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Endocrinology Vol. 138, No. 8 3112-3118
Copyright © 1997 by The Endocrine Society

ARTICLES

Effect of Fasting on Insulin-Like Growth Factor (IGF)-IA and IGF-IB Messenger Ribonucleic Acids and Prehormones in Rat Liver1

Jihui Zhang, Robert E. Whitehead, Jr.2 and Louis E. Underwood

Department of Pediatrics, Division of Endocrinology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Dr. Louis E. Underwood, Pediatric Endocrinology, CB 7220 509 Burnett-Womack Building, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7220.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin-like growth factor I (IGF-I) gene generates by alternative splicing two IGF-I messenger RNAs (mRNAs) coding for IGF-I prehormones with different E domain sequences. In rats, these two mRNAs differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain coding region. The purpose of this study was to investigate the effect of nutritional perturbation on IGF-IA and -IB expression in rat liver. Northern blot analysis of liver mRNA revealed that the 1.5–1.9 kb and 0.9–1.2 kb IGF-I mRNA species were decreased in rats fasted for 48 h compared with either fasted-refed (48 h of each) or control-fed rats (each, P < 0.01), whereas the 7.5 kb IGF-I mRNA was decreased only when compared with the fasted-refed animals. Using semiquantitative RT-PCR, the IGF-IA transcript (114 bp amplicon) was not altered, whereas the IGF-IB transcript (166 bp amplicon) was decreased in fasted rats compared with the other two groups (both P < 0.01). We confirmed the RT-PCR results by RNase protection assay (RPA), observing that the IGF-IA (224 and 100 bases protected) was not decreased and that the IGF-IB transcript (376 bases protected), accounting for only 23% of the total IGF-I transcripts of control fed rats, was decreased by fasting. Because the results from RT-PCR and RPA do not necessarily predict full-length translatable mRNA, we subjected hepatic IGF-I transcripts to in vitro translation, and we immunoprecipitated IGF-IA and -IB prehormones. Both prehormones were translated principally from exon 1-containing mRNAs, with molecular weights of about 17K and 18K, representing 80% and 20% of the total IGF-I prehormones observed in control fed rats, respectively. Both peptides were reduced in fasted rats compared with controls (P < 0.01), and refeeding restored both. By immunoblotting of the protein extract from liver of fasted rats, IGF-IA was decreased by 77% compared with control-fed animals. Refeeding returned IGF-IA to normal. The lack of reduction of IGF-IA transcript at the alternative splice site suggests that posttranscriptional mechanisms are responsible for the reduction in steady-state IGF-I mRNAs that occurs during fasting. Additionally, we present evidence that biosynthesis of IGF-IA and -IB prehormones by liver is impaired at a posttranscriptional level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPRESSION OF the insulin-like growth factor (IGF)-I gene is influenced by nutritional status (1, 2). Decreased concentrations of IGF-I in serum and decreased abundance of IGF-I messenger RNAs (mRNAs) in liver result from fasting or from protein or energy restriction (3, 4, 5, 6, 7, 8, 9, 10). Although fasting reduces binding of GH to its receptor (11), down-regulation of the GH receptor does not occur with dietary protein restriction (12), and it is believed that neither reduced GH binding nor reduced GH receptor (13) are principal causes of reduced IGF-I. Rather, decreased hepatic IGF-I mRNA abundance in fasted or energy restricted rats is proposed to result from decreased gene transcription (5, 6, 9), whereas posttranscriptional mechanisms also may be involved when dietary protein is restricted (7, 10). The mechanisms resulting in decreased hepatic IGF-I mRNA in undernutrition have not been defined completely, and the translational and posttranslational regulation of IGF-I gene expression is poorly understood.

The rat IGF-I gene contains six exons spanning almost 100 kb of genomic DNA (14, 15, 16). Exons 1 and 2 encode distinct 5'-untranslated regions (UTRs) and some signal peptide sequences. Exon 3 encodes the remainder of the signal peptide and the first part of the B domain of IGF-I peptide. Exon 4 encodes the remainder of the B, C, and D domains, as well as the first part of the E peptide moiety of the prohormones. Exon 5 is an alternatively spliced "cassette" exon of 52 bp. Inclusion of this exon changes the reading frame and the remainder of the E peptide sequence, which differs from that encoded by IGF-I mRNAs derived by splicing exon 4 directly to exon 6. Specifically, alternative splicing results in two mRNAs coding for IGF-I prehormones. In rats, these two mRNAs differ by the presence (IGF-IB) or absence (IGF-IA) of the 52-bp insert. Exon 6 encodes the carboxyl-terminus of the E peptide, and long 3'-UTRs with multiple polyadenylation sites. Thus, a combination of leader exons containing multiple transcription start sites, differential splicing, and the presence of multiple polyadenylation sites results in a variety of IGF-I mRNAs (17). These diverse mRNAs provide numerous possible mechanisms to influence IGF-I gene expression.

To investigate the effect of fasting on IGF-IA and IGF-IB mRNAs and prehormones, we employed semiquantitative RT-PCR and RNase protection assay (RPA) to measure hepatic IGF-IA and -IB transcripts. Because both RPA and RT-PCR amplification of IGF-I sequences may include some partially degraded mRNAs that cannot be translated into polypeptides, we assessed the biosynthesis of the IGF-IA and -IB prehormones by in vitro translation of hepatic mRNAs, followed by immunoprecipitation. We also measured IGF-I prehormones in extracts of liver tissue by immunoblotting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design
Five-week-old male Sprague-Dawley rats (n = 18, Charles River, Wilmington, MA), weighing 140–160 g, were housed in our animal care facility in 12-h light, 12-h dark cycles. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill. Animals were randomly divided into three groups of six. They were either fasted for 48 h, fasted for 48 h then refed on standard laboratory chow for 48 h, or fed ad libitum for 96 h. Each animal was allowed free access to water. Body weight was measured daily. At the end of the experiment, blood was collected by cardiac puncture under ether anesthesia, and serum was stored at -20 C. The livers were dissected quickly from the rats after decapitation, weighed, flash-frozen in liquid nitrogen, minced on dry ice, and stored at -70 C.

Measurement of IGF-I in serum
IGF-I serum concentrations were measured by RIA after removal of IGF binding proteins using ODC-silica cartridge chromatography (C-18 Sep-Pac, Waters Associates, Milford, MA) (18). Purified human plasma-derived IGF-I (PS III) was used as a standard in the RIA.

Liver RNA extraction and Northern blot analysis
Total RNA was isolated from each liver (19), and poly(A)+-enriched RNA was obtained by one passage through an oligo(dT)-cellulose column. Five micrograms of poly(A)+-enriched RNA from each sample were denatured in glyoxal dimethylsulfoxide, size fractionated by electrophoresis on an 1% agarose gel, and transferred to nylon membranes by capillary blotting. After prehybridization, the blot was hybridized with a 194-base rat IGF-I exon 3-specific riboprobe (20) at 65 C for 16 h and washed at 75 C as described previously (21). Abundance of IGF-I mRNA transcripts were determined from densitometric scanning of autoradiograms using the Image-Pro Plus system (Media Cybernetics, Silver Spring, MD). The results, means of six values from each group, are reported in optical density units (DU).

RT-PCR
The absence (IGF-IA) or presence (IGF-IB) of a 52-bp insert in rat IGF-I transcripts (22) allows amplification of both IGF-IA and IGF-IB by RT-PCR with one primer pair. To normalize the amount of complementary DNA (cDNA) template, we used a primer pair for rat ß-actin to amplify ß-actin sequences simultaneously. We designed the primers for IGF-I and ß-actin (Table 1Go) in our laboratory using the WI Sequence Analysis Package (Genetic Computer Group, Madison, WI). In preliminary experiments using RNA from livers of control fed rats, we determined that there was no involvement of target sequence amplification from genomic DNA; that the two sets of primer pairs were specific by performing PCRs with different combinations of the four primers; and the amplification limit (PCR plateau phase) for each primer pair. We used the same amount of cDNA, primers, and Taq DNA polymerase in each experiment. We also determined the range of RNA input which led to proportional PCR product yield before the plateau phase.


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  		Table 1. Primers used in RT-PCR for IGF-I and ß-actin

 
We subjected liver total RNAs (2.5 µg) from each sample (n = 18) to reverse transcription with random hexamers and MMLV-RTase (Life Technologies, Gaithersburg, MD) at 37 C for 1 h. After heat-inactivation of the reverse transcriptase, one tenth of the mix was used in PCR to amplify ß-actin, IGF-IA, and -IB. Other components used in the PCR were 2 U Taq DNA polymerase (Promega, Madison, WI), 0.75 µM each sense and antisense primers for both IGF-I and ß-actin, 200 µM dNTPs, and 2.5 µCi [{alpha}-32P]-dCTP. We carried out PCRs in a Perkin-Elmer Thermocycler as follows: 1 min denaturation at 94 C, 45 sec annealing at 57 C, and 45 sec extension at 72 C for 28 cycles. We analyzed PCR products on a 10% polyacrylamide gel and visualized them by autoradiograghy. We measured the relative amount of the PCR products by densitometry as for the Northern analysis.

To further verify the IGF-I transcripts amplified by RT-PCR, we used restriction analysis to analyze IGF-IA (114 bp) and -IB (166 bp) amplicons. Sequence data show a HinfI site in the common sequences of IGF-IA and -IB, and a TaqI site in the 52-base insert of IGF-IB. IGF-IA and -IB amplicons were digested by either HinfI or TaqI, electrophoresed on a 10% polyacrylamide gel, and visualized by silver staining (24).

RNase protection assay (RPA)
A rat riboprobe designed to allow simultaneous detection of IGF-IA (protected bands of 224 and 100 bases) and IGF-IB (one protected band of 376 bases) mRNAs (25) was generously provided by Dr. Charles T. Roberts, Jr. of the Oregon Health Sciences University. Labeled IGF-I antisense RNAs were synthesized with T7 polymerase using Biotin-14-CTP (Ambion, Austin, TX). A rat ß-actin riboprobe (giving one protected band of 126 bases), was obtained from Ambion (Austin, TX) and labeled in a fashion similar to IGF-I riboprobe except that a 1:3 dilution of Biotin-14-CTP was used to reduce the ß-actin signal. Ten micrograms of total RNAs from each sample (n = 18) were hybridized overnight at 45 C with 2 fmol of labeled IGF-I antisense RNAs and 3 fmol of labeled ß-actin antisense RNAs. After RNase A/RNase T1 digestion, protected bands were separated on a 5% acrylamide/8 M urea/1X TBE gel and transferred onto nylon membrane by electroblotting. Protocols for washing and detection recommended by manufacturer (Ambion) were followed. The relative amount of each band was quantified desitometrically as for Northern analysis.

In vitro translation and immunoprecipitation
Poly(A)+-enriched RNA (4 µg) from each liver sample was translated in a nuclease-treated rabbit reticulocyte lysate system (Promega, Madison, WI). Translated products were labeled with 35S-cysteine (Amersham, Arlington Heights, IL). We used three polyclonal antibodies (UBK487, EaAb, and EbAb) in immunoprecipitation. UBK487 is an antiserum raised against mature human IGF-I peptide, which has 80–90% cross-reactivity with rat IGF-I. The EaAb and EbAb antisera were raised in our laboratory against rat IGF-IA and -IB, respectively (26), using oligopeptides unique to the E domains of IGF-IA and -IB.

We diluted the translation mix (50 µl) with 0.4 ml of NET-gel buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris·Cl, pH 7.5, 0.05% NP-40, 0.25% gelatin). The diluted mix was incubated at 4 C with normal rabbit serum (preimmune serum) for 2 h to reduce nonspecific binding, and then with 25 µl of Pansorbin (Calbiochem, San Diego, CA) for 2 h. After centrifugation, we divided the supernatant into three aliquots, and incubated with either EaAb (1:250), EbAb (1:250), or UBK487 (1:100) at 4 C overnight. We precipitated the immune complex by incubating with 25 µl of Pansorbin at 4 C on a rotator for 2 h (27). We washed the pansorbin pellets twice with NET-gel buffer, and once with 10 mM Tris·Cl pH 7.5 and 0.1% NP-40. Immunoprecipitates were solubilized in 1 x SDS gel loading buffer and denatured by boiling for 5 min. After centrifugation, we electrophoresed samples on a reduced 15% tricine-SDS-polyacrylamide gel (28). Gels were subsequently fixed in 10% acetic acid/25% isopropanol, rinsed, treated with Amplify (Amersham, Arlington Heights, IL), dried, and analyzed by autoradiography. The autoradiographs were scanned densitometrically as for Northern blotting.

Liver protein extraction and immunoblotting
Proteins were extracted from each frozen liver sample (n = 18) using a single-detergent lysis buffer (50 mM Tris·Cl, pH 8.0, 150 mM NaCl, 100 µg/ml PMSF, 1 µg/ml aprotinin, 1% NP-40) (29). Liver extracts were stored at -20 C overnight, then thawed and centrifuged at 12,000 x g for 5 min at 0 C to remove aggregates of cytoskeletal elements. Protein concentrations of each liver extract were measured by the Bradford method (30). Ninety micrograms of each liver extract were denatured, electrophoresed on a reduced 15% SDS polyacrylamide gel in a tricine-buffered system (28), and transferred onto nitrocellulose membrane. Immunodetection of IGF-I prehormones was conducted with either EaAb, EbAb, or UBK487 using an ECL kit (Boehringer Mannheim, Indianapolis, IN). Autographs were analyzed by densitometric scanning.

Statistics
All values are presented as mean ± SEM. Results were evaluated using a program for statistical data analysis (SPSS). The Levene test was performed to evaluate the homogeneity of variances. Data were log-transformed when necessary. Statistical differences were determined by one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of fasting and refeeding on growth and serum IGF-I concentration
Fasting for 48 h decreased body weight by 10% (80% of the control-fed animals at day 2), and refeeding increased body weight by 23.5% (92% of control-fed at day 4; Table 2Go). Fasting reduced serum IGF-I concentrations to 46% of control fed rats (P < 0.01, Table 2Go), and this was restored by refeeding.


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  		Table 2. Changes of rat body weight and serum IGF-I concentrations during fasting and refeeding

 
Alteration of liver IGF-I mRNAs abundance
We quantified the 7.5 kb, 1.5–1.9 kb, and 0.9–1.2 kb size species of IGF-I mRNA transcripts by Northern blotting (Fig. 1Go). After fasting, the 7.5 kb IGF-I mRNA transcript (400 ± 59 DU) was lower than that in fasted-refed animals (653 ± 119 DU; P < 0.05) but was not different from control fed (446 ± 71 DU). Fasting caused a marked reduction in the 1.5–1.9 kb species (812 ± 33 DU) compared with either control fed (2703 ± 449 DU, P < 0.01) or fasted-refed rats (1645 ± 130 DU, P < 0.01). Fasting also reduced the 0.9–1.2 kb species (1978 ± 203 DU) compared with control fed (5746 ± 1007 DU, P < 0.01) and with fasted-refed rats (6276 ± 1013 DU, P < 0.01).



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  		Figure 1. Liver IGF-I mRNA expression in fasted, fasted-refed, and control fed rats. Top panel, Northern blot showing the 7.5, 1.5–1.9, and 0.9–1.2 kb IGF-I transcripts. Autoradiography is done with intensifying screens for 20 h at -80 C. At bottom of the upper panel is ethidium bromide stained 18S ribosomal RNA in each sample. Bottom panel, Quantitation in densitometric units of the three species of transcripts of IGF-I, expressed as mean ± SEM (n = 6, *, P < 0.05; ** P < 0.01 vs. fasted group).

 
Changes of IGF-IA and -IB transcripts in liver
To confirm the specificity of the IGF-I and ß-actin primers, we performed 15 RT-PCRs with either a single primer (4 PCRs), two primer combinations (6 PCRs), three primer combinations (4 PCRs), or four primers together (1 PCR). Amplified IGF-I target sequences (114 bp for IGF-IA and 166 bp for -IB) were seen only when the two primers for IGF-I were present; amplified ß-actin sequence (194 bp) was only seen when the two primers for ß-actin were present; and target sequences of IGF-IA, -IB, and ß-actin were seen when all four primers were used. Studies of the amplification limit of parameter-defined RT-PCR revealed that a plateau phase for ß-actin was reached after 30 cycles and for IGF-IA after 33 cycles (Fig. 2Go). We chose 28 cycles for the routine RT-PCR. Amplified IGF-IB sequence was below the limit of detection by ethidium bromide staining. Furthermore, we observed that the PCR product at 28 cycles was proportional to the RNA added over the range from 50–400 ng (data not shown). The preliminary PCRs were done using liver RNA from control fed rats.



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  		Figure 2. Determination of the plateau phases for IGF-IA and ß-actin in a parameter-defined RT-PCR (detailed in Materials and Methods). Top panel, Ethidium bromide stained gel showing RT-PCR products for ß-actin (194 bp) and IGF-IA (114 bp) with 22–37 cycles. The IGF-IB (166 bp) band is too faint to be seen in this gel. Lane A, 25 bp DNA ladder. Lane B-Q, RT-PCR with 22–37 cycles. Lower panel, Quantitation by densitometry of Polaroid 55 negative of ethidium bromide stained gel. The arrows indicate the cycle number at which the PCR enters the plateau phase.

 
The ß-actin amplicon (194 bp) was not changed by dietary manipulation (fasted, 4181 ± 190 DU; fasted-refed, 4093 ± 92 DU; control-fed, 4038 ± 160 DU; Fig. 3Go). The 114-bp IGF-IA band was not lower in the fasted (3180 ± 115 DU) than in fasted-refed (2915 ± 53 DU) or control fed animals (2835 ± 91 DU). The 166-bp IGF-IB band was decreased by 55% in fasted (60.6 ± 4.0 DU) compared with control fed (135.1 ± 11.6 DU; P < 0.01), and refeeding caused an exaggerated rise in IGF-IB (196.1 ± 5.2 DU; P < 0.05 vs. control fed). We observed the same RT-PCR results in experiments using three fewer cycles (25 cycles). We also observed similar RT-PCR alterations of IGF-IB mRNA using IGF-IB specific primers whose upstream primer is residing in the 52-base exon 5 sequences (data not shown).



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  		Figure 3. RT-PCR amplification of IGF-IA and -IB hepatic transcripts in fasted, fasted-refed, and control fed rats. Top panel, Amplified IGF-IA (114 bp), -IB (166 bp), and the ß-actin internal control (194 bp). Autoradiography was done at room temperature for 12 h. Bottom panel, Quantitation in densitometric units of each amplicon from each group expressed as mean ± SEM (n = 6; **, P < 0.01 vs. fasted group).

 
We verified the IGF-I amplicons by restriction analysis (Fig. 4Go). HinfI cut IGF-IA amplicon into two bands of 58 bp and 56 bp, and cut IGF-IB amplicon into two bands of 58 bp and 108 bp. TaqI cut only IGF-IB into 86 bp and 80 bp bands. Double digestion of IGF-IA and -IB amplicons with HinfI and TaqI produced four bands of 86 bp, 58 bp, 56 bp, and 22 bp. These patterns conform to those deduced from the known IGF-I sequence (22).



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  		Figure 4. Restriction analysis for verification of IGF-IA and -IB amplicons from hepatic total RNA by RT-PCR. Lane A, 25 bp DNA ladder. Lane B, intact amplicons of IGF-IA (114 bp) and IB (166 bp). Lane C, IGF-IA and -IB digested with HinfI. IGF-IA is cut into 58 bp and 56 bp bands and IGF-IB is cut into 58 bp and 108 bp bands. Lane D, IGF-IA and -IB digested with TaqI. IGF-IB is cut into 86 and 80 bp bands. Lane E, double digestion of IGF-IA and -IB with HinfI and TaqI. As expected, this produces bands of 86, 58, 56, and 22 bp.

 
Using the RPA assays, both IGF-IA and -IB transcripts were present in each liver sample, and the changes caused by fasting and refeeding were consistent with the RT-PCR results (Fig. 5Go). Four protected bands of the expected sizes were detected in each sample: 224 nt and 100 nt (IGF-IA), 376 nt (IGF-IB), and 126 nt (ß-actin). When normalized to ß-actin, which did not change, fasting did not change the 224 nt IGF-IA transcript (fasted: 11.2 ± 1.3, fasted-refed: 9.6 ± 1.2, control-fed: 11.0 ± 1.2), or the 100 nt transcript (fasted: 4.2 ± 0.6, fasted-refed: 4.7 ± 0.5, control-fed: 5.6 ± 0.4). Fasting, however, decreased the IGF-IB transcript (fasted: 0.55 ± 0.14, fasted-refed: 3.2 ± 0.4, control-fed: 3.3 ± 0.2; P < 0.01, fasted vs. fasted-refed or control fed rats). Refeeding restored the IGF-IB transcript. The relative abundance of IGF-IA and -IB in control fed rat liver were 77% and 23% of the total IGF-I transcripts, respectively.



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  		Figure 5. RNase protection assay of liver IGF-IA and -IB mRNAs in fasted, fasted-refed, and control-fed rats. Lane A and E, RNA ladder. Lane B, undigested riboprobes for IGF-I (410 nt) and ß-actin (165 nt). Lane C and D, ten micrograms of liver RNA from control-fed rats hybridized with either ß-actin riboprobe (lane C, 126 nt protected) or IGF-I riboprobe (lane D, 3 protected bands, 376 nt for IGF-IB, 224 nt and 100 nt for IGF-IA). The remaining lanes contain ten micrograms of liver RNAs from each rat of the three groups hybridized with both IGF-I and ß-actin riboprobes. The ß-actin riboprobe was labeled with 1:3 diluted Biotin-14-CTP compared with the labeled IGF-I riboprobe. Chemiluminesence was done by exposing membrane to Kodak Biomax film for 10 min.

 
Biosynthesis of liver IGF-IA and IB prehormones
After in vitro translation of liver mRNA and immunoprecipitation, pre-pro-IGF-IA (17kDa band, Fig. 6AGo) was observed to be decreased by 92% in fasted (31.3 ± 10.9 DU) compared with control-fed animals (421.8 ± 96.0 DU; P < 0.01). Refeeding increased the liver pre-pro-IGF-IA level to 94% of the control fed value (Fasted-refed: 397.6 ± 190.0 DU; P < 0.01, vs. fasted). This peptide (17 kDa) corresponds to the translation product of class 1 mRNA (17,059 Da), containing a 48-amino acid signal peptide (31, 32). Pre-pro-IGF-IB (18-kDa band, Fig. 6BGo) is reduced by 87% in fasted (74.9 ± 35.0 DU) compared with control-fed animals (597.4 ± 49.9 DU; P < 0.01), and is restored by refeeding (fasted-refed: 590.8 ± 160.4 DU). It is also translated from class 1 mRNA (17,923 Da) containing a 48 amino acid signal peptide. Both pre-pro-IGF-IA and -IB are immunoprecipitated with the antiserum (UBK487) against mature IGF-I (Fig. 6CGo), and the effects of dietary manipulation using this antibody are similar to those produced by the Ea and Eb antibodies (Fasted: 316.0 ± 94.5 DU, fasted-refed: 1109.8 ± 307.6 DU, control-fed: 1154.2 ± 233.7 DU). In control-fed rats, pre-pro-IGF-IA (926.8 ± 192.6 DU) accounts for more than 80% of the total IGF-I prehormones, whereas less than 20% of the total IGF-I prehormones was represented by IB (227.4 ± 46.2 DU).



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  		Figure 6. In vitro translation and immunoprecipitation of IGF-I prehormones from livers of fasted, fasted-refed, and control-fed rats. A–C, Results of in vitro translation of hepatic mRNA followed by immunoprecipitation of pre-pro-IGF-IA by the Ea antibody designated EaAb (17 kDa; A), pre-pro-IGF-IB by the Eb antibody designated EbAb (18 kDa: B), and precipitation of both pre-pro-IGF-IA and -IB by UBK487 (C). Lane A, negative control without liver RNA in the translation system (background translation). Lanes B–E, fasted rats. Lane F-I, fasted-refed rats. Lanes J–M, control fed rats. Fluorography was done with intensifying screen at -80 C for 160 h (A and B) or 120 h (C). D, Quantification of panels A–C. Each of the three panels were scanned densitometrically with respective calibration. Results are reported in densitometric units as mean ± SEM (n = 4; **, P < 0.01 vs. fasted group).

 
Only the IGF-IA prehormone (29 kDa) was detected by immunoblotting of liver extract (Fig. 7Go). It was decreased by 77% in fasted (58.2 ± 11.6 DU) compared with control fed animals (248.4 ± 32.2 DU; P < 0.01), and increased to 106% of the control fed value by refeeding (fasted-refed: 264.0 ± 45.0 DU). This relatively large IGF-IA prehormone has two potential N-glycosylation sites (26) and can be deglycosylated by N-glycosidase F (31). We were unable to detect IGF-IB prehormone by either EbAb or UBK487 in liver extract.



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  		Figure 7. Immunoblotting of IGF-IA prehormones in liver extracts of fasted, fasted-refed, and control-fed rats. Top panel, Liver IGF-IA prehormone (29 kDa band) in each of the three groups. Blot is exposed to Kodak ECL film for 15 min. Bottom panel, Quantitation in densitometric units of IGF-IA from each group expressed as mean ± SEM (n = 6; **, P < 0.01 vs. fasted group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Alterations in nutritional status are reported to cause differential regulation of IGF-I mRNAs by transcription initiation from two different leader exons (33, 34). Little attention, however, has been directed at the nutritional regulation of IGF-I gene expression by alternative RNA splicing or differential polyadenylation. Likewise, minimal study has been directed at the effects of nutrients on translation of IGF-I mRNA. In the present study, we examine mechanisms for the decreased IGF-I during fasting, using semiquantiative RT-PCR and RPA to measure alternatively spliced IGF-IA and -IB mRNA in liver, and immunoprecipitation following in vitro translation to measure hepatic IGF-IA and -IB prehormones.

As reported by others (4, 5, 6), we observe by Northern blotting that steady-state IGF-I mRNA in liver is decreased by fasting. In our animals, this decrease results from reduction of the 1.5–1.9 kb and 0.9–1.2 kb size species, as the fasting-related reduction in the relatively smaller quantities of the 7.5-kb species was more equivocal. Most reports on the effect of diet restriction indicate that there is coordinate reduction in each of the major IGF-I mRNA species (5, 6, 8, 9). In protein restriction, however, the 7.5 kb species shows a greater decrease than the other size classes (7, 35, 36). We speculate that the difference we observe might be related to minor differences in strains of rats used, the conditions of fasting, the handling of the tissue, etc. The observation of a net decrease in IGF-I mRNA does not reveal whether the alternative splice site is involved mechanistically, because the IGF-IA and -IB mRNAs are distributed through each of the size species (25, 37, 38).

Using both semiquantitative RT-PCR and RPA, we observe that fasting decreases IGF-IB, but not IGF-IA mRNA. Because the latter does not decrease, and IGF-IA and -IB mRNAs are encoded by one single-copy gene and result from alternative splicing after transcription, we conclude that there is no appreciable decrease in IGF-I gene transcription or in IGF-I mRNAs at the alternative splice site. The decrease in total IGF-I mRNAs during fasting, therefore, must result from other mechanisms. Furthermore we surmise that the apparent decrease in IGF-IB transcript during fasting might be due to posttranscriptional pre-mRNA processing. We also observe that more IGF-IB transcript is present in fasted-refed rats than in control-fed animals (by RT-PCR). This pattern of change is similar to the change of the 7.5 kb IGF-I transcript in Northern blotting and suggests that the increase in 7.5 kb IGF-I transcript during refeeding might result from the increase in IGF-IB transcript. The absence of a decline in the IGF-IA transcript, which accounts for 77% of IGF-I mRNAs might seem inconsistent with reduced total IGF-I mRNAs. This is possible, however, because neither the protected bands in RPA nor the RT-PCR amplicons are full-length messengers/cDNAs and don’t necessarily represent the abundance of intact mRNAs.

To assess the functionality of IGF-I mRNAs, we carried out in vitro translation followed by immunoprecipitation of pre-pro-IGF-IA or IB peptides. Using antibodies specific for IGF-IA prehormone (EaAb) and IGF-IB prehormone (EbAb), we observed that fasting caused a decrease in both peptides, and refeeding caused both peptides to be restored to normal. These results were confirmed using an antibody against mature IGF-I (UBK487), which precipitates both prehormones. The observation that pre-pro-IGF-IA accounts for 80% of the total IGF-I prehormones confirms a previous report (36). The pre-pro-IGF-IA and IB from in vitro translation have molecular weights of 17K and 18K, respectively. These correspond to IGF-I prehormones translated from class 1 mRNAs (IA: 17,059 Da; IB: 17,923Da) (31) and are consistent with greater transcription activity from leader exon 1 than from leader exon 2 (39). The decreased peptide product from in vitro translation suggests that fasting reduces the translatable IGF-I mRNA, or attenuates the translation efficiency of IGF-I mRNA. There are at least two possible reasons for the discrepancy between decreased liver IGF-IA peptides measured by immunoblotting and the normal abundance of hepatic IGF-IA transcripts measured by both RT-PCR and RPA. First, some of these IGF-IA transcripts could be present in the untranslated free messenger ribonucleoprotein particle (mRNP). This has been reported for IGF-II mRNA in fetal liver and some cell lines (40) but does not seem to occur with IGF-I mRNA in liver of protein-restricted rats (41). Our in vitro translation results seem to exclude this possibility, because messengers in mRNPs should be translated in the in vitro system. Second, the IGF-IA transcripts may have a shorter half-life, being degraded quickly. Although these degraded transcripts are not translatable, they may be detected by either RT-PCR or RNase protection assay. Our results seem to support this possibility indirectly.

The immunoblot results from liver extracts showing that IGF-IA prehormone is reduced by fasting and returned to normal by refeeding are consistent with the in vitro translation results. The IGF-IA prehormone in liver extract is a 29 kDa N-glycosylated protein that is able to be deglycosylated (31). IGF-IB is not detected by immunoblotting with either Eb antibody or UBK487, perhaps because its concentration is too low or because the antigen determinants of denatured IGF-IB protein are not recognized by our antibodies.

The results of these studies add additional insight into the complexity and redundancy of the response of the IGF system to nutrient restriction. We have reported that during fasting or dietary protein restriction a variety of mechanisms act to attenuate the production and action of IGF-I (1). These include postreceptor resistance to GH action (42), evidence for translational stalling of IGF-I mRNAs (35), accelerated clearance of IGF-I (43), and resistance to the growth promoting effect of IGF-I (44). The present study indicates that fasting differentially regulates IGF-IA and -IB transcripts in rat liver. Reduction of hepatic IGF-I mRNAs by fasting is likely caused by posttranscriptional mechanisms, such as nuclear RNA splicing and/or RNA degradation, which attenuates translation of IGF-I mRNAs.


   Acknowledgments
 
We acknowledge the excellent technical assistance of Ms. Eyvonne Bruton and Mr. Ward Jarvis. We thank Dr. Dionisios Chrysis for help with statistical analysis and Dr. Billie Moats-Staats for helpful advice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant HD-26871. Presented in part at the 10th International Congress of Endocrinology at San Francisco, June 12–15, 1996. Back

2 Present address: Integrated Science and Technology Program, James Madison University, Harrisonburg, Virginia 22807. Back

Received January 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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