1 Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, Sydney, New South Wales 2065, Australia; and 2 Institute of Experimental Clinical Research, Aarhus University Hospital, DK-8000 Aarhus, Denmark
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ABSTRACT |
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There is little information on free insulin-like growth factor I (IGF-I) and its regulatory proteins during fasting and refeeding. Therefore, we examined rats during fasting (0, 1, 2, and 3 days) and refeeding (3, 6, and 12 h and 1, 2, 3, and 7 days) (n = 6-9). Serum was analyzed for insulin, C-peptide, growth hormone (GH), free and total IGF-I, IGF-binding protein (IGFBP)-1 and -3, and the acid-labile subunit (ALS). Additionally, liver mRNA for IGF-I, IGFBP-1, and ALS was determined. Fasting reduced serum levels of GH, free and total IGF-I, IGFBP-3, and ALS, whereas IGFBP-1 was increased (P < 0.0001). Refeeding normalized IGFBP-1 at 3 h and GH at 12 h. Free IGF-I changed in parallel with total IGF-I, ALS, and IGFBP-3, being normalized at 48 h of refeeding. IGFBP-1 (peptide and mRNA) correlated inversely with insulin and C-peptide (P < 0.001). The correlation between peptide and mRNA was relatively strong for IGFBP-1 (r2 = 0.36; P < 0.0001), moderate for IGF-I (r2 = 0.18; P < 0.0005), and insignificant for ALS. In conclusion, insulin appears to regulate IGFBP-1 in fasted and refed rats. However, the normal inverse relationship between free IGF-I and IGFBP-1 was absent, and free IGF-I changed in parallel with total IGF-I and thus ALS and IGFBP-3. Finally, the regulation of the hepatic synthesis of IGF-I, IGFBP-1, and ALS seems to differ substantially.
free and total insulin-like growth factor I; insulin-like growth factor-binding protein-1 and -3; acid-labile subunit
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INTRODUCTION |
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THE INSULIN-LIKE GROWTH FACTOR (IGF) system is extremely sensitive to metabolic alterations, and changes within it are believed to play a key role in the processes that link nutrition and growth (29). During nutritional deprivation, the first observed change is an increase in IGF-binding protein-1 (IGFBP-1), an in vivo inhibitor of IGF-I action (7). IGFBP-1 is inversely regulated by portal insulin, and an increased hepatic production and secretion of IGFBP-1 are detectable within a few hours of fasting in rats (15, 17, 20, 21). Because serum IGFBP-1 binds only a minor portion of the circulating IGF-I pool, short-term changes in IGFBP-1 do not affect the total (extractable) levels of IGF-I (11). However, an increase in levels of IGFBP-1 is usually related to a reduction in free IGF-I, and this may serve as a mechanism to adjust IGF-I bioactivity in relation to the actual fuel supply (1, 29).
Sustained nutritional deprivation causes profound changes in the IGF system. In the rat, fasting for 48-72 h markedly reduces the circulating levels of total IGF-I (14, 18, 32). This appears to be a direct effect on the production of IGF-I by the liver (27), the primary source of circulating IGF-I. However, the concomitant reduction in serum levels of IGFBP-3 (29) and acid-labile subunit (ALS) (8) may also be of importance. IGFBP-3 is the predominant IGFBP and carries as much as 80% of the circulating IGF-I pool in a ternary complex with ALS. Formation of the ternary complex prolongs the half-life of IGF-I and is considered to be a prerequisite for maintenance of high and stable plasma levels of total IGF-I (1).
In rats, nutritional rehabilitation normalizes the IGF system in the same order as the abnormalities appeared during fasting: in 2 h, refeeding has reduced levels of IGFBP-1 to normal values (17, 20), whereas between 1 and 4 days (8, 14, 18, 32) are required to normalize serum total IGF-I. Interestingly, serum levels of ALS show an even later response to refeeding, and changes in IGFBP-3 remain to be studied, because quantitative rat IGFBP-3 assays were not available until recently (8).
Studies based on nonfasting and overnight fasting serum samples have shown an inverse relationship between levels of IGFBP-1 and free IGF-I (11, 12). However, it remains to be investigated whether the response of IGFBP-1 to fasting and refeeding is accompanied by oppositely directed changes in free IGF-I. Therefore, we compared serum levels of immunoreactive IGFBP-1 and ultrafiltered free IGF-I in rats during fasting and refeeding. Additionally, measurements of serum levels of total (extractable) IGF-I, ALS, IGFBP-3 (determined by use of a recently developed competitive binding assay; Ref. 10), growth hormone (GH), insulin, and C-peptide were included. Finally, we determined liver mRNA levels of IGF-I, ALS, and IGFBP-1.
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MATERIALS AND METHODS |
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Protocol. Adult male Wistar rats
(~300 g) were randomized to 12 groups containing two control groups
at day
0 (n = 9) and day 10 (n = 8) and 10 groups
(n = 6) of rats, which were studied after 1, 2, and 3 days
of fasting and after 3 days of fasting plus refeeding for 3, 6, and 12 h and 1, 2, 3, and 7 days. During the 10 days of study, all animals had
free access to water, and during the refeeding period, animals had free
access to fodder. At the time of killing, animals were anesthetized by
use of NO-O2-halothane and bled by
cardiac puncture. The liver was quickly removed, weighed, and
snap-frozen in liquid nitrogen for later mRNA determination. All blood
samples were collected within 5 min after anesthesia, and serum samples
were kept at 80°C for later analysis. The study was approved
by the Animal Care and Ethics Committee, Royal North Shore Hospital,
Sydney, Australia.
Assays. All samples were analyzed in duplicate within the same assay unless otherwise stated. IGF-I was determined by an in-house IGF-I RIA with a polyclonal rabbit antibody (Nichols Institute Diagnostics, San Capistrano, CA) and recombinant human (rh) IGF-I as standard (Amgen Biologicals, Thousand Oaks, CA). Monoiodinated rhIGF-I [125I-(Tyr31)-rhIGF-I] was obtained from Novo Nordisk A/S, Bagsværd, Denmark. Total IGF-I was determined in acid-ethanol serum extracts with a within-assay and between-assay coefficient of variation (CV) averaging 5 and 10%, respectively (12). Free IGF-I was determined with ultrafiltration by centrifugation at conditions approaching those in vivo (13). In brief, Amicon YMT 30 membranes and MPS-1 supporting devices were used (Amicon Division, Beverly, MA). Before centrifugation, serum samples were adjusted to pH 7.4 by being gassed with CO2, after which aliquots of 400 µl were applied to the membranes, incubated (30 min at 37°C), and centrifuged (1,500 rpm at 37°C; model Rotixa/RP, Hettich Zentrifugen, Tuttlingen, Germany). Serum free IGF-I was determined directly in the ultrafiltrates with a within-assay CV that, including ultrafiltration and RIA, averaged 19%.
Rat IGFBP-3 was determined by a recently developed competitive binding assay (10). The assay is based on the ability of IGFBP-3 to form a ternary complex with ALS in the presence of IGF-I, and it fails to detect any interaction between IGFBP-3 and ALS in the absence of IGF-I. A defined amount of human ALS (hALS) was bound to Maxisorb test tubes (Roskilde, Denmark) that were precoated with hALS antibody. The assay depends on competition between a covalent complex of 125I-hIGF-I and hIGFBP-3, added as tracer, and hIGFBP-3 or rat IGFBP-3 (rIGFBP-3) in standard and test samples, for binding to the immobilized hALS. Purified natural hIGFBP-3 served as standard. hIGFBP-3 and rIGFBP-3 were able to compete for tracer binding in the presence, but not in the absence, of IGF-I, and they diluted in parallel. Before assay, rat serum samples were acidified to denature endogenous ALS. Samples were analyzed in two assays with a within-assay and between-assay CV of <13%.
Rat ALS and rat IGFBP-1 were determined by specific RIA methods as previously described (2, 17). Rat insulin was determined by RIA (Novo Nordisk A/S) with iodinated recombinant human (rh)-insulin (125I-rh-insulin) as tracer, purified rat insulin as standard, and a polyclonal guinea pig antibody. Samples were analyzed in triplicate in one assay with a mean CV of <5%. Rat C-peptide was determined by RIA (Linco Research, St. Charles, MO) with a within-assay CV averaging <5%. Rat GH was determined by RIA (Amersham, Bucks, UK). Serum samples were analyzed in duplicate in two assays with a within-assay and between-assay CV of <5 and 13%, respectively. Serum glucose was determined in duplicate by the oxidase method.
RNA extraction and Northern analysis.
Total RNA was extracted from rat livers with the guanidine
isothiocyanate/acid-phenol technique (4). Total RNA samples (20 µg)
were electrophoresed in 1% agarose gels containing 2.2 mol/l
formaldehyde. The integrity of the ethidium bromide-stained RNA samples
was confirmed on an ultraviolet light-box. The RNA was then transferred
by capillary blotting to Zetaprobe GT membranes (Bio-Rad, Richmond, CA)
and cross-linked by heat treatment at 80°C in a gel drying
apparatus. A 350-bp rat ALS cDNA probe was generated by PCR from a
genomic DNA construct containing exon 2 of the rat ALS gene, with
oligodeoxynucleotides described previously (Genbank accession no.
AF006203; Ref. 8). The rat IGFBP-1 cDNA probe was prepared from an
800-bp EcoR
I/Hind III fragment derived from a PCR
product generated with the rat decidual IGFBP-1 cDNA sequence (19). An
800-bp Xho
I/EcoR I fragment of clone IGF-IB2
pBluescript KS (Genbank accession no. X06108) was used to generate the
IGF-I cDNA probe. The rat IGFBP-1 and IGF-I cDNA probes were kindly
provided by Dr. S. Shimasaki (Scripps Research Institute, LaJolla, CA)
and Dr. P. Rotwein (Oregon Health Science University, Portland, OR), respectively. A rat cyclophilin cDNA probe was generated by reverse transcriptase-PCR from rat liver total RNA (with oligodeoxynucleotides: cyclophilin 1, 5'-ACCGTGTTCTTCGACATCACG-3', cyclophilin 2, 5'-ACATGAATCCTGGAATAATTCTG-3'). The cDNA was
171-bp long, and the oligonucleotides were based on sequence in Genbank
accession no. M19533 for the rat cyclophilin cDNA. All cDNAs were
labeled with a Ready-to-Go random-priming kit (AMRAD-Pharmacia, Sydney,
Australia) and
([32P]dCTP; AMRAD-NEN,
Sydney, Australia). Filters were prehybridized and hybridized (2 × 106
counts · min1 · ml
1)
as described previously and then washed with 0.1× saline sodium citrate (SSC) at 42°C. Filters were then quantified with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The blots were
stripped in 0.01× SSC, 0.5% SDS at 80°C and then rescreened
sequentially with the IGFBP-1, IGF-I, and cyclophilin cDNA probes. Data
were normalized by expressing the ratio of ALS, IGF-I, and IGFBP-1 to
cyclophilin mRNA. Two different gels were needed to run all samples
from fasted and refed animals, and care was taken to distribute samples
from all groups on each gel. However, because of the limited capacity of the gels, the control group was reduced to include six randomly selected animals from day
10. The fasted and refed groups
consisted of RNA from six animals. A third gel was used for comparison
of day
0 and
day
10 control animals. All data are
expressed as the percentage of the mean of the
day
10 controls (~ 100%).
Statistics. The 12 groups were compared by one-way ANOVA. If significant, the fasted and refed groups were compared with day 0 controls with Bonferroni's unpaired t-test for multiple comparisons. Linear regression analysis was used to assess the relationship between the measured variables. All data were log transformed before analysis to improve normality and variance homogeneity. Data are given as means ± SE, and P values <0.05 were considered statistically significant.
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RESULTS |
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During the 10 days of study, freely fed control rats gained 13% in body weight from 308 ± 5 to 347 ± 4 g (P < 0.05). Fasting for 3 days reduced the body weight by 12% (to 271 ± 5 g; P < 0.05), compared with day 0. However, at the end of the study, after 7 days of refeeding, the body weight of the fasted rats was fully restored (344 ± 5 g) and similar to that of day 10 controls.
Serum glucose decreased gradually during fasting, and at
day 3 levels were reduced by 34%, compared with fed controls
(P < 0.05; Fig.
1A).
Refeeding caused an initial increase in serum glucose at 3 h (+25%;
P < 0.05) and 6 h (+22%;
P < 0.05), but apart from this
levels remained within the range of fed controls. Serum levels of
insulin (Fig. 1B) and C-peptide
(Fig. 1C) were markedly reduced
during the 3 days of fasting (P < 0.05). Refeeding for 3 h increased serum C-peptide and insulin
by ~160 and 80%, respectively, compared with the fasting level at
day
3, but both peptides remained below
the level of the controls until 6 h of refeeding. A difference in
levels of serum C-peptide was observed when day 0 and
day 10 controls were compared
(P < 0.05).
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Serum GH was significantly reduced in the groups being fasted for 2 days and refed for 3 and 6 h (P < 0.05; Fig.
2A). A
tendency toward low levels of GH in rats being fasted for 3 days was
observed (P < 0.06).
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Serum IGFBP-1 increased significantly during fasting (P < 0.05; Fig. 2B), and although levels reached a maximum on the second day of fasting, the increases in serum IGFBP-1 were statistically similar when rats fasted for 1, 2, and 3 days were compared. Three hours of refeeding was sufficient to normalize serum IGFBP-1, which however continued to decline, and at 6 h the level was below that of day 0 controls (P < 0.05). After 12 h of refeeding, levels of IGFBP-1 were not different from fed controls.
During the study, serum levels of free IGF-I (Fig.
2C), total IGF-I (Fig.
2F), IGFBP-3 (Fig.
2E), and ALS (Fig.
2D) changed in parallel. When the
relative reductions from day
0 to
day 3 in ALS (46 ± 3%), IGFBP-3 (
47 ± 6%),
total IGF-I (
40 ± 3%), and free IGF-I (
35 ± 7%)
were compared, they were statistically similar
(P = 0.4). However, minor differences
were observed. Free and total IGF-I were both significantly reduced at
the second day of fasting (P < 0.05), and refeeding for 2 days was needed to restore levels. As was
the case for IGF-I, levels of ALS were reduced from the second day of
fasting (P < 0.05), but in contrast to IGF-I, 3 days of refeeding were necessary to increase levels to that
of the controls. Levels of IGFBP-3 were reduced in rats being fasted
for 3 days and refed for 3 and 6 h (P < 0.05). The absolute changes from baseline (
values) in serum
levels of ALS, IGFBP-3, total IGF-I, free IGF-I, IGFBP-1, insulin,
C-peptide, and GH have been summarized in Table
1.
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The hepatic mRNA expression of ALS (Fig.
3A) and
IGF-I (Fig. 3B) showed a delayed
response to fasting, and both mRNA levels continued to decrease during
the refeeding period (P < 0.05). In
contrast, IGFBP-1 mRNA levels were increased severalfold during the
fasting period (P < 0.05; Fig.
3C). When the hepatic mRNA expression of ALS, IGF-I, and IGFBP-1 in controls at
day 0 and day
10 was compared, a significant
difference was observed for ALS, whereas no changes were observed for
IGF-I and IGFBP-1 (Fig. 3,
A-C).
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Linear regression analysis was used to assess the relationship between
the different variables. The three GH-dependent peptides ALS, IGFBP-3,
and IGF-I were all positively correlated (0.38 r2
0.64;
P values < 0.0001). Free IGF-I
correlated positively with ALS, IGFBP-3, and total IGF-I (0.28
r2
0.48;
P < 0.0001), whereas IGFBP-1 was
inversely correlated with free IGF-I during the fasting period only
(r2 = 0.38;
P < 0.001). The correlation between
serum peptide and mRNA levels was relatively strong for IGFBP-1
(r2 = 0.36;
P < 0.0001) but only moderate for
IGF-I (r2 = 0.18;
P < 0.0005) and insignificant for
ALS. Serum levels of C-peptide and insulin were positively correlated
(r2 = 0.41;
P < 0.0001), and for both peptides
we observed an inverse association with serum IGFBP-1
(C-peptide: r2 = 0.38; P < 0.0001; insulin:
r2 = 0.15; P < 0.001) and liver
IGFBP-1 mRNA (C-peptide:
r2 = 0.50;
P < 0.0001; insulin:
r2 = 0.21;
P < 0.0001). However, it is
noteworthy that both liver IGFBP-1 mRNA and serum IGFBP-1 were
significantly more strongly correlated with serum C-peptide than
insulin (P < 0.05).
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DISCUSSION |
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It has been suggested that the ternary complex plays a major role in regulating IGF-I bioactivity over the long term, whereas the short-term regulation of IGF-I not sequestered in the ternary complex is provided by IGFBP-1 (1). However, as we show here in rats, the situation appears to be different when the IGF system has been suppressed for some days by nutritional deprivation. In this case, the rapid normalization of IGFBP-1 after refeeding did not result in any detectable increase in free IGF-I, which changed in parallel with ALS, IGFBP-3, and total IGF-I. This finding suggests that the role of IGFBP-1 in the regulation of IGF-bioactivity is restricted to situations where the circulating levels of IGF-I are maintained at a constant level.
In the rat, unlike in humans, the secretion of GH becomes markedly suppressed by fasting (28, 29). Tannenbaum et al. (28) studied the secretion of GH in conscious male rats fasted for 1, 2, and 3 days and after refeeding for 3 days. They observed a progressive reduction in the amplitude and duration of GH pulses during fasting, whereas refeeding resulted in a rebound response with an increased number of secretory pulses and a shorter duration of the GH rhythm (28). The present study did not include determination of GH pulsatility. However, as judged from the single sample GH measurements, the secretion of GH changed in accordance with the aforementioned observations, i.e., levels were low during fasting, and showed a rebound phenomenon with a tendency toward higher levels in rats refed for 2 days.
Fasting increases the circulating levels of IGFBP-1 in humans as well as in rats (6, 15, 17). Because insulin markedly suppresses the synthesis of IGFBP-1 in human fetal liver explants in organ culture (17) and the expression of IGFBP-1 in rat hepatocytes (24), the stimulatory effect of fasting on IGFBP-1 was believed to be a consequence of reduced insulin levels in both humans and rats (20). However, Murphy and et al. (20) questioned this view by reporting that in rats GH deficiency rather than insulin deficiency was responsible for the fasting-induced increase in IGFBP-1, and they speculated that the regulation of IGFBP-1 was subject to species differences. The authors, however, did not consider differences between portal and peripheral insulin concentrations, which may be important. In fact, because the liver is the primary source of circulating IGFBP-1, serum IGFBP-1 has been suggested to reflect portal rather than peripheral insulin levels (3, 15). In accordance with previous observations, liver mRNA and serum peptide levels of IGFBP-1 increased during fasting and normalized within 3 h of refeeding (17, 20, 31). It is noteworthy that serum insulin remained markedly suppressed during the first 3 h of refeeding, whereas serum glucose was significantly elevated, compared with fed controls. These observations are in accordance with findings by Ziegler et al. (32), and they point to an impaired secretion of insulin in the postfasted state. On the other hand, in situations with major nutritional changes, the peripheral levels of insulin may not be representative for the pancreatic secretion of insulin, and, therefore, we included determinations of C-peptide, which is not removed by the liver. These results showed a marked increase in serum C-peptide in rats refed for 3 h, and although the level was not fully restored to that of day 0 controls, the changes in C-peptide clearly show that the pancreatic secretion of insulin must have increased markedly during the first hours of refeeding. Furthermore, levels of C-peptide were strongly correlated with both IGFBP-1 mRNA (r2 = 0.50) and serum IGFBP-1 (r2 = 0.38). Therefore, we suggest that insulin is indeed an important regulator of the hepatic synthesis and secretion of IGFBP-1 in rats, as also observed in humans (15). In contrast to C-peptide and insulin, GH did not correlate with liver mRNA and serum peptide levels of IGFBP-1 (data not shown), and although our data do not preclude the possibility that hyposomatotropinemia rather than hypoinsulinemia is responsible for the fasting-induced stimulation of IGFBP-1 (20), any direct role of GH on IGFBP-1 during refeeding seems to be more questionable.
Numerous in vitro and in vivo studies show an inhibiting effect of IGFBP-1 on IGF-I stimulated growth and differentiation. Similarly, IGFBP-1 is able to counteract the insulin-like hypoglycemic activity of IGF-I in experimental in vivo studies (for review see Refs. 1, 7, 15, 25). Considering the anabolic and hypoglycemic actions of IGF-I, the increase in IGFBP-1 during fasting (6, 17, 26) may be regarded as a protective mechanism serving to mobilize energy. Conversely, during nutritional rehabilitation the reduction in IGFBP-1 may serve to promote the metabolic actions of IGF-I. Although it remains to be proven, the effects of IGFBP-1 on IGF-I action are most likely mediated through changes in free IGF-I. This view is supported by observations of an inverse correlation between levels of IGFBP-1 and free IGF-I in nonfasting as well as in overnight fasting serum samples (11-13). However, it has not previously been investigated whether the response of IGFBP-1 to fasting and refeeding is accompanied by oppositely directed changes in free IGF-I. The present data show that during fasting the inverse relationship between free IGF-I and IGFBP-1 was maintained, whereas during refeeding it was absent. As judged from the circulating levels of free and total IGF-I, the hepatic secretion of IGF-I was progressively reduced during fasting and it remained suppressed for at least 24 h during refeeding. Together, these observations suggest that reductions in IGFBP-1 may only allow an increase in serum free IGF-I in situations when IGF-I is being synthesized normally, and we hypothesize that the role of IGFBP-1 as a dynamic regulator of serum free IGF-I is restricted to noncatabolic situations. Whether the delayed increase in serum free IGF-I during refeeding is potentially beneficial is not clear, but it may represent a mechanism serving to regenerate energy storage rather than protein storage and to avoid IGF-I-induced hypoglycemia.
The observed changes in serum total IGF-I are in agreement with earlier findings in rats (18), and ALS showed the same delayed response to fasting and refeeding as originally described (8), i.e., levels continued to decrease during the first hours of refeeding, and normalized later than total IGF-I. So far, nutritional studies of circulating IGFBP-3 in rats have been performed by use of ligand blotting, and with this technique reduced levels of IGFBP-3 have been observed in protein-restricted (16, 30), protein-deprived (5), and caloric-restricted rats (23). Interestingly, in one of these studies, a positive correlation between IGFBP-3 and total IGF-I was observed (30). In the present study, serum levels of IGFBP-3 were determined by a recently developed quantitative competitive binding assay (10), and the observed changes were in good agreement with those mentioned above. Furthermore, in broad outline, IGFBP-3 changed in parallel with ALS and IGF-I, as indicated by the positive relationship among the three peptides. However, the time of refeeding needed to normalize the three peptides differed; thus refeeding for 1 day was sufficient to normalize levels of IGFBP-3, whereas total IGF-I and ALS were normalized after 2 and 3 days, respectively. As suggested from the GH determinations, the secretion of GH was increased if not restored 12 h after refeeding was initiated, and the different temporal changes of IGFBP-3, IGF-I and ALS may reflect real differences in the sensitivity to GH. However, further studies are needed to determine this.
IGFBP-3 correlated positively with free IGF-I, in accordance with previous findings and in clear contrast to IGFBP-1 (11, 12). This strongly supports the view that IGFBP-1 and IGFBP-3 possess different biological roles in the regulation of IGF bioactivity. IGFBP-3 stabilizes levels of IGF-I within the ternary complex, but how this affects the access of IGF-I to the target tissues remains unknown. The ternary complex is thought to cross the capillary barrier only poorly, and it has therefore been speculated that IGF-I bioavailability is controlled in some way through a release of IGF-I from the circulating ternary complex to the extravascular compartment. Given the present findings, our study implies that the ternary complex controls IGF-I bioavailability at least partly through regulation of free IGF-I.
To examine the basis for the observed changes in serum levels, we measured the hepatic mRNA expression of the predominantly liver-derived proteins ALS, IGF-I, and IGFBP-1. ALS mRNA was determined, because in a smaller study of fasted (24 and 48 h) and refed (2 and 24 h) rats, serum ALS changed significantly, whereas steady-state hepatic ALS mRNA levels remained unchanged (8). Accordingly, in this much larger study, we found no correlation between serum peptide and mRNA levels of ALS and suggest that ALS synthesis in the fasted state is regulated primarily at the posttranscriptional level, in accordance with recent findings in vitro (9). Zhang et al. (31) studied the hepatic gene expression of IGF-I in fasted rats. Their results suggested that fasting was regulating the hepatic IGF-I gene expression mainly at the posttranscriptional level by delaying the IGF-I pre-mRNA splicing, which attenuated mature IGF-I mRNA generation. Furthermore, the degradation of cytoplasmic mature IGF-I mRNA was accelerated (31). In the present study, the hepatic expression of IGF-I mRNA showed a delayed response to fasting and refeeding, compared with the concomitant changes in serum IGF-I. Accordingly, the correlation between serum peptide and mRNA levels of IGF-I was weak (r2 = 0.18), albeit significant. Thus our results support the concept that the regulation of the hepatic expression of IGF-I in fasted and refed rats takes place predominantly at the posttranscriptional level.
To our knowledge, this is the first study to compare changes of immunoassayable serum IGFBP-1 and hepatic IGFBP-1 steady-state mRNA levels in the rat. The positive correlation between IGFBP-1 peptide and mRNA levels (r2 = 0.36) indicates that the hepatic production of IGFBP-1 is at least partly transcriptionally regulated, and this view is in accordance with findings in diabetic (22) and fasted rats (31). However, our study does not preclude the possibility that posttranscriptional mechanisms participate in the regulation of IGFBP-1 synthesis and secretion.
In conclusion, during fasting and refeeding of adult male rats, insulin appears to be a decisive regulator of the hepatic expression and secretion of IGFBP-1, in accordance with observations in humans. During refeeding of fasted rats, serum IGFBP-1 was rapidly suppressed to the normal range within 3 h of refeeding. However, the inverse relationship between free IGF-I and IGFBP-1 previously observed in nonfasting and overnight fasting serum samples was absent, and free IGF-I levels remained low despite the reduction in IGFBP-1. Instead, serum free IGF-I changed in parallel with serum total IGF-I, both being normalized at day 2 of refeeding, and ALS and IGFBP-3. We therefore suggest that IGFBP-1 is not a dynamic regulator of free IGF-I in severe catabolic situations. Instead, the ternary complex appears to be more important for free IGF-I in situations with marked nutritional changes. Whether these considerations are also valid in humans remains to be investigated. However, there are no apparent species differences, when changes in the IGF system during fasting and refeeding are compared (13, 15, 29), and we therefore believe our data may be extrapolated to humans, despite the diversity of the GH secretion. Finally, our study indicates that IGFBP-1 is regulated predominantly at the transcriptional level, whereas both IGF-I and ALS appear to be regulated significantly by posttranscriptional mechanisms.
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ACKNOWLEDGEMENTS |
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We are indebted to V. Baxendale, Sunita Babu, K. Nyborg Rasmussen, S. Sørensen, J. Hansen, and I. Bisgaard for skilled technical assistance.
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FOOTNOTES |
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This study was supported by grants from the Danish Health Research Council (Grant no. 9602012), Aarhus University-Novo Nordisk Centre for Research in Growth and Regeneration (Danish Health Research Council Grant no. 9600822), the Institute of Experimental Clinical Research, University of Aarhus, and the National Health and Medical Research Council, Australia (Grant no. 960875).
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: J. Frystyk, Institute of Experimental Clinical Research, Aarhus Kommune Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark (E-mail: jan{at}frystyk.dk).
Received 28 July 1998; accepted in final form 20 April 1999.
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