Istituto di Fisiologia Generale, Università degli Studi di Genova, I-16132 Genoa, Italy
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ABSTRACT |
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The insulin-like growth factor (IGF) binding proteins (IGFBPs) are important regulators of cell growth produced by different tissues. The IGFBPs regulate cell growth by modulating the activity and bioavailability of IGFs. The evidence that IGFBP-1 is a liver-specific immediate-early gene highly induced after 70% partial hepatectomy (PHx) suggests a role for the IGF-IGFBP system in hepatic regeneration. In this work we analyzed the effect of PHx on the expression of IGFBP-4, which is highly produced by the liver and very abundant in rat serum. Our results show a marked increase in hepatic IGFBP-4 mRNA levels 6-12 h after PHx and no significant change in sham-operated control animals. A parallel rise in IGFBP-4 transcript abundance was observed in the kidneys of PHx rats but not in sham-operated animals. Moreover, ligand blot analysis demonstrated that serum IGFBP-4 levels began to increase 12-24 h after surgery, consistent with the rise in the corresponding mRNA. This enhancement in IGFBP-4 production after PHx could be part of a fine regulatory mechanism to modulate IGF activity during liver regeneration.
insulin-like growth factor; hepatic growth; kidney
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INTRODUCTION |
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THE INSULIN-LIKE GROWTH FACTORS (IGF-I and -II) are mitogenic peptides that promote the growth and differentiation of tissues (13). The availability of IGFs to interact with their specific receptors and trigger biological responses is regulated by the levels of high-affinity binding proteins (IGFBPs), a family of bifunctional adaptor proteins that specifically bind the IGFs and interact with the cell surface, extracellular matrix, or other protein components. The IGFBPs regulate cell growth by modulation of the bioactivity of IGFs and by mechanisms independent of their effects on IGF availability. The abundance of IGFBPs is regulated by hormonal, nutritional, and developmental factors that act at the gene expression or protein synthesis level as well as through specific proteases. This complex mechanism may, in turn, regulate IGF activity (19, 26).
Although the liver is the main site of biosynthesis for IGF-I and some IGFBPs, the role played by the IGF-IGFBP system in the regulation of hepatic growth is not obvious. Liver regeneration after partial hepatectomy (PHx) has been used as a model in experimental animals to study the response of the hepatic IGF-IGFBP system to internal changes and to investigate the possible role of IGFs and IGFBPs in the regeneration process. In fact, it has been demonstrated that, unlike normal rat hepatocytes, cells isolated from regenerating rat liver and proliferating HepG2 cells have high levels of IGF binding sites (4). Nonparenchymal cells such as Kupffer, Ito, and endothelial cells possess the type I IGF receptor (3, 37), and recent evidence supports a role for IGF-I in regulating the proliferation of nonparenchymal cells within the liver (1, 15, 16, 30). A role for the IGF-II receptor in liver regeneration has been suggested as well, because its levels appear to be increased in hepatocytes isolated from hepatectomized rats, which are more sensitive to IGF-II for the stimulation of DNA synthesis (31, 32). However, a rise in IGF-II production by the regenerating liver after PHx has not been observed (31), and several reports demonstrate a decrease in circulating IGF-I levels in PHx animals (28, 31, 36).
Concerning IGFBPs, it is well established that the IGFBP-1 gene is one of the most highly expressed liver-specific immediate-early genes, because it is rapidly and dramatically induced in rat liver soon after PHx (14, 23). Less is known about the effect of PHx on the other IGFBPs of hepatic origin. Ghahary et al. (14) observed a decline in hepatic IGFBP-3 mRNA and serum IGFBP-3 levels both in hepatectomized and sham-operated animals, which is likely caused by changes in nutrient intake and reduced pituitary growth hormone secretion. No data are available as yet about the effect of PHx on IGFBP-4, the second most abundant IGFBP in adult rat serum after IGFBP-3. IGFBP-4 is synthesized mainly in the liver and exhibits a high affinity for IGF-I and -II that is nearly equal to that of IGFBP-3 (9). In this work we investigated the expression of hepatic IGFBP in rats subjected to two-thirds hepatectomy and demonstrate an increase in IGFBP-4 expression during liver regeneration.
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MATERIALS AND METHODS |
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Animals. Adult male Wistar rats (250-300 g) were housed under conditions of controlled temperature and light. The animals were anesthetized by intraperitoneal injection of Ketalar (Parke-Davis, Barcelona, Spain) at the dose of 15 mg/100 g body wt and subjected to PHx or sham hepatectomy (SHx) between 800 and 1200. For PHx, the left lateral and median lobes (~2/3 of total liver mass) were ligated and excised through a median incision (17). This tissue was used as the zero time control. SHx animals underwent surgery and manipulation of the liver but no tissue resection. After surgery, the rats were warmed under a heat lamp until they regained consciousness and were allowed free access to water. Food was provided daily at 1800. To control for the animals' nutritional status, in additional experiments SHx rats were fed the average intake of PHx animals, but no significant differences were seen in respect to freely feeding animals.
Rats were killed by cervical dislocation 3, 6, 9, 12, 24, 48, 72, and 96 h after surgery. Blood was collected and allowed to clot, and serum was stored atRNA isolation.
Total RNA was prepared using acid guanidinium
thiocyanate-phenol-chloroform extraction (7). RNA was quantified by
measuring absorbance at 260 nm. Samples were stored at 80°C
until used.
cDNA probes. The rat IGFBP-1, -2, -3 and -4 cDNA probes were kindly provided by Dr. S. Shimasaki (Department of Cell Biology, Scripps Research Institute, La Jolla, CA). The rat 18S rDNA probe (BamH I-Hind III fragment; Ref. 5) was generously provided by Dr. I. G. Wool (Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL). The probes were labeled to high specific radioactivity by multiprime DNA labeling systems (Amersham International, Amersham, UK) using [32P]dCTP (Du Pont NEN, Bad Homburg, Germany; specific radioactivity 3,000 Ci/mmol).
Northern blot analysis.
Total RNA (20 µg) was subjected to electrophoresis on 1% agarose gel
containing 2.2 M formaldehyde in 20 mM MOPS (pH 7.0), 5 mM sodium
acetate, and 1 mM disodium EDTA. RNA was stained with ethidium bromide
to check the integrity of the samples and to ensure equal loading in
each lane. After transfer to nylon membrane by capillary blotting,
prehybridization was performed for 2 h at 42°C in a solution
containing 5× SSPE (1× SSPE = 0.15 M NaCl-0.01 M
NaH2PO4-1 mM EDTA), 50% formamide, 5×
Denhardt's solution, 0.5% SDS, and 100 µg/ml denatured salmon sperm
DNA (29). Hybridization was carried out for 20 h at 42°C in fresh
buffer containing the 32P-labeled probe. After
hybridization, the filters were washed sequentially in 2×
SSPE-0.1% SDS, 1× SSPE-0.1% SDS, and 0.1× SSPE-0.1% SDS
at 42°C, with each wash lasting for 20 min. Filters were then exposed to Amersham Hyperfilm-MP films at 80°C using
intensifying screens.
Measurement of serum IGF-I. Serum IGF-I was measured by RIA (Medgenix Diagnostics, Fleurs, Belgium) after acid-ethanol extraction according to Daughaday et al. (10).
Tissue extraction for IGFBPs.
Liver tissue was homogenized in 20 mM Tris-2% Triton X-100 buffer (pH
7.4), and IGFBPs were extracted following the method of Flyvbjerg et
al. (12). Aliquots of extracts were stored at 80°C. Protein
content of the extracts was measured using the bicinchoninic acid
protein assay (Pierce Chemical, Rockford, IL).
Ligand blotting and immunoblotting.
For ligand blotting, sodium [125I]iodide
(carrier free) was purchased from Amersham. Recombinant human IGF-I was
a gift from Ciba Geigy (Basel, Switzerland). IGF-I was iodinated by the
chloramine-T method to a specific activity of ~60-80 µCi/µg.
Samples were analyzed according to Hossenlopp et al. (18), with slight
modifications. Briefly, aliquots of tissue extracts equivalent to 300 µg of protein or 5 µl of serum were separated on a 12% SDS-PAGE
under nonreducing conditions and transferred to nitrocellulose.
Membranes were blocked overnight at 4°C in 0.9% NaCl-50 mM
Tris · HCl (pH 7.4)-0.5% fish gelatin and then
incubated with 125I-IGF-I (5-10 × 106 cpm) at 4°C for 24 h in 0.9% NaCl-50 mM
Tris · HCl (pH 7.4)-0.5% fish gelatin-0.2% NP-40.
After washing, membranes were air-dried and exposed for 24 h at
80°C to X-ray films.
Statistical analyses. Data are presented as means ± SE. Statistical significance was analyzed using ANOVA with the multiple-group comparison test of Bonferroni. Differences were regarded as significant when P < 0.05.
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RESULTS |
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Serum thyroid hormone levels.
The sera from PHx rats were analyzed for thyroid hormone levels because
it has been reported that PHx is associated with reduced serum
T3 and T4 during the first 24-48 h after
surgery (20, 21). Our results reported in Table
1 are in agreement with these reports. It
can be observed that serum T3 and T4
significantly declined soon after PHx and remained low until 48 h after
surgery, returning to control values thereafter. SHx did not cause
significant changes in serum thyroid hormone levels (data not shown).
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Effect of PHx on hepatic IGFBP mRNA levels.
Northern blot analyses were carried out on liver RNA samples to
determine the changes in IGFBP mRNA levels after PHx. The results are
depicted in Fig. 1. The immediate-early
increase in IGFBP-1 mRNA abundance during liver regeneration is in
total agreement with data reported by others (14, 23). Hybridization of
liver RNA with IGFBP-3 cDNA revealed a faint signal that was not
significantly affected by PHx. In contrast, hepatic IGFBP-4 expression
was markedly increased within 6 h, peaked at 9-12 h, and
began to decrease at 24 h after PHx. As shown in Fig.
2, no significant increase in the abundance
of hepatic IGFBP-4 mRNA was observed after SHx. IGFBP-2 transcript was
not detected in the liver of intact rats and remained undetectable
after PHx (data not shown).
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Effect of PHx on renal IGFBP-4 mRNA levels.
PHx also influenced kidney IGFBP-4 mRNA expression similarly to that
observed in regenerating liver. As shown in Fig.
3, renal IGFBP-4 mRNA levels increased
within 3 h, peaked at ~9 h, and returned to control values
at 24 h after PHx. No significant change in renal IGFBP-4 mRNA
expression was seen in the SHx rats (data not shown).
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Effect of PHx on serum IGF-I and IGFBP levels.
Serum IGF-I levels after PHx are shown in Table
2. Our data are in line with previous
results by others reporting a decrease in serum IGF-I levels and
activity after PHx (28, 31, 35, 36). In our experiments, a decline in
circulating IGF-I was evident from 12 h after liver resection. Serum
IGF-I levels were ~60% of normal at 24 h after surgery and returned
to control values by 72 h after PHx.
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Effect of PHx on liver IGFBP levels.
Figure 6 is a representative autoradiogram
of ligand blot performed on liver extracts. IGFBPs with molecular
masses similar to those observed in serum were detected. A depletion of
liver IGFBP content was observed within 24- 48 h after PHx,
followed by a progressive return to control levels.
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DISCUSSION |
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In this work, the expression of IGFBPs in the regenerating liver was investigated. Classic reports demonstrated that IGFBP-1 is an immediate-early gene, whose expression is rapidly and dramatically increased after 70% PHx (14, 23). In our experiments this pattern was confirmed. However, serum IGFBP-1 levels appeared to be decreased during the first day of liver regeneration, returning to control values thereafter. Similarly, a decline in IGFBP-3 serum levels was observed within 24 h after PHx, without significant differences in the hepatic mRNA abundance. A rapid decrease in circulating IGFBP levels also was reported by Phillips et al. (25), who proposed that this was caused by a rapid clearance of the binding proteins from blood once the main site of production was partially removed by two-thirds hepatectomy. Moreover, as observed by Ghahary et al. (14), a decrease in circulating IGFBP-3 during liver regeneration may be caused by a drop in nutrient intake and growth hormone secretion.
Our original observation concerns an enhanced expression of IGFBP-4 during liver regeneration. The hepatic IGFBP-4 mRNA abundance was increased about fourfold over control values 9-12 h after PHx and returned to basal levels 24-48 h after surgery. Consistent with what was observed at the mRNA level, serum IGFBP-4 concentration rose 12-24 h after PHx and remained high thereafter.
Although a reduction in food intake occurs in rats after PHx (28, 35), the enhancement in IGFBP-4 expression during liver regeneration was not caused by changes in nutritional status, because it did not occur in food-restricted SHx rats. Moreover, fasting was reported to reduce rather than increase IGFBP-4 mRNA levels in the liver (6). Finally, the changes in IGFBP-4 expression were limited to the first 24 h after PHx, a time at which nutritional inputs should not have great influence.
Interestingly, IGFBP-4 transcript levels also were elevated in the kidney of PHx rats. This is in line with the notion that the kidney is among the organs that are mostly affected by PHx; in fact, compensatory renal hypertrophy during liver regeneration in rats has been described (24), and elevated IGFBP-1 mRNA levels have been seen in both liver and kidney of PHx rats (14).
The increase in both hepatic and renal IGFBP-4 expression would suggest the involvement of some local and/or systemic factors induced by PHx and responsible for the enhanced IGFBP-4 expression during liver regeneration. Recently, we (11) demonstrated a stimulation by thyroid hormone, which is able to increase IGFBP-4 production by rat hepatocytes, both in vitro and in vivo. However, in the case of liver regeneration after PHx, an involvement of thyroid hormone in enhancing IGFBP-4 production should be ruled out, because we observed a decline in T3 and T4 serum levels within 24-48 h after liver resection, confirming data reported by others (20, 21).
What is the physiological significance of the rise in IGFBP-4 production after hepatectomy? So far, IGFBP-4 has been described only as an inhibitor of the mitogenic actions of IGF-I on several cell types (22, 27, 34). It is known, and also documented in this paper, that serum IGF-I levels and activity are decreased after PHx (28, 31, 35, 36). The classic actions of circulating IGFBP-4 in this context would be to bind IGF-I, prolong its half-life, and affect its activity by preventing or delaying its binding to the receptors. Because high levels of IGF-I and -II receptors are present on hepatocyte membranes during liver regeneration (4, 31, 32), IGF-I-mediated effects on the hepatocyte itself cannot be excluded. Paracrine effects are also possible, because recent evidence suggests an important role for IGF-I in regulating the proliferation of nonparenchymal liver cells (particularly Ito cells) that possess the type I IGF receptor (1, 15, 16, 30). The depletion of liver IGFBP-4 protein content after PHx, as judged by ligand blot analysis of cell proteins, appears to be in line with an enhanced action of IGF-I on liver cells during regeneration. The finding that IGFBP-4 does not accumulate in the liver, despite the increase in mRNA, could indicate its prompt release and/or cellular catabolism. In fact, it is well established that an important site of regulation for IGFBP-4 is its proteolysis, resulting in fragments that do not bind IGF. Within the liver, the soluble lysosomal aspartic protease cathepsin D might cleave IGFBP-4 and be involved in the clearance of IGFBPs or IGFs (8).
Finally, the possibility exists that the IGFBPs exert IGF-independent effects. This has been validated for IGFBP-1, -3, and -5, which can bind the cell surface or the extracellular matrix (19). Although such interactions have not been documented for IGFBP-4, inhibitory action of the protein through an as yet unknown IGF-independent process has been suggested (33).
In conclusion, our finding of an increase in liver and kidney IGFBP-4 production in PHx rats, resulting in elevated serum IGFBP-4 levels, adds proof of the involvement of the IGF-IGFBP system in liver regeneration. The regulation of IGFBP expression could be part of a mechanism to modulate local and peripheral IGF activity after liver resection.
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ACKNOWLEDGEMENTS |
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The authors are indebted to Prof. M. Orunesu for encouragement and support during this study. We thank V. Capicchioni for excellent technical assistance and Dr. A. Barreca for help with IGF-I RIA measurements. We are grateful to Dr. J.-G. Scharf for help with immunoblot analyses.
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FOOTNOTES |
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This work was supported by a grant from Minestero dell'Università e della Ricerca Scientifica e Technologica, Rome, Italy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. Fugassa, Istituto di Fisiologia Generale, Corso Europa 26, I-16132 Genova, Italy (E-mail: fugassae{at}unige.it).
Received 16 November 1998; accepted in final form 6 October 1999.
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