/ml in a 150-ml conical flask. The mixture was incubated at
37 C in a shaking water bath (100 cycles/min) under continuous gassing.
After 60 min, the cell suspension was washed with Krebs bicarbonate
buffer containing 2.55 mM Ca2+ and
then centrifuged at 35 g for 5 min and filtered through a nylon
mesh (500 µm). The washing step was repeated with a nylon mesh of 100
µm. During washings, at very low speed, separation occurred between
parenchymal and hematopoietic cells, the latter mostly remaining in
suspension. By counting under a microscope, hematopoietic cell
contamination was shown to be lower than 5%. The procedure produced
approximately 1.5 x 107 cells/g of fetal liver,
representing about a 15% recovery yield. Cell viability (Trypan blue
exclusion) for fetal hepatocytes was always higher than 95%.
Adult hepatocytes. Isolation of adult hepatocytes was carried out from 3-month-old male rats by perfusion with collagenase in Krebs-bicarbonate buffer under continuous gassing with carbogen (O2/CO2, 19:1). The hepatocyte suspension was washed twice with sterile DMEM and then resuspended in this medium supplemented with 50 mg/ml gentamicin, 50 µg/ml penicillin G, and 50 µg/ml streptomycin.
Cell culture
Fetal hepatocytes. For culture of fetal cells, sterile
techniques were used throughout the procedure, and media was
supplemented with 120 µg penicillin G/ml, 100 µg streptomycin/ml.
The isolated cells were plated in 100-mm-diameter plastic dishes
containing 8 ml medium 199 with Earle’s salts supplemented with 10%
(vol/vol) FCS and antibiotics as described above. Each dish was
inoculated with 6 x 106 cells, and the
primary culture was kept at 37 C under an atmosphere of 5%
CO2 in air with 80% humidity in a cell incubator
for 4 h. Then the attached monolayer of cells was washed with
serum-free medium, and fresh FCS-free medium supplemented with the
various different conditions was added and the dishes incubated for
established time periods. This procedure ensures a fairly pure culture
of fetal hepatocytes in which the fibroblast-like cells comprise less
than 10% of the total cells (6).
Adult hepatocytes. A total of 3 x 106 hepatocytes were plated in 100-mm-diameter tissue culture dishes in a medium containing 8 ml DMEM supplemented with 10% FCS. After a 4-h incubation to facilitate cell attachment to the matrix, the medium was aspirated and the plates were washed twice with PBS to remove the nonadherent cells and filled with 8 ml of DMEM lacking serum. Additions were made so that the changes in the total incubation volume were less than 2%.
Iodination, purification, and determination of IGF-I and -II
Recombinant human IGF-I and -II were labeled by a modified
chloramine T method (2, 3). The specific activity achieved
with this method was approximately 90–175 µCi/µg for both
peptides.
Before IGF-I and -II determination, serum IGF binding proteins (IGFBPs) were removed by standard acid gel filtration. This method has proved to be the most reliable one for use with rat serum (2, 3).
The RIA for IGF-I and rat liver membrane receptor assay for IGF-II were carried out as previously described (2, 3). The coefficients of variation within and between assay were 8.0% and 12.4%, respectively.
Preparation of RNA
Total RNA. Cultured hepatocytes were separated from the
plastic substrate with a rubber policeman and total RNA was prepared by
homogenization of cells in guanidinium thiocyanate as originally
described (16). RNA was reprecipitated for
purification and its concentration determined by absorbance at 260 nm.
Samples were electrophoresed through 1.1% agarose/2.2 M
formaldehyde gels and stained with ethidium bromide to render the 28S
and 18S rRNA visible and thereby confirm the integrity of the RNA and
normalize the quantity of RNA in the different lanes.
Nuclear-enriched RNA. Nuclear pellets were extracted from 10 x 106 cells (5). Hepatocytes were removed from the plates with a rubber policeman in PBS and centrifuged at 15,000 rpm for 15 sec. Cell pellet was resuspended in 10 mM HEPES-KOH (pH 7.9) at 4 C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulphonil fluoride, allowed to swell on ice for 15 min, and then vortexed for 10 sec. Samples were centrifuged for 15 sec at 15,000 rpm and the nuclear pellets homogenized on ice in a solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, and 0.1 M 2-mercapthoethanol. Purification, precipitation, and quantification of the nuclear- enriched RNA were as described above for total RNA.
Riboprobes
Rat IGF-I and -II cDNAs used for the RNase protection assay of
total RNA were kindly provided by Dr. C. T. Roberts and Dr. D.
LeRoith (NIH, Bethesda, MD). Rat IGF-I cDNA ligated into a pGEM-3
plasmid (Promega Corp. Biotech) was linearized with
HindIII and an antisense riboprobe was produced by T7 RNA
polymerase. Rat IGF-II cDNA ligated into a pGEM-3 plasmid was
linearized with HindIII and incubated with T7 RNA polymerase
to generate a riboprobe. A pT7 RNA 18S antisense control template
(Ambion, Inc., Austin, TX) was used for lane-loading
control. The riboprobe was incubated with T7 RNA polymerase to produce
a 109-nucleotide runoff transcript, 80 nucleotides of which are
complementary to human 18S rRNA.
Rat IGF-I and albumin cDNAs containing intronic and exonic sequences to be used for the RNase protection assay of nuclear transcripts were kindly provided by Dr. Daniel Straus (University of California, Riverside, CA), and an intron-exon-containing clone for rat IGF-II was kindly provided by Dr. Peter Rotwein (Oregon Health Sciences University, Portland, OR). Intron-exon containing clones were processed as indicated by the providers to obtain riboprobes complementary only to nuclear pre-mRNA (unspliced RNA). These riboprobes were used in an RPA of nuclear transcripts to give an appropriate estimation of gene transcription. Rat albumin cDNA was used for lane-loading control.
All riboprobes were labeled with [32P]UTP. More than 3 million dpm were usually incorporated per microliter of mixture, and the mixtures were adjusted for size variations and diluted to a final specific activity of 600,000–800,000 dpm/µl. Finally, 1 µl was incubated with the RNA samples.
RNA stability assay
The effect of insulin on IGF-I and -II transcript stability was
determined in RNA transcript decay assays. Fetal hepatocytes were
cultured for 16 h in plain medium M199 with 5 mM
glucose in the absence or presence of 0.1 µM insulin.
Then, at time 0, 60 µM DRB was added to the medium and
cultures stopped at different time periods up to 6 h; total RNA
was extracted and mRNA expression of IGF-I and -II determined by RNase
protection assay (RPA). To rule out nonspecific effects of DRB on gene
expression, a set of plates with cultures of fetal hepatocytes was left
DRB free and treated with or without insulin for the same experimental
period. The 18S ribosomal antisense assayed in the same samples was
used for lane-loading control.
Solution hybridization/RPA
Solution hybridization/RPAs were performed as previously
described (17). Briefly, 20 mg total liver RNA were
hybridized with 600,000–800,000 dpm of the
32P-labeled riboprobes described above for
18 h at 45 C in 75% formamide and 400 M NaCl. After
RNase digestion with a buffer containing 40 mg/ml RNase A and 2 mg/ml
RNase T1 for 1 h at 37 C, protected RNA-RNA hybrids were resolved
on denaturing 8% polyacrylamide and 8 M urea gels.
Autoradiography was performed at -70 C against a Hyperfilm MP film
between intensifying screens. Bands representing protected probe
fragments were quantified using a scanning densitometer and
accompanying software (Molecular Dynamics, Inc.,
Sunnyvale, CA).
Statistical analysis
Data are presented as means ± SD. Statistical
comparisons were performed by one-way ANOVA, followed by the protected
least significant difference test (2, 3).
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Results |
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). Dose-response curves to increasing
doses of insulin (0.1–5 µM) for 16 h were obtained
both in the absence and presence of 5 mM glucose in the
serum-free medium. In general, intensity of the IGF-I and -II bands was
2- to 4-fold greater in the presence than in the absence of glucose,
and, to obtain clearly visible bands, films with experiments of the
latter condition required longer exposure times. Similar results were
obtained when the cultures were treated only for 6 h (data not
shown). Furthermore, in a time-course experiment (Fig. 2A), 0.1 µM insulin in the
presence of 5 mM glucose stimulated mRNA expression of
IGF-I and -II as soon as 2 h after the onset of treatment and
remained significantly elevated throughout the experiment (Fig. 2A).
Because the insulin doses necessary to evoke an IGF response in these
fetal cultures were significantly higher than those reported to be
physiological in adult animals and that have been currently used in
adult cultures, the observed effects could be mediated by the insulin
receptor or another receptor (i.e. IGF-I receptor). IGF-I
receptors have been known to bind and respond to insulin with fair
efficiency owing to their similar molecular and conformational
structure and signaling pathways. To rule out such a possibility,
cultures of fetal hepatocytes were treated with similar doses of
standard IGF-I and the gene expression of IGF-I and -II was evaluated.
No significant changes in the mRNA expression of both IGFs were evoked
by the IGF-I doses (Fig. 2B). In all experiments, at similar time
exposures of films, IGF-II transcript abundance in fetal hepatocytes
was at least 5-fold higher than that of IGF-I, in agreement with the
fact that IGF-II is considered to be the major IGF in fetal stages.
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). To evaluate the IGF peptide
secretion, fetal hepatocytes were treated with 0.1–5 µM
insulin for 16 h or with 1 µM insulin for different
time periods in the presence of 5 mM glucose and the
conditioned medium assayed for IGF-I (RIA) and IGF-II (RRA). A
significant dose-response increase in peptide levels in the conditioned
medium was found with 1 and 5 µM insulin for IGF-I and
with 0.1–5 µM for IGF-II. Besides, a time-dependent
increase in IGF-I and -II peptide levels in the medium was observed
1 h (for IGF-II) and 2 h (for IGF-I) after the onset of the
treatment with 1 µM insulin (Table 1). IGF-II values in
the conditioned medium were approximately 10-fold higher than those of
IGF-I, which agrees with the higher mRNA expression of IGF-II in these
fetal hepatocyte cultures.
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Mechanism of action of insulin on IGF-I and -II gene
expression
Characterization of the IGF response to glucose. As a first
approach to investigate the mechanism of action of insulin on IGF mRNA
expression in fetal hepatocytes, use was made of cycloheximide.
Incubation for 6 h with 10 µg/ml of cycloheximide, a protein
synthesis inhibitor, did not obliterate but significantly reduced the
0.1 µM insulin-induced mRNA signal of both IGF-I and -II
(Fig. 3). Cycloheximide had no effect
per se on the level of transcripts.
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. A significant increase in IGF-I and
-II transcript stability was observed during the 6 h of treatment
with insulin plus DRB vs. treatment with the RNA synthesis
inhibitor alone. A transient increase of the IGF-I and -II transcript
levels was observed during the first 2 h of DRB treatment, but
from that time on, the decrease of IGF transcripts was faster in the
absence than in the presence of insulin. In the presence of insulin, no
statistical differences were observed between 0 and 6 h for IGF-I
and -II, whereas both IGFs were approximately 50% reduced at 6 h,
compared with 0 h in the absence of insulin. Quantitative analysis
of the data were performed and the correlation coefficients and
probabilities for the different conditions were calculated. The results
were as follows: r = 0.84 and P < 0.0367 for
IGF-I -insulin, r = 0.95 and P < 0.0038 for
IGF-I + insulin, r = 0.88 and P < 0.0204 for
IGF-II -insulin and r = 0.63 and P < 0.1736 for
IGF-II + insulin. Statistical significance was found between -insulin
and + insulin assays only for IGF-I but not for IGF-II. However, when
comparisons were established between the different time periods,
statistical significance between -and + insulin was obtained for
IGF-II only in the period between 4 and 6 h. Similar results were
obtained when actinomycin D and doxorubicin were used as RNA synthesis
inhibitors (data not shown). To rule out nonspecific effects of DRB on
gene expression, fetal hepatocytes were left DRB free and treated with
or without insulin for the same experimental period. No significant
differences were observed between 0 and 6 h in the RNA transcripts
extracted from cells treated with or without insulin in the absence of
DRB in the media (Fig. 4).
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). The use of this method
revealed no significant increase of IGF-I and -II gene transcription
when fetal hepatocytes were treated with 0.01–1
µM insulin in the presence of 5
mM glucose for 3 h (Fig. 5B). Contrary to
what was observed in RPAs of total RNA, greater transcription levels of
IGF-I than those of IGF-II were observed in RPAs of nuclear
transcripts, probably indicating a distinct specificity/sensitivity of
the intron-exon-containing riboprobe.
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Discussion |
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Experimental models in vivo have been widely used for the study of IGF regulation and have shown the important contribution of factors such as nutrients and insulin involved in liver IGF synthesis and secretion (1, 2, 3, 4). But to delineate direct from indirect unspecific effects of such factors on the regulation of liver mRNA synthesis of IGFs, in vitro systems are required. The study of the molecular pathways and mechanism of action of the different factors on the IGF regulation has been carried out mainly in cultures of adult hepatocytes in which insulin (12, 13, 14, 15), GH (18, 19), glucocorticoids (13, 14, 19), and amino acid availability (14, 18, 20) have been reported to regulate IGF-I gene expression.
Although the factors involved in the IGF regulation seem to be the same during lifetime, the relative contribution is different depending on the stage of development (21). The study of the regulation of IGFs is particularly interesting in stages of immaturity when such regulation is GH independent (2, 3, 22) and other factors may play a decisive role. However, the research on IGF synthesis and secretion in vitro in stages of development is scant, mainly owing to changes in the differentiating pattern and to the very low synthesis of these peptides by fetal hepatocytes in plastic substratum. Two technical approaches have been used in our laboratory to overcome these difficulties: a primary culture of late fetal hepatocytes with a maximum incubation time of 24 h and a high sensitivity RPA for IGF-I and -II mRNA transcripts (5, 23). Because gluconeogenesis is very inefficient in fetal stages (24), fetal hepatocytes were cultured in a serum-free basis for no longer than 24 h to prevent cell starvation; in this condition, control hepatocytes cultured in a serum- and glucose-free medium still showed a high viability (over 85%).
As already described for fetal stages of rat development (1, 2, 3), gene expression and peptide synthesis of IGF-II were greater than those of IGF-I in all experiments, supporting both the reliability of the model and the main role of IGF-II on fetal growth and differentiation (5). Because 10% FCS evoked a significant and steady increase in IGF-I and -II transcript expression (5), all experiments were carried out in serum-free conditions. In this experimental model, we have recently shown a stimulating effect of physiological doses of glucose on IGF-I and -II gene expression in the presence of a basal nonstimulating concentration (0.01 µM) of insulin (5). However, in the same model, we observed no response of IGFs to GH (5), supporting the GH-independent IGF regulation at stages of development already demonstrated in vivo by us (2, 3) and other authors (1, 22). The role of insulin, both as a permissive factor for the glucose effect in cultured fetal rat hepatocytes and as the other potential regulatory factor during fetal stages, remained to be studied. Therefore, in the present manuscript, we have investigated whether insulin regulates IGF gene expression and secretion as shown for glucose because both are necessary for the system in vivo (1, 2, 3).
A transcriptional effect of insulin on IGF-I in cultures of adult hepatocytes has been reported (14, 25). Although insulin is a regulatory factor for liver IGFs during perinatal stages (1, 2, 3), no data showing a specific effect of insulin on IGFs in the developing liver have been reported to date. The physiological concentration of insulin during the late fetal stages is higher than that of adulthood, showing elevated values at the end of gestation that decrease immediately after birth, contrary to that of glucagon, which follows the opposite pattern (26). Supraphysiological doses of insulin have been reported to be necessary to evoke a phosphoenolpyruvate carboxykinase gene expression response in primary cultures of fetal hepatocytes (27). This response has been explained by a lower sensitivity of the insulin receptors during the fetal stages, perhaps owing to immaturity of the signaling pathways (26, 28). The fact is that higher concentrations of insulin are needed to evoke cellular responses in fetal hepatocytes (0.1–1 µM) than in adult hepatocytes (0.01–0.1 µM). Therefore, the response of IGFs in fetal hepatocytes to insulin doses higher than those considered physiological does not seem to be an unique response but a regular one at these stages.
The fact that addition of 5 mM glucose to the medium potentiated the effect of insulin on both transcripts suggests a permissive effect of glucose as a fuel because gluconeogenesis in fetal hepatocytes is basically nonexistent (24). Therefore, in the absence of glucose, the metabolic status of the cultured hepatocyte is that of a fasting cell, focused on housekeeping tasks rather than those related to growth, such as growth factor synthesis. Previous studies have shown that glucose regulates the gene expression of glycolytic and lipogenic related proteins (29, 30). More recently, glucose has been reported to stimulate IGF-I gene expression in C6 glioma cells (31) and both IGF-I and -II in fetal hepatocytes (5); but the concentrations needed to evoke significant responses were over 10 mM. Thus, the potentiating effect of 5 mM glucose on the IGF response to insulin supports the role of this carbohydrate as a fuel, ameliorating the metabolic condition of the otherwise fasting fetal hepatocyte.
Because the insulin doses necessary to evoke the IGF response in these fetal cultures of hepatocytes were slightly higher than those commonly used in adult cultures, the observed effects could be mediated by the insulin receptor or throughout another receptor with a relevant affinity for insulin (i.e. IGF-I receptor). IGF-I receptor is known to bind and respond to insulin with fair efficiency owing to their similar molecular structure and signaling pathways (1). Indeed, a great quantity of IGF-I receptor (type I receptor for IGFs) has been reported to be present in the developing liver (1). To rule out such a possibility, standard IGF-I in a concentration similar to that of insulin was added to cultures of fetal hepatocytes, and the gene expression of IGF-I and -II was measured. The absence of changes in the mRNA expression of both IGFs after IGF-I treatment indicates that IGF-I receptor is not involved in the response of IGFs to insulin in cultures of fetal hepatocytes. Although a fair cross-reactivity of the high doses of IGF-I with the insulin receptor should be expected, the lack of IGF-I and -II response in fetal hepatocytes after IGF-I treatment supports the suggested low responsiveness of the insulin receptor in these stages of development. Besides, IGFBPs produced and released by the fetal hepatocyte to the culture medium could inhibit the effect of exogenous IGF-I, but we have observed in our laboratory (our unpublished data) that IGFBP synthesis is very low in these fetal cells and a small amount of these proteins was detected in the culture medium by Western ligand blot.
The concentration of IGFs was determined in the culture medium of fetal hepatocytes to investigate whether doses of insulin within the fetal physiological range regulate not only IGF transcript abundance but also, in a parallel manner, IGF peptide secretion. An excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to different doses of insulin, suggesting that all steps of the synthetic and secretory pathway in response to insulin are fully functional at these immature stages of development.
Because an increase of transcript expression might result from transcription induction, transcript stabilization or both, the two possibilities were tested. Transcript stability was determined by transcript abundance decay assays in the presence of the transcription inhibitor DRB (32). The results showed that IGF-I and -II mRNA transcript stability was higher in the presence than in the absence of insulin, especially 6 h after the onset of DRB treatment. Transient increases of IGF-I and -II mRNA transcripts both in the absence and presence of insulin were observed during the first 2 h of DRB treatment, an effect that was also observed when use was made of other mRNA synthesis inhibitors such as actinomycin D and doxorubicin. Perhaps the stabilizing effect of the drug during the first 2 h results from a balance between a positive effect by inhibiting the synthesis of multiple RNases and the destabilizing effect on the overall RNA synthesis. The latter is dominant over the former after the first 2 h of treatment, and the insulin effect can then be observed. Although the decay experiment cannot unequivocally show the RNA stability effect during the first 2 h, this does not imply a lack of effect of insulin during this short term. It is worth mentioning that the increase of transcript abundance observed during the first 2 h in time-course experiments could still be explained by the effect of insulin on transcript stability, which is supposed to start upon addition of the hormone to the cell culture. In any case, the fact that three different RNA synthesis inhibitors with distinct mechanisms of action led to the same result strongly supports the stabilizing effect of insulin on IGF-I and -II mRNA transcripts in cultures of fetal hepatocytes. A specific effect of insulin downregulating RNase activity or the presence of alternative 3' untranslated terminal repeats in these IGF mRNA transcripts (33) could explain this increase in transcript stability, but this remains a subject for further research. The present results, however, do not rule out a more general effect of insulin to stabilize several liver gene transcripts, perhaps as part of mechanism by which insulin maintains hepatocyte viability.
Recent work has pointed to RPA of nuclear transcripts as a reliable method to assess transcriptional activity in cultured cells (23). Although a transcriptional effect of insulin on IGF-I has been found in other experimental models, especially primary cultures of adult hepatocytes or transformed cell lines (14, 25), our results showed no significant effect on IGF-I and -II transcription in fetal hepatocytes. These results suggest that insulin stimulates IGF-I and -II gene expression by increasing transcript stability rather than by inducing gene transcription. Perhaps the transcriptional machinery of immature hepatocytes is more responsive to simple nutritional signals, such as glucose (5, 29, 30), and becomes more sensitive to insulin throughout development. This would agree with the higher levels of insulin circulating during fetal stages than in adulthood (26). Indeed, responsiveness of the hepatocyte IGF system to glucose is lost during development and adult hepatocytes are insensitive to physiological doses of glucose when GH and insulin become the main regulators of the system (5). In any case, insulin could still play a crucial role in fetal growth by regulating transcript stability of mitogens of many cell types, such as IGFs. In fact, the stabilizing effect of insulin on the IGFs transcripts appears to be rather rapid because significant increases of the mRNA expression levels of both IGFs are observed in time-course experiments as soon as 2 h after the onset of insulin treatment. In addition, the partial inhibitory effect of the protein synthesis inhibitor cycloheximide suggests the need of other protein factors induced by insulin for its complete effect on IGF gene expression. Further investigation is needed to expose the protein factor(s) required for the mechanism of action of insulin.
In summary, the results support the cultures of fetal rat hepatocytes as a reliable model to address specific effects and molecular mechanisms when studying IGF regulation in perinatal stages. In this model, glucose plays a dominant role on the regulation of the IGF secretion at transcriptional and posttranscriptional levels (5), and insulin stimulates IGF-I and -II gene expression by increasing transcript stability.
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Acknowledgments |
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Footnotes |
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Abbreviations: DRB, 5,6-Dichlorobenzimidazole riboside; IGFBP, IGF binding protein; RPA, RNase protection assay.
Received May 9, 2001.
Accepted for publication August 8, 2001.
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, P < 0.05, compared with 0
µM insulin; 
, P < 0.05, compared
with prior dose of insulin.
