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Endocrinology Vol. 143, No. 12 4505-4512
Copyright © 2002 by The Endocrine Society

ARTICLE

Age-Dependent Regulation of the Acid-Labile Subunit in Response to Fasting-Refeeding in Rats

Sung-Eun Kong, Robert C. Baxter and Patric J. D. Delhanty

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Dr. P. J. D. Delhanty, Kolling Institute of Medical Research, Royal North Shore Hospital, Pacific Highway, St. Leonards, New South Wales 2065, Australia. E-mail: delhanty{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The GH-dependent, hepatocyte-derived acid-labile subunit (ALS) regulates IGF release from the serum by forming ternary complexes containing IGF binding protein (IGFBP)-3 or IGFBP-5. Malnutrition suppresses ALS and IGF-I expression in a development-dependent manner. Our aim was to investigate whether the effect of feeding following fasting was similarly age dependent. We fasted juvenile and adult rats for 48 h and then refed them, collecting serum and liver tissue at 8, 24, and 48 h. These were compared with rats before fasting (0 h controls) and animals fed throughout the study (free-fed controls). During fasting, serum ALS fell to 25 ± 5.3% of 0 h controls in juveniles but only 56 ± 6% in adults. Within 24 h of refeeding, ALS in juveniles had returned to 0 h control levels, and by 48 h to free-fed levels, whereas there was no significant refeeding response in adults during this period. Circulating IGF-I and IGFBP-5 showed similar age-dependent responses to refeeding, rising significantly faster in juveniles. IGFBP-3 did not show this response. Furthermore, hepatic ALS and IGF-I mRNA showed no age-differential response to fasting and refeeding, suggesting posttranscriptional regulation. Neither regulation of hepatic GH receptor nor ALS clearance rates could explain the age-dependent effect. We hypothesize that development-dependent regulation of ALS and IGF-I during refeeding may involve a posttranscriptional hepatic response that is not GH dependent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I AND IGF-II HAVE developmental and growth stimulatory effects, as well as insulin-like metabolic actions. In the circulation, the IGFs are stabilized in 130- to 150-kDa ternary complexes with IGF binding protein (IGFBP)-3 or IGFBP-5 and the acid-labile subunit (ALS) (1, 2, 3, 4). ALS is expressed primarily in the liver and its expression is regulated by GH (5, 6). By stabilizing ternary complexes in the circulation, ALS appears to modulate the retention of IGFs within the blood compartment because mice in which the ALS gene has been inactivated have significantly reduced circulating levels of IGF-I (7). Furthermore, mice with combined inactivation of ALS and hepatic IGF-I are severely growth retarded, suggesting that maintenance of ternary complexes in the circulation may be important for normal growth (8).

During nutritional deprivation, circulating levels of IGF-I are markedly decreased (9). This can partly be attributed to reduced hepatic gene expression (10, 11); however, another possible cause may be the reduction in levels of the circulating ternary complex due to a decrease in either circulating IGFBP-3, IGFBP-5, or ALS, effectively reducing the IGF holding capacity of the blood. Such changes might be expected to contribute to the increased clearance rate of IGF-I observed in protein-restricted 5-wk-old rats, concomitant with a shift in distribution of IGF-I into approximately 40-kDa complexes with IGFBPs rather than approximately 150-kDa ternary complexes (9). This corresponds with the finding that both acute fasting and chronic malnourishment in rats markedly decreases levels of serum ALS and IGFBP-3 (12, 13, 14, 15).

Malnourishment probably acts at multiple levels in the liver. Altered serum GH (16) and desensitization to GH are major mediators of the response to fasting (17), and probably contribute to the observed reduction in hepatic gene expression of IGF-I (10). In the liver, a major mediator of the response to starvation is cAMP. Using isolated hepatocytes, we have shown that cAMP can have a posttranscriptional effect on ALS expression (18). Additionally, there is a posttranscriptional component to the effect of malnutrition on IGF-I because GH induces hepatic IGF-I gene expression in severely protein-restricted rats but does not increase plasma IGF-I levels (19). Furthermore, it has been shown that there is an age-dependent component to the regulation of IGF-I and other ternary complex proteins, which are more sensitive to malnourishment in juvenile rats (14, 20) and to some extent in children (21).

In contrast to malnourishment, refeeding restores IGF-I levels in humans (9) and rats (13). Similarly, we have previously shown that refeeding of fasted adult rats restores serum levels of ALS (and IGF-I) only relatively slowly (13). Because the regulation of ALS and other ternary complex proteins by malnourishment appears to be age dependent and may be modulated at transcriptional and posttranscriptional levels in the liver, we were interested whether similar mechanisms of regulation may occur during refeeding. Accordingly, we have compared the effect of refeeding subsequent to fasting on the hepatic gene expression and serum levels of ALS and IGF-I in postweaning juvenile (4 wk) and adult (10 wk) rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal preparation and experimental protocol
Postweaning juvenile (75–95 g) and adult (280–300 g) male Sprague Dawley rats were individually housed at a constant temperature and 12-h light, 12-h dark cycle. They had free access to rat chow and water ad libitum for 5 d before the study commenced. After acclimatization, animals were divided into seven groups of similar mean body weight (83.0 ± 0.53 g for juvenile rats and 288.7 ± 1.06 g for adult rats for the entire group). Groups consisted of 1) free-fed animals culled immediately following acclimatization (0 h controls); 2) 24-h fast; 3) 48-h fast; 4) 48-h fast then 8 h refeed; 5) 48-h fast then 24 h refeed; 6) 48-h fast then 48 h refeed; and 7) freely fed for 96 h after acclimatization (n = 5 juvenile and 5 adult rats in each group). During the 4 d of study, all animals had free access to water, and during the refeeding period all animals had free access to standard rat chow. At the end of the experiment, animals were anesthetized and blood was collected by cardiac puncture. Serum samples were prepared and stored at -20 C until analysis. The liver was quickly removed and snap frozen in liquid nitrogen. All tissues were stored at -80 C until analysis. Experiments were conducted with the approval of the Institutional Animal Care and Ethics Committee.

RIAs
All samples were analyzed in duplicate within the same assay unless otherwise stated. Rat ALS and IGFBP-5 were measured by specific RIAs, which have been described previously (4, 22). Rat IGFBP-3 was measured using a competitive binding assay that detects both IGFBP-3 and IGFBP-5 on the basis of their binding to immobilized ALS (23). Because IGFBP-3 and IGFBP-5 reacted similarly in the assay when expressed in mass units (equivalent to ~70% IGFBP-5 cross-reactivity on a molar basis), IGFBP-3 concentrations were corrected by subtracting IGFBP-5 concentrations in ng/ml measured by RIA, from IGFBP-3 values in ng/ml measured in the competitive binding assay. Rat IGF-I was measured in acid-ethanol extracted and neutralized whole sera with an IGF-I specific RIA using des[1–3] IGF-I as tracer (24). A rat insulin RIA kit (Linco Research, Inc., St. Charles, MO) was used to measure serum insulin levels, whereas serum glucose concentration was measured using the Advantage blood glucose monitor (Roche Diagnostics Australia Pty. Ltd., Castle Hill, Australia). Rat IGFBP-1 was measured by a modification of a previously described method to enhance sensitivity (25). Briefly, rat serum samples were incubated overnight at room temperature with the primary antibody but without the tracer. The tracer was added next morning, incubated for 7 h at room temperature, and samples were then treated as previously described.

RNA extraction, cloning of cDNAs, and Northern blotting
Total RNA was extracted from snap frozen rat livers using the guanidine isothiocyanate/cesium chloride extraction method, as described previously (26). The RNA pellets were briefly dried, resuspended in ribonuclease-free water and stored at -80 C. Total RNA samples (20 µg) from all animals were electrophoresed in 1% agarose gels containing 2.2 M formaldehyde. The integrity of the ethidium bromide-stained RNA samples was confirmed on an UV light box. RNA was then transferred by capillary blotting to Zetaprobe GT membranes (Bio-Rad Laboratories, Inc., Richmond, CA) and cross-linked by heat treatment at 80 C in a gel drying apparatus for 2 h.

A cDNA probe for GHR mRNA was generated by RT-PCR from rat liver total RNA and sequenced to confirm its identity. The ALS cDNA has been described previously (13) and the IGF-I cDNA was kindly provided by Dr. P. Rotwein (Portland, OR). cDNAs were labeled with a Ready-To-Go random-priming kit (AMRAD-Pharmacia, Uppsala, Sweden) and [32P]deoxy-CTP (AMRAD-NEN Life Science Products, Uppsala, Sweden). Northern blot analyses for each gene were performed sequentially as described previously (13). Northern blots were quantified with a PhosphorImager and the ImageQuant computer software package (Molecular Dynamics, Inc., Sunnyvale, CA). Expression of the genes was normalized relative to 36B4 mRNA levels.

GH binding studies
Liver microsomal membrane fractions were prepared from animals at all time points (n = 5). Briefly, liver tissue was homogenized in 0.25 M sucrose then fractionated by differential centrifugation at 4 C as previously described (27). Two hundred micrograms of membrane protein were incubated with 20,000 cpm [125I] human (h) GH (70–100 Ci/g), for 16 h at 22 C in 25 mM Tris-HCl, 10 mM CaCl2 (pH 7.4), 0.5% BSA in a total volume of 300 µl. After the incubation period bound and free GH were separated by adding 1 ml of ice-cold buffer and centrifuged for 30 min at 4 C. The supernatants were discarded and membrane pellets were counted in a {gamma}-counter. Nonspecific binding was determined in the presence of excess unlabeled hGH (1 µg/tube).

Intravenous cannulation and infusion of radiolabeled ALS
Four adults and four juvenile rats were anesthetized and catheters were inserted into the left internal jugular vein. The free end of the catheter was tunneled sc and exteriorized between the shoulder blades. The catheter was flushed twice daily with heparinized saline (500 U/ml, porcine grade 1-A; Sigma, St. Louis, MO) to maintain patency. Following surgery, animals were fasted for 2 d and then refed for 1 d until infusion of radiolabeled purified rat serum-derived ALS (22). One baseline blood sample was taken before the infusion of radiolabeled ALS commenced. Each rat was infused via the cannula with 4 x 106 cpm of rat [125I]-ALS tracer (~80 µl). The cannulae were then flushed with 100 µl of 0.9% sterile saline followed by 50 µl of heparinized saline. At 1, 15, 30, 60, 180, 300, and 1440 min after infusion, samples of 300 µl and 100 µl of blood from adult and juvenile rats, respectively, were taken. Blood was placed in tubes containing 0.01 vol of heparinized saline and immediately spun in a microcentrifuge for 2 min. Plasma was collected and kept on ice until stored at -20 C. Red blood cells were resuspended in the original volume of 0.9% sterile saline and reinjected into the rats to prevent anemia and hypovolemia.

To examine the clearance characteristics of the ALS tracer, 50 and 17 µl of plasma (volumes related to mean weight of the animal groups) from adult and juvenile rats, respectively, was counted in a {gamma}-counter before and after trichloroacetic acid precipitation (TCA; 10 volumes of 20%, ice-cold). After centrifugation, 1.1 ± 0.6% of total counts remained in the supernatant. At the end of the experiment, 50 µl of urine was TCA precipitated in the same manner and only 3.0 ± 1.3% of total counts were measurable in the pellets.

To confirm that circulating rat ALS tracer was still intact and functional, ternary complex formation assays were performed. For each time point, 20 ng of hIGFBP-3, 50 ng of hIGF-I, and 50 mM Na phosphate (pH 6.5), 1% BSA was added to 10 µl of plasma in a total volume of 400 µl. The tubes were mixed and incubated for 2 h at room temperature. Subsequently, 25 µl of hIGFBP-3 antiserum (1/50) were added and incubated at room temperature for 1 h, followed by the addition of 25 µl of secondary antibody (1/10 dilution of goat antirabbit IgG), which was incubated at room temperature for 30 min. Ternary complexes were precipitated with 1 ml of ice-cold 6% polyethylene glycol in 0.15 M NaCl for 15 min, then pellets were counted in a {gamma}-counter. To assess whether the rat ALS tracer was becoming degraded in the serum, 2 µl of plasma from each time point were run on a 6% SDS-polyacrylamide gels. ALS tracer (10,000 cpm) was run as a size marker. The gel was then dried and autoradiographed.

Statistics
Comparative data were analyzed by one-way ANOVA using Statview version 5 (SAS Institute, Cary, NC). Linear regression analysis was used to assess the relationship between the measured variables in the juvenile and adult groups separately. Because of their wide range of variation IGFBP-1 serum and mRNA data were log-transformed before statistical analysis. All data are provided as means ± SEM and P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of fasting and refeeding on body weights
Fasting for 48 h reduced the body weight of juvenile and adult rats by 19% and 12%, respectively, compared with 0 h controls (P = 0.0073 and P < 0.0001, respectively; data not shown). Refeeding for 48 h restored their weights to original values (juveniles 108%; adults 98%), although not to those of free-fed control rats (juveniles 82%, P < 0.007; adults 91.5%, P < 0.0001).

Age-dependent effect of fasting and refeeding on ternary complex components
ALS expression.
Fasting for 48 h caused rapid and marked suppression of serum ALS levels in both adult and juvenile animals, falling from 54.5 ± 3.5 to 30.4 ± 3.3 µg/ml in adults and from 17.5 ± 1.3 to 4.4 ± 0.9 µg/ml in juveniles (Fig. 1AGo). Fasting suppressed ALS serum levels to a significantly greater extent in juveniles (25 ± 5.3% of 0 h levels) than adults (56 ± 6% of 0 h levels; Fig. 1BGo). During subsequent refeeding, serum ALS in juveniles recovered above 0 h levels to free-fed control concentrations. However, this did not occur in adults where ALS concentrations remained below both initial and free-fed control levels. When serum levels are normalized as a percentage of 0 h control levels in both age groups (Fig. 1BGo), it becomes clear that the juveniles respond rapidly to refeeding with circulating levels returning to 0 h control levels within 24 h, whereas adult animals do not recover circulating levels for the entire 48-h period.



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  		Figure 1. Age-dependent differential regulation of ALS serum levels but not gene expression during refeeding. A, ALS serum levels. B, ALS serum levels in each age group expressed as percentage of own 0 h controls. C, Steady-state hepatic ALS mRNA levels expressed as percentage of 0 h adults (normalized for 36B4 mRNA). Triangles, Juveniles; open triangles, free-fed juveniles; squares, adults; open squares, free-fed adults. Values are mean ± SEM; n = 5 per group. *, P < 0.05 vs. 0 h juveniles; #, P < 0.05 vs. 0 h adults; **, P < 0.05 vs. adults at same time point; ##, P < 0.05 vs. free-fed controls.

 
Fasting has recently been shown to suppress hepatic GH sensitivity through modulation of components of its signal transduction pathway (17), and ALS has been shown to be regulated at the transcriptional level by GH in the liver (5, 6). Consequently, we examined hepatic ALS mRNA levels to determine if gene expression could explain the age-dependent differential effect of refeeding on serum ALS levels. Hepatic ALS gene expression was lower in juvenile than adult animals consistent with the lower levels of circulating ALS in juveniles (Fig. 1CGo). Like circulating ALS, hepatic gene expression was rapidly suppressed by fasting in both juvenile (45 ± 6.5% and 33 ± 7.4% of 0 h controls at 24 and 48 h, respectively; P < 0.01) and adult rats (74 ± 12.3% and 47 ± 5.6% of 0 h controls at 24 and 48 h, respectively, P < 0.01). Similar to serum levels, gene expression was more sensitive to fasting in juveniles than adults. This finding was surprising because we have previously shown that fasting in Wistar rats had no significant impact on hepatic ALS gene expression (12, 13). This may be related to the strain of rat used because ALS gene expression has been shown to be modulated by nutrition in other animal species, e.g. sheep (28). Remarkably, unlike circulating protein levels, refeeding restored hepatic ALS mRNA to 0 h control and free-fed levels within 48 h in adults as well as juveniles (Fig. 1CGo).

IGF-I expression
After 48 h of fasting, there was a significant decrease in serum IGF-I levels in both juvenile and adult rats (39 ± 4.4% and 73 ± 14.1% of 0 h controls, respectively, P < 0.04) (Fig. 2Go, A and B). However, the effect of re-feeding on serum IGF-I levels between juvenile and adult rats was quite different (Fig. 2Go, A and B). In juvenile rats, 8 h of refeeding was sufficient to increase serum IGF-I levels close to those of 0 h controls, whereas IGF-I levels continue to drop in adult rats at this time point. Even after 48 h of refeeding, serum IGF-I increased to only 84% of 0 h controls in adult rats (P < 0.05). ALS regulates IGF-I concentrations in the serum and a functional molar excess appears to be required for this to occur (29). We found that ALS maintained a consistent approximately 4-fold molar excess over IGF-I throughout the periods of fasting and refeeding in this study, and correlated strongly with IGF-I (r2 = 0.477, P < 0.0001; Fig. 2CGo).



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  		Figure 2. Age-dependent differential regulation of IGF-I serum levels but not gene expression during refeeding. A, IGF-I serum levels. B, IGF-I serum levels in each age group expressed as percent of own 0 h controls. C, Regression analysis of circulating ALS vs. IGF-I. The slope of regression is approximately 4, suggesting maintenance of a 4-fold molar excess of ALS over IGF-I (r2 = 0.477). D, Steady-state hepatic IGF-I mRNA levels expressed as percentage of 0 h adults (normalized for 36B4 mRNA). Triangles, Juveniles; open triangles, free-fed juveniles; squares, adults; open squares, free-fed adults. Values are mean ± SEM; n = 5 per group. *, P < 0.05 vs. 0 h juveniles; #, P < 0.05 vs. 0 h adults; **, P < 0.05 vs. adults at same time point; ##, P < 0.05 vs. free-fed controls.

 
During refeeding, unlike circulating IGF-I, hepatic gene expression of IGF-I followed similar rates of increase in both adults and juveniles with levels of mRNA after 48 h of refeeding remaining significantly different from free-fed controls in both groups (Figs. 2DGo). Hepatic IGF-I mRNA was expressed as multiple transcripts of approximately 7.5 kb and 0.8–2.0 kb, as previously described (30). The approximately 7.5-kb transcript has previously been shown to be differentially regulated relative to the other low molecular weight transcripts by protein malnutrition and fasting (31, 32). However, we found no significant differential regulation of transcripts during refeeding (data not shown).

IGFBP-5 and IGFBP-3
Because of the strong relationship that we found between circulating concentrations of IGF-I and ALS throughout fasting and refeeding, we examined the regulation of IGFBP-5, another component of the ternary complexes. Circulating IGFBP-5 showed a virtually identical pattern of response to fasting and refeeding as ALS (Fig. 3AGo) with a refeeding driven induction of circulating levels in juveniles above 0 h controls, and even slightly above free-fed animals (Fig. 3BGo). In adults, serum IGFBP-5 showed the same blunted response to refeeding as ALS and IGF-I. IGFBP-5 mRNA was not detectable in the liver (data not shown), as also described by others (33). We have recently shown that circulating levels of IGFBP-5 and ALS are significantly associated in humans (4). This was also confirmed in the rats used in this protocol in which IGFBP-5 showed an even tighter correlation with ALS (r2=0.776, P < 0.0001; Fig. 3CGo) than with IGF-I (r2 = 0.498, P < 0.0001).



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  		Figure 3. Age-dependent regulation of circulating IGFBP-5 during refeeding. A, IGFBP-5 serum levels. B, IGFBP-5 serum levels in each age group expressed as a percentage of own 0 h controls. C, Regression analysis of ALS vs. IGFBP-5 (r2 = 0.776). D, IGFBP-3 serum levels. E, Regression analysis of ALS vs. IGFBP-3 (r2 = 0.455). F, Regression analysis of IGFBP-5 vs. IGFBP-3 (r2 = 0.59). Triangles, Juveniles; open triangles, free-fed juveniles; squares, adults; open squares, free-fed adults. Values are mean ± SEM; n = 5 per group. *, P < 0.05 vs. 0 h juveniles; #, P < 0.05 vs. 0 h adults. **, P < 0.05 vs. adults at same time point; ##, P < 0.05 vs. free-fed controls.

 
IGFBP-3 is the primary carrier of IGFs in the postnatal circulation. However, accurate assessment of its regulation in the circulation of rodents has been hampered by the lack of a specific IGFBP-3 assay. We have used our competitive binding assay using immobilized ALS (23) to determine total IGFBP-3 plus IGFBP-5 in serum samples and have then calculated IGFBP-3 levels by subtracting IGFBP-5 concentrations determined by RIA. Reactivity of IGFBP-5 in the assay was identical to IGFBP-3 on a mass basis (~70% of IGFBP-3 reactivity on a molar basis). Overall, we found that IGFBP-3 levels were not significantly affected by the cycle of fasting-refeeding, although there was a significant increase relative to 0 h control levels in 48-h refed adults, which corresponded to an increase in the free-fed animals (Fig. 3DGo). ALS showed an approximately 6-fold molar excess over IGFBP-3 with an intermediate level of correlation (r2 = 0.455, P < 0.0001; Fig. 3EGo). IGFBP-3 occurred in an approximately 1:1 molar ratio with IGF-I but was poorly correlated (r2 = 0.26, data not shown). In humans, IGFBP-5 circulates at approximately 10% of the molar concentration of IGFBP-3 (4). Remarkably, in rats from this study we found that IGFBP-5 occurs consistently at about 25% of the total level of circulating IGFBP-3 (r2 = 0.59, P < 0.0001; Fig. 3FGo).

Age-dependent effect of fasting and refeeding on GH receptor expression
Because IGFBP ternary complexes are dependent on GH, and because malnutrition has a marked effect on hepatic GH sensitivity (17), which may also be age dependent (20), we examined the effect of fasting and refeeding on the expression of the GH receptor.

Hepatic gene expression of GHR was rapidly suppressed by 24 h and 48 h of fasting in both juvenile (43 ± 5.9% and 39 ± 2.8% of 0 h controls, respectively, P < 0.0002) and adult rats (64 ± 4.2% and 59 ± 8.6% of 0 h controls, respectively, P < 0.002) and reached a nadir after 48 h of fasting (Fig. 4AGo). Refeeding resulted in increased gene expression, adults being slightly more sensitive, reaching 0 h and free-fed control levels within 24 h compared with 48 h in juveniles.



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  		Figure 4. GHR expression is not age dependently regulated during refeeding. A, GHR mRNA levels in each age group expressed as percentage of own 0 h controls. B, GH binding to liver microsomal membranes in each age group expressed as percentage of own 0 h controls. Triangles, Juveniles; open triangles, free-fed juveniles; squares, adults; open squares, free-fed adults. Values are mean ± SEM; n = 5 per group. *, P < 0.05 vs. 0 h juveniles; #, P < 0.05 vs. 0 h adults.

 
GH binding to hepatic microsomal membranes showed similar trends to that of GHR mRNA levels with no significant differences between adults and juveniles at any time point when expressed as percentages of 0 h controls (Fig. 4BGo).

Age-dependent effect of refeeding on ALS clearance and ability to form ternary complexes
Because refeeding induced changes in hepatic ALS and IGF-I mRNA levels and expression of GHR could not easily explain the age-dependent response of serum ALS and IGF-I, we examined the clearance of ALS from the circulation of adult and juvenile rats following 48 h of fasting and during a 24-h period of refeeding. IGF-I has increased clearance from the circulation of malnourished rats (9), and we reasoned that there may also be age-related differences between fasted and refed animals in the release of ALS from the circulation and/or its rate of degradation.

The clearance of radiolabeled purified rat ALS in cannulated adult and juvenile rats was examined during a 24-h period following 48 h of fasting and 24 h of refeeding (Fig. 5AGo). Data were fitted to a two-phase model and half-lives of release from the circulation were calculated in three independent experiments (Prism, GraphPad Software, Inc., San Diego, CA). Mean half-lives for ALS were not significantly different between adult and juvenile rats (214 ± 44 min and 232 ± 25 min, respectively).



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  		Figure 5. Effect of refeeding on ALS clearance and functional activity. A, TCA-precipitable radioactivity in serial samples of plasma following bolus injections of rat [125I]-ALS into cannulated rats that had been fasted for 48 h then refed for 24 h. Volumes of plasma were adjusted for animal weight. B, Ability of rat [125]-ALS in the serial samples of plasma in (A) to form ternary complexes in vitro. Values are mean ± SEM (n = 4 per group). Triangles, Juveniles; squares, adults. C, Representative autoradiograph of a 6% SDS-PAGE analysis of rat ALS tracer (on left) and 2 µl of serial plasma samples from refed adult and juvenile animals (times in hours except 1-min sample; arrow indicates ~85-kDa rat ALS band).

 
To assess the functionality of the infused ALS over the period of study, we measured its ability to form ternary complexes by binding to hIGFBP-3 in the presence of IGF-I (Fig. 5BGo). There was no significant change in binding ability during the first 300 min of infusion, although by 24 h binding had dropped to 57% and 50% of initial values in adult and juvenile animals, respectively. In addition, during the first 300 min the ability of radiolabeled ALS from juvenile animals to form ternary complexes was significantly greater than that in adult animals (P < 0.008). This may be related to the lower levels of endogenous ALS in juvenile plasma and consequently reduced competition for binding sites in the assay.

Because of the loss of ternary complex forming ability after 24 h in the circulation, we examined whether ALS had remained intact in the plasma by running samples on SDS-polyacrylamide gels (Fig. 5CGo). Although the amount of radio-labeled ALS had decreased, we could discern no apparent change in molecular weight and no break-down products were visible on the gels. However, at 24 h the urine contained significant levels of non-TCA-precipitable radioactivity, indicating degradation and concomitant rapid release of protein fragments from the circulation.

Developmental effect on insulin and hepatic response to insulin
Insulin induced by refeeding has been implicated in initiating protein synthesis, and furthermore, its development-dependent rate of release into the circulation may explain the age-dependent response to refeeding (34). We examined insulin levels in juvenile and adult rats and assessed the respective physiological response to these changes by measuring serum glucose and circulating IGFBP-1.

Insulin levels were found to be significantly suppressed by fasting only in adult rats during the 48-h period of fasting and 48 h of refeeding was required to restore levels to those of controls (Fig. 6AGo). In juvenile animals, serum insulin levels did not deviate statistically from controls during the entire protocol. Serum glucose levels fell below 0 h control levels during fasting in both age groups (Fig. 6BGo). However, during the refeeding period, adults were transiently hyperglycemic at 24 h, whereas juveniles showed a more controlled response, increasing to control levels during the first 24 h and then stabilizing.



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  		Figure 6. Circulating profiles of insulin, glucose, and IGFBP-1 during fasting and refeeding. A, Insulin concentrations were significantly suppressed by fasting, relative to 0 h controls, only in adults and responded more rapidly to refeeding in juvenile animals. B, Glucose levels were similarly suppressed in both juveniles and adults, but on refeeding rose above control levels only in adults. C, Serum IGFBP-1 was hyperinduced by fasting and markedly suppressed by refeeding in juveniles but responded relatively modestly in adults. Values are mean ± SEM; n = 5 per group. *, P < 0.05 vs. 0 h juveniles; #, P < 0.05 vs. 0 h adults.

 
IGFBP-1 is produced predominantly by the liver, and its expression is markedly inhibited by insulin making it a useful marker of hepatic insulin sensitivity (25, 35). Fasting has been demonstrated to increase serum IGFBP-1 levels in adult rats (25). We found that fasting hyperinduced serum IGFBP-1 in juvenile animals being increased to 45-fold of 0 h control levels by 48 h, compared with only 3-fold in adult rats (Fig. 6CGo). Refeeding restored IGFBP-1 to control levels in both age groups within 24 h, although because of the greater circulating levels attained in juveniles at 48 h of fasting, the rate of fall was more marked in this age group than in the adults.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found that the ternary complex components ALS and IGF-I responded more rapidly to refeeding following a period of fasting in juvenile than adult rats. Serum levels of IGFBP-5 showed a similar age-dependent response to refeeding and was significantly correlated with ALS and IGF-I in the circulation. However, circulating IGFBP-3 was not significantly altered during the fasting-refeeding cycle, suggesting that its regulation is not central to the age-dependent alterations in ALS and IGF-I that we observed. This finding suggests that its concentration in the rodent circulation is effectively buffered by ALS in the ternary complexes during acute fasting. Remarkably, IGFBP-5 was found to occur at about 25% of circulating IGFBP-3 molar concentrations. We have recently determined in humans that about 45% of circulating IGFBP-5 is free or in binary complexes, compared with only approximately 10% of IGFBP-3, suggesting that IGFBP-5 plays an important role in regulating the tissue availability of IGFs (4). Our data suggest that this potential role of IGFBP-5 may be of even greater importance in rats. The endocrine IGF-axis in humans is differentiated from that in rats by having both circulating IGF-I and -II. However, the total concentration of IGF in the circulation of both species (~100 nM) and the apparent size distribution and circulating half-lives are very similar in both species (9, 36). These data suggest that the endocrine IGF-axis is regulated by ternary complexes in the same way in rats and humans. Although it is difficult to extrapolate precisely, these characteristics of the IGF axis suggest that our findings in rats are applicable to humans.

Hepatic steady-state levels of ALS and IGF-I mRNA were suppressed by fasting and increased by refeeding. However, the age-dependent differential response of the circulating proteins to refeeding did not correspond with their hepatic patterns of gene expression, suggesting that this developmental effect was posttranscriptional. Hepatic expression of ALS and IGF-I are markedly GH dependent. However, no development-dependent differences in GHR expression or GH-binding in response to refeeding were observed, further suggesting that the mechanism involved in their age-dependent regulation is not at the transcriptional level.

It has been previously suggested that, regardless of changes in IGF production and secretion, a change in clearance rates may also contribute to developmental increases in circulating concentrations of IGFs (37). Additionally, fasting has been shown to alter not only the rate of release of IGF-I from the circulation but also its compartmentalization among 40-kDa binary and 150-kDa ternary complexes in the serum (9), suggesting that there may be differences in the clearance or degradation of IGF-I. Age-dependent nutritional regulation of tissue IGF receptors and cell-surface IGFBPs could conceivably also affect the clearance of IGFs. To determine whether ALS was also regulated by its clearance, we measured clearance rates of ALS in both juvenile and adult rats after 48 h of fasting and 24 h of refeeding. However, our study showed no age-dependent differences in the clearance of ALS. Furthermore, during the first 5 h following infusion, the ability of ALS to form ternary complexes was unaffected.

Starvation is associated with the suppression of protein synthesis in the liver that is rapidly restored by refeeding. The acute increase in protein synthesis, in this case, occurs through stimulation of mRNA translation by increasing the number of newly initiated polypeptide chains, rather than the rate of polypeptide elongation (38). The postnatal period of rapid growth is characterized by high rates of protein turnover. However, this is age dependent, and as development progresses, the rates of growth and protein synthesis fall. Similarly, the relative response of protein synthesis to refeeding declines with increasing age (39). Therefore, it is possible that the age-dependent posttranscriptional effect of fasting on ALS and IGF-I may be linked more to global changes in hepatic protein synthesis than to specific effects on their translation.

ALS is transcriptionally regulated by GH (6), but we have evidence that its expression is also influenced by posttranscriptional mechanisms involving cAMP and insulin (5, 18). Because of this, we examined whether refeeding induced changes in circulating insulin and its downstream effects on serum glucose and the hepatic insulin-sensitive protein IGFBP-1 were indicative of development-dependent responses. We found that there was an age-dependent effect with a more robust response to insulin in juveniles, indicated most strongly by the suppression of IGFBP-1 from markedly raised fasting levels. Insulin has been demonstrated to modulate the signaling pathway involved in initiation of protein synthesis by stimulating phosphorylation of 4E-BP1 (40), releasing it from complexes with eukaryotic initiation factor 4E and allowing this latter factor to initiate formation of an active ribosomal preinitiation complex (41). However, the acute effects of insulin on initiation of protein synthesis may not explain the age-dependent induction of ALS and IGF-I that we have observed. In perinatal pigs, the acute age-dependent protein synthetic response to refeeding in the liver has been linked primarily to nutritional composition (amino acid) rather than an inductive effect of insulin (42, 43). Furthermore, in the liver (unlike muscle) refeeding does not seem to modulate acute phosphorylation events in either the proximal insulin-signaling pathway (44) or the pathway involved in initiation of translation (41) in a markedly development-dependent way. However, the pigs in these studies were developmentally younger than the rats used in the current study, and it is possible that the precise mechanisms are different. Additionally, it is presently unclear how much can be inferred from the acute studies in pigs in relation to the longer-term effects that we have described in rats.

Clearly, more work needs to be done to determine the mechanisms involved in the development-dependent response to refeeding that we have observed in the hepatic ALS-IGF axis. Although questions remain concerning the mechanisms involved, our results show an exaggerated response of specific components of the IGF axis to refeeding in juvenile animals, possibly providing a stimulus for catch-up growth after fasting during this rapid growth phase.


   Acknowledgments
 
We gratefully acknowledge Ms. Fiona McDougall for her excellent technical assistance.


   Footnotes
 
This study was supported by Grant 990424 from the National Health and Medical Research Council, Australia (to P.J.D.D. and R.C.B.).

Abbreviations: ALS, Acid-labile subunit; h, human; IGFBP, IGF binding protein.

Received May 20, 2002.

Accepted for publication August 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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