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Reduction of Hepatic Insulin-Like Growth Factor I (IGF-I) Messenger Ribonucleic Acid (mRNA) during Fasting Is Associated with Diminished Splicing of IGF-I Pre-mRNA and Decreased Stability of Cytoplasmic IGF-I mRNA¹

Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599-7220

Address all correspondence and requests for reprints to: Louis E. Underwood, M.D., Department of Pediatrics, 509 Burnett-Womack Building, CB# 7220, University of North Carolina, Chapel Hill, North Carolina 27599-7220.

	Abstract

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The mechanisms by which fasting decreases liver insulin-like growth factor I (IGF-I) messenger RNA (mRNA) abundance have not been defined completely. In the present study, we have examined the effects of fasting in rats on hepatic IGF-I gene transcription, IGF-I pre-mRNA splicing, and cytoplasmic IGF-I mRNA stability. Using the in vitro nuclear run-on transcription technique, we observed that fasting did not change IGF-I gene transcription activity [76 ± 32 densitometric units (DU) for fasted vs. 58 ± 23 DU for control-fed rats; P = 0.1], whereas IGF-binding protein-1 (IGFBP-1) gene transcription, a positive control, was increased more than 2-fold (729 ± 157 DU for fasted vs. 261 ± 56 DU for control-fed rats; P < 0.05). This implies that fasting-induced reduction of liver IGF-I mRNA is due to events other than a decreased rate of IGF-I gene transcription. By measuring nonspliced (pre-mRNA) and spliced IGF-I transcripts in liver nuclear RNA using ribonuclease protection assays, we found that IGF-I pre-mRNA was increased in fasted rats (measured as the percentage of ß-actin: 34.0 ± 5.5% for fasted vs. 8.1 ± 3.8% for control-fed rats; P < 0.01), whereas spliced IGF-I transcript remained unchanged (measured as the percentage of ß-actin: 60.9 ± 9.2% for fasted vs. 79.0 ± 6.2% for control-fed rats; P = 0.75). We then compared this pattern of splicing to IGF-I pre-mRNA splicing in hypophysectomized rats subjected to GH stimulation and to IGFBP-1 pre-mRNA splicing in the same fasting experiment. One hour after GH injection, we observed a coordinate increase in both nonspliced and spliced IGF-I transcripts in liver nuclei of hypophysectomized rats. Fasting increased both IGFBP-1 pre-mRNA and spliced transcript. Taken together, these results indicate that the increase in IGF-I pre-mRNA in liver nuclei during fasting is caused by delayed pre-mRNA splicing, rather than increased IGF-I gene transcription. To examine the possible effect of fasting on hepatic IGF-I mRNA stability, we used an in vitro model of nutrient deprivation (fewer amino acids in culture medium) of rat hepatocyte primary culture. Each of the three major IGF-I mRNA species exhibited a shortened half-life in the amino acid-deprived media. The 7.5-kb IGF-I mRNA, however, was degraded faster than the two smaller IGF-I mRNA species. This may indicate that fasting decreases the stability of liver IGF-I mRNA in vivo. In summary, these results suggest that fasting regulates hepatic IGF-I gene expression mainly at the posttranscriptional level by delaying IGF-I pre-mRNA splicing, which attenuates mature IGF-I mRNA generation, and by accelerating the rate of degradation of IGF-I mRNA in cytoplasm.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
REGULATION of insulin-like growth factor I (IGF-I) gene expression by nutrients involves a wide range of mechanisms at multiple levels (1, 2, 3, 4). In undernourished animals these include alteration of IGF-I gene transcription (5, 6, 7, 8), decreased stability of the 7.5-kb IGF-I messenger RNA (mRNA) (9, 10), stalling of the translation of IGF-I mRNA (11), postreceptor resistance to GH action (12), accelerated clearance of serum IGF-I (13), and resistance to the action of IGF-I in target tissues (14). The many complex steps involved in IGF-I synthesis, including differential transcription initiation and polyadenylation (15, 16, 17), alternative splicing of IGF-I primary transcript (18, 19), multiple cytoplasmic IGF-I mRNA species (20, 21), and differential translation initiation (22, 23) also provide potential points at which IGF-I gene expression may be affected when nutrient intake is altered. The effects of nutritional perturbation on the differential transcription initiation and the alternative splicing of IGF-I gene have been recently investigated (24, 25, 26).

The decreased hepatic IGF-I mRNA abundance in fasted or energy-restricted rats is reported to be caused by decreased IGF-I gene transcription (5, 7). There are, however, inconsistent data on the transcriptional mechanisms involved in this event and on the factors that regulate posttranscriptional expression of this gene (8, 26, 27). We have reported that IGF-I gene transcription in rat liver is not decreased by short term fasting (26). This conclusion, derived using ribonuclease protection assays (RPAs) and RT-PCR, is based on the observations that one of the products of alternative splicing at exon 4 to exon 6, IGF-IA mRNA, is not decreased. The IGF-IB mRNA, however, is reduced during fasting, but this could not be taken as evidence of reduced transcription, because production of IGF-IA and -IB mRNAs results from alternative splicing of a single IGF-I primary transcript synthesized from the same single-copy gene (16, 19, 28). To extend the search for the mechanisms involved in decreased steady-state abundance of IGF-I mRNA in fasting, we have examined the effect of fasting on IGF-I gene expression at three different levels: we assessed transcription directly using nuclear run-on transcription assays, we monitored IGF-I pre-mRNA splicing using ribonuclease (RNase) protection assays, and we evaluated the effects of nutrient deprivation on IGF-I mRNA stability using primary cultures of hepatocytes. In both nuclear run-on assays and RNase protection assays, a rat IGF-binding protein-1 (IGFBP-1) probe generated by PCR in our laboratory was used as a positive control in this fasting study.

	Materials and Methods

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Probes and reagents
A 786-bp rat IGF-I complementary DNA (cDNA) in pGEM-4Z (29), provided by Dr. P. Kay Lund (University of North Carolina, Chapel Hill, NC), was used for nuclear run-on assays. A 345-bp rat IGF-I genomic DNA in pBluescript SK (30) containing exon 4 (182 bp) and a portion of intron 3 (163 bp), a gift from Dr. Peter Rotwein (Oregon Health Sciences University), was used for RNase protection assays. Rat genomic probes for IGFBP-1 and ß-actin were produced by PCR in our laboratory. A rat ß-actin cDNA clone used for RPAs, biotin-14-CTP, and biotin detection reagents were purchased from Ambion, Inc. (Austin, TX). Restriction enzymes and DNA polymerase were obtained from Promega Corp. (Madison, WI). RNA polymerases, digoxigenin (DIG)-11-UTP, and DIG detection reagents were purchased from Boehringer Mannheim (Indianapolis, IN). Other reagents were recombinant human (h) GH (Genentech, Inc., South San Francisco, CA), 5,6-dichlorobenzimidazole (DRB; Sigma Chemical Co., Inc., St. Louis, MO), Matrigel basement membrane matrix (Becton Dickinson Co. Labware, Bedford, MA), and two basic DMEM-Ham’s F-12 media containing either 1 or 0.2 times the peripheral arterial plasma amino acid concentration in normal rats (prepared through the Tissue Culture Facility of Lineberger Cancer Center of the University of North Carolina).

PCR
Rat genomic DNA was isolated from liver tissue (31). Primers were designed according to the published sequences (32, 33). For IGFBP-1, the sense oligo was 5'-TGCCCAGATTCTCATCCAC-3' [nucleotides (nt) 113–131], and antisense oligo was 5'-GCTAATGGCATTCCACAGG-3' (nt 1965–1983). For ß-actin, the sense oligo was 5'-ATAGCCCTCTTTTGTGCC-3' (nt 1211–1228), and the antisense oligo was 5'-TTGCCGATAGTGATGACC-3' (nt 2530–2547). PCRs were carried out at 94 C for 1 min, 57 C (ß-actin) or 59 C (IGFBP-1) for 1 min, and 72 C for 1.5 min, for total of 40 cycles. PCR products of IGFBP-1 and ß-actin were subcloned individually into the pNoTA/T7 vector (5 Prime3 Prime, Inc., Boulder, CO). To assure that the probes were free of other rat genomic sequences that might influence hybridization in nuclear run-on assays, IGFBP-1 and ß-actin were reamplified by PCR from their corresponding plasmids.

For RPAs, another pair of IGFBP-1 primers was designed to amplify the last 210 bp of the above IGFBP-1 clone (76 bp from intron 1 and 134 bp from exon 2). The sense oligo was 5'-TTAAGGCGGTAGACGTGAG-3' (nt 1774–1792), with the T3 promoter consensus sequence linked to its 5'-end. The antisense oligo was the same as the one used for the previous clone, except the T7 promoter consensus sequence was linked to the 5'-end. This modification made the full-length riboprobe bigger than IGFBP-1 pre-mRNA and thus facilitated the separation of probe from protected IGFBP-1 pre-mRNA band in RPAs.

Animals and experimental design
Eight five-week-old male Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA), weighing 145–155 g, were delivered to our animal care facility and housed in 12-h light, 12-h dark cycles. One group of four was fasted, and the other group of four was fed ad libitum for 48 h. Each animal was allowed free access to water during fasting. Liver tissue was collected from each rat under ether anesthesia after blood was depleted by cardiac puncture (this procedure greatly reduced the amount of blood cells left in liver tissue). Approximately 2–3 g fresh liver tissue were used for isolation of nuclei, and the remainder was flash-frozen in liquid nitrogen and stored at -80 C.

We employed a GH-treated hypophysectomized rat model, in which IGF-I gene transcription was enhanced, to monitor IGF-I pre-mRNA splicing. Five-week-old male Wistar rats, hypophysectomized at 4 weeks of age, were purchased from Charles River Laboratories. Completeness of hypophysectomy was confirmed by lack of weight gain over 2 weeks. At 6 weeks of age the rats were injected ip with 1.5 µg/g BW recombinant hGH or vehicle only. Livers were excised 1 h later under ether anesthesia, and nuclei were isolated.

Measurement of IGF-I in serum
IGF-I serum concentrations were measured by RIA after removal of IGFBPs using ODC-silica cartridge chromatography (C₁₈ Sep-Pak, Waters Corp., Milford, MA). Purified human plasma-derived IGF-I (PS III) was used as a standard in the RIAs (34).

Isolation of nuclei and nuclear run-on assay
Rat liver nuclei were isolated using the method described by Blobel and Potter (35) with minor modifications. Briefly, up to 3 g fresh liver tissue were homogenized in a Dounce homogenizer (Kontes Co., Vineland, NJ) in 2 vol ice-cold 0.25 M sucrose in TKM [50 mM Tris-HCl (pH 7.5), 25 mM KCl, and 5 mM MgCl₂]. The homogenate was filtered through a 41-mesh nylon membrane (Spectrum, Houston, TX), and mixed with 2 vol 2.3 M sucrose in TKM. The mixture was then layered onto a cushion of 2.3 M sucrose in TKM and centrifuged at 30,000 x g for 1 h at 4 C. The nuclear pellet was resuspended in 0.25 M sucrose in TKM by gently mixing with a glass rod at 0–4 C, flash-frozen on dry ice, and stored at -80 C. We chose this isoosmotic sucrose medium to resuspend liver nuclei, because it was reported to better maintain morphological integrity and transcriptional activity of the stored frozen nuclei (36). To prepare blots for nuclear run-on transcription, Equimolar amount of probes (0.6 pmol each) for IGF-I (gel-purified cDNA), IGFBP-1, and ß-actin (genomic DNA generated by PCR) were denatured with alkali and individually blotted onto positively charged nylon membranes.

Nuclear run-on assays were performed as described previously (37, 38, 39), except that DIG-11-UTP was incorporated into newly transcribed RNA in nuclei instead of [-³²P]UTP. Nuclei (5 x 10⁷) in a 110-µl volume were mixed with an equal volume of 2 x reaction buffer [10 mM Tris-Cl (pH 8.0), 5 mM MgCl₂, 0.3 M KCl, 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.6 mM UTP, 0.4 mM DIG-11-UTP, and 5 mM dithiothreitol]. In vitro transcription was carried out at 30 C for 30 min. The reaction was quenched by adding 3 vol 4 M guanidine thiocyanate, and nuclear RNA was extracted from disintegrated nuclei (40). Sixty micrograms of RNAs were heat-denatured and added to standard hybridization buffer with formamide [5 x SSC (standard saline citrate), 0.1% N-lauroylsarcosine, 0.02% SDS, 1% blocking reagent, and 50% formamide). Hybridization of DIG-labeled nascent nuclear RNAs to DNA blots harboring rat IGF-I, IGFBP-1, and ß-actin genes was accomplished in 2-ml screw cap microtubes at 42 C overnight with rotation. Blots were washed twice in 2 x SSC-0.1% SDS at room temperature for 5 min each, then twice in 0.1 x SSC-0.1% SDS at 68 C for 15 min each. Chemiluminescent detection of DIG-labeled RNA was carried out using the protocol recommended by the manufacturer (41). Hybridization increased linearly as the amount of DIG-labeled nuclear RNA was increased. Target DNA sequences on blots were in excess. Blots were scanned, and the densitometric signals were normalized to the ß-actin signal.

Nuclear RNA extraction and RNase protection assay
Nuclear RNA was extracted from liver nuclei as described for the nuclear run-on assays. To remove any residual genomic DNA, nuclear RNA samples were treated with RQ1 RNase-free deoxyribonuclease (Promega Corp.). RPAs were carried out with either DIG-labeled or biotin-labeled riboprobes as reported previously (26). Briefly, IGF-I and IGFBP-1 antisense RNAs (345 and 210 nt) were synthesized with T7 RNA polymerase from linearized plasmid (IGF-I) and purified PCR products (IGFBP-1), respectively. Probes were gel purified and quantified by UV spectrophotometry. Hybridization was accomplished at 45 C overnight in 20 µl hybridization buffer. After RNase digestion, RNAs were resolved on 5% denaturing polyacrylamide gels and electroblotted onto positively charged nylon membranes. Signals were revealed by chemiluminescent detection (DIG or biotin system) and scanned densitometrically. A rat ß-actin riboprobe was included in each RPA to assure uniform RNA input and uniform loading for electrophoresis.

Rat hepatocyte primary culture
Hepatocytes were prepared from 7-week-old male Sprague-Dawley rats by nonrecirculating collagenase perfusion of the liver in situ (42, 43, 44). The viability of freshly isolated hepatocytes was greater than 90%, as judged by trypan blue exclusion. We used the thin coating method recommended by the manufacturer to spread 350 µl of 1:2.5 diluted Matrigel evenly onto 100-mm plastic dishes (45). Cells were seeded at a density of 1.0 x 10⁷/dish in 10 ml serum-free DMEM-Ham’s F-12 medium with supplements (44) and were cultured at 37 C in a humidified atmosphere of air containing 5% CO₂.

To mimic the in vivo nutrient deprivation, we cultured hepatocytes in two basic DMEM-Ham’s F-12 media differing only in their amino acid concentration, as prepared previously (44). The media contained either 0.8 or 4.0 mM of each essential and nonessential amino acid, corresponding, respectively, to 0.2 or 1 times the peripheral arterial plasma amino acid concentration in the normal rat (42). Experiments were started 40 h after inoculation and after medium had been changed twice at 8 and 32 h. The culture media were changed to either 0.2 or 1 x serum-free DMEM-Ham’s F-12 medium containing 50 mM DRB. IGF-I mRNA abundance was determined after incubation of the cells in each of the two media for 3, 6, 12, or 24 h.

Extraction of whole cellular RNA and Northern blotting
Whole cellular RNAs were extracted either from liver tissue or cultured hepatocytes (40), fractionated on 1% agarose gels containing formaldehyde, transferred onto positively charged nylon filters, and fixed by UV cross-linking. Riboprobes of rat IGF-I (345 nt), IGFBP-I (210 nt), ß-actin (126 nt, Ambion, Inc.), and human 28S ribosomal RNA (1.5 kb; American Type Culture Collection, Manassas, VA) were labeled with DIG-11-UTP using the appropriate RNA polymerase (T7 or T3). Hybridization was completed overnight at 68 C. Blots were washed twice for 5 min each time at room temperature in 2 x SSC-0.1% SDS, then twice at 68 C for 15 min each time in 0.1 x SSC-0.1% SDS. The chemiluminescent detection protocol recommended by the vendor was followed (41).

Statistics
Blots were scanned as reported previously (26), with individual calibration for each blot. Data are reported in densitometric units (DU) as the mean ± SE. We used an independent t test to determine the statistical significance of differences between the fasted and control groups. Linear regression analysis was performed, after rectification of our data by transformation of logarithm, to determine the statistical difference between the rate of IGF-I mRNA degradation in hepatocytes cultured in 0.2 x medium and that in hepatocytes cultured in 1 x medium.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of rat IGFBP-1 and ß-actin genomic DNA probes
Our rat IGFBP-1 probe migrated on electrophoresis at the anticipated size of 1871 bp containing the last 412 bp of exon 1, intron 1 (1325 bp), and the first 134 bp of exon 2. This sequence specificity was observed by mapping the HindIII site shown in the published IGFBP-1 sequences (32) (Fig. 1, lanes 2 and 3). The ß-actin probe was compatible with an anticipated size of 1337 bp containing the last 154 bp of exon 1, intron 1, exon 2, intron 2, and the first 392 bp of exon 3. This was confirmed by mapping the BglI site indicated in the known ß-actin sequences (33) (Fig. 1, lanes 4 and 5). Another IGFBP-1 genomic probe designed for RPAs showed the exact size we designed (Fig. 1, lane 8), containing the last 210 bp of the previous IGFBP-1 clone, 19 bp of T3 promoter sequences, and 19 bp of T7 promoter sequences.

View larger version (35K): [in this window] [in a new window]   Figure 1. PCR amplification of rat IGFBP-1 and ß-actin probes. Both IGFBP-1 and ß-actin probes were amplified from rat genomic DNA as indicated in Materials and Methods. According to their sequence specificity, the IGFBP-1 probe was 1871 bp (lane 2) and could be cut by HindIII into two bands of 1056 and 815 bp (lane 3). The ß-actin probe was 1337 bp (lane 4) and could be cut by BglI into two bands of 991 and 346 bp (lane 5). Another IGFBP-1 probe designed for RPAs was amplified from the above 1871-bp IGFBP-1 clone. It contained 210 bp of IGFBP-1 sequences and 38 bp of both T3 and T7 promoter sequences (lane 8). Lanes 1, 6, and 7 were DNA markers.

View larger version (51K): [in this window] [in a new window]   Figure 2. Effect of fasting on liver IGF-I gene transcription. Nuclear run-on transcription assays were accomplished by hybridization of DIG-labeled newly transcribed RNA from liver nuclei of fasted or control fed rats to denatured IGF-I, IGFBP-1, and ß-actin DNA sequences. Blots prepared from each of the four study animals and representing 1-h exposure by chemiluminescent detection with substrate CSPD are in the top panel. As indicated in the bottom panel, fasting did not change IGF-I gene transcription when the data were normalized to the ß-actin signal. IGFBP-1, which served as a positive control, increased more than 2-fold. *, P < 0.05. ß-Actin, which is not changed by fasting, was used to assure that the input of DIG-labeled RNA was uniform.

View larger version (38K): [in this window] [in a new window]   Figure 3. A, The effect of fasting on IGF-I pre-mRNA splicing assessed by RPAs that measure nonspliced (pre-mRNA) and spliced IGF-I transcripts in liver nuclei of fasted and control rats. Hybridization of biotin-labeled IGF-I antisense RNA (396 nt) to hepatic IGF-I nuclear transcripts (20 µg nuclear RNA used) gave two protected bands: a 345-nt pre-mRNA band and a 182-nt band of spliced transcript. Fasting caused a significant increase in IGF-I pre-mRNA, whereas spliced IGF-I transcript did not change significantly. The ß-actin probe (165 nt synthesized with T7 RNA polymerase), which gave one protected band of 126 nt, was used to assure equal nuclear RNA input. B, A similar experiment was performed with 5 µg liver total RNA. Fasting decreased cytoplasmic IGF-I mRNA (182-nt protected band) abundance by 52% (6.6 ± 0.4 vs. 13.7 ± 1.9 DU; P = 0.01). Chemiluminescent detection was performed using substrate CDP Star for 10-min (B) or 45-min (A) exposure of Kodak BioMax film. Lane M, Biotinylated RNA marker lane. Lane P, Full-length riboprobes.

View larger version (63K): [in this window] [in a new window]   Figure 4. Assessment of nonspliced pre-mRNA and spliced RNA for IGF-I after injection of GH into hypophysectomized rats. Nuclear RNA (20 µg) from liver of hypophysectomized rats given a single ip injection of 1.5 µg GH/g BW 1 h previously (lanes 2 and 3) or vehicle (lane 1) or from normal rats (lanes 4 and 5) were hybridized with biotin-labeled IGF-I (396 nt) and ß-actin (165 nt) riboprobes. Both IGF-I pre-mRNA (345 nt) and spliced transcript (182 nt) levels were increased in response to the augmented IGF-I gene transcription caused by GH. The ß-actin probe protected a 126-nt sequence (lanes 1–5). Blot results from chemiluminescence after 30-min exposure to BioMax film source are shown. Lane M, Biotinylated RNA marker. Lane P, Full-length riboprobes.

View larger version (67K): [in this window] [in a new window]   Figure 5. Coordinate increase in IGFBP-1 nonspliced pre-mRNA and spliced mRNA transcripts in liver nuclei of fasted rats. Liver nuclear RNA (10 µg) from each fasted or control rat were hybridized to DIG-labeled IGFBP-1 probe (232 nt) and ß-actin probe (181 nt synthesized with T3 RNA polymerase). The IGFBP-1 riboprobe gives two protected bands: 210 nt from IGFBP-1 pre-mRNA and 134 nt from spliced transcript. The ß-actin probe gave one protected band of 126 nt. To avoid the influence of excessive ß-actin activity on the adjacent IGFBP-1 band, the specificity of DIG-labeled ß-actin was greatly reduced by using 1:4 diluted DIG-11-UTP in the labeling reaction. Fasting increased the abundance of both IGFBP-1 pre-mRNA and spliced transcript. Results were derived from chemiluminescence with 3-min exposure to BioMax film. Lane M, Biotinylated RNA marker (detected separately with 45-min exposure). Lane P, Full-length riboprobes.

View larger version (31K): [in this window] [in a new window]   Figure 6. Pre-mRNA splicing patterns for IGF-I and IGFBP-1 in liver nuclei of fasted rats. IGF-I pre-mRNA is increased in fasted rats compared with control rats, whereas spliced IGF-I transcript in fasted rats is not significantly different from that in control rats (A). Both IGFBP-1 pre-mRNA and spliced transcript are significantly increased in fasted rats (B), which is caused by up-regulated IGFBP-1 gene transcription. *, P < 0.01.

View larger version (65K): [in this window] [in a new window]   Figure 7. Northern blots of IGFBP-1 transcript. Five micrograms of liver nuclear RNA from four fasted rats (mixed sample, lane F) or four control rats (mixed samples, lane C) were probed with the DIG-labeled IGFBP-1 riboprobe (A). A Northern blot with 10 µg liver total RNA from four fasted or four control rats (mixed samples) is shown in B. Fasting increased IGFBP-1 steady state mRNA (1.7 kb) abundance in cytoplasm as well as the size-heterogeneous IGFBP-1 transcripts in nuclei. The 1.7-kb mature IGFBP-1 mRNA was barely detected in nuclear RNA preparations. Lane M, DIG-labeled RNA marker.

View larger version (60K): [in this window] [in a new window]   Figure 8. Decreased IGF-I mRNA stability in cultured hepatocytes subjected to amino acid restriction. A, Northern blot of IGF-I cytoplasmic mRNAs in hepatocytes cultured in either 1 x (lanes 2, 4, 6, and 8) or 0.2 x (lanes 3, 5, 7, and 9) medium. Whole cellular RNA (15 µg/lane) was isolated from hepatocytes at 3 h (lanes 2 and 3), 6 h (lanes 4 and 5), 12 h (lane 6 and 7), and 24 h (lanes 8 and 9) after the addition of DRB. Lane 1, RNA at the starting time, when the culture medium was changed to either 1 or 0.2 x, and DRB was added. Lane L, Fifteen micrograms of RNA from normal rat liver. Lane M, DIG-labeled RNA marker. Hybridization was performed using DIG-labeled IGF-I antisense RNA (345 nt). A representative blot by chemiluminescence of 40-min exposure to BioMax film is shown. The blot inserted in B shows the two smaller IGF-I mRNA species with shorter (20-min) exposure to the imaging film. B, Linear regression analysis of the decay of IGF-I mRNAs in hepatocytes cultured in 1 or 0.2 x medium. IGF-I mRNA abundance was determined by scanning the blots densitometrically. Data were log transformed before plotting on a linear scale. Each point represents the mean ± SE of three determinations. The half-life (t1/2) was measured as 0.693/, where is the slope of the lines obtained by plotting log(IGF-I mRNA level) vs. time. Analysis of the slopes of both RNA species shows that decay of RNA in cells cultured in 0.2 x medium is significantly faster than that in cells cultured in 1 x medium (P < 0.0001 for both species).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We used an in vitro nuclear run-on transcription assay to evaluate the effect of fasting on IGF-I gene transcription. This assay allows the concurrent measurement of transcription of other genes (51), in this case IGFBP-1 and ß-actin. As reported by others (7, 48), we observed a 2-fold increase in IGFBP-1 gene transcription during fasting. This confirms that our fasting model and the nuclear run-on transcription assay worked satisfactorily. Fasting did not alter the rate of IGF-I gene transcription. This result is consistent with previous reports on the effects of fasting (8, 26) and dietary protein restriction (6, 50) on IGF-I gene transcription. Previous reports, however, have shown a remarkable variance in IGF-I gene transcription among rats of the same group and when different IGF-I probes are used for hybridization (7, 8). To avoid these problems, we normalized IGF-I gene transcription hybridization results to those of ß-actin gene transcription. Perhaps more importantly, we used a full-length IGF-I cDNA probe to hybridize the newly transcribed IGF-I RNA. Such a probe is less likely to miss hybridization signals, as might occur with probes containing only one exon sequence (8, 30). Our findings using these nuclear run-on assays indicate that fasting does not impair IGF-I gene transcription and that posttranscriptional events are involved in the decline in hepatic IGF-I mRNA abundance.

Posttranscriptionally, pre-mRNA is spliced, and the mRNA is translocated from nucleus to cytoplasm (52, 53, 54, 55). We sought to assess the splicing of IGF-I pre-mRNA by measuring nonspliced (pre-mRNA) and spliced IGF-I transcripts in nuclear RNA. This approach is based on the assumption that the relative abundance of spliced and nonspliced IGF-I transcripts in nuclei reflects the success of the splicing process. We observed that fasting decreased the spliced IGF-I transcript slightly (P = 0.75), but greatly increased IGF-I pre-mRNA level. This accumulation of IGF-I pre-mRNA could result either from an increase in IGF-I gene transcription that might be underestimated by nuclear run-on assays or from defective posttranscriptional mechanisms, such as a block in pre-mRNA splicing or in mRNA nucleocytoplasmic export. We conclude that a principal cause of the accumulation of IGF-I pre-mRNA is attenuated splicing. We base this conclusion on observations made in two situations. First, in liver nuclei of hypophysectomized rats injected with GH, we observe a coordinate increase in nonspliced and spliced IGF-I transcripts. Consistent with a previous report by others (30), this pattern of IGF-I pre-mRNA splicing is an indication of increased IGF-I gene transcription by GH. Secondly, in nuclei from our fasted rats, we observe a coordinate increase in both nonspliced and spliced transcripts of IGFBP-1. This confirms our observation by nuclear run-on assays and the observations of others (46, 47, 48) that fasting increases liver IGFBP-1 gene transcription. This also indicates that fasting does not impair IGFBP-1 pre-mRNA splicing and that the alteration in IGF-I pre-mRNA splicing is gene specific. Thus, we exclude the possibility that an alteration in IGF-I gene transcription causes the accumulation of IGF-I pre-mRNA in liver of fasted rats. If the mechanisms involved in nucleocytoplasmic export of the mature IGF-I transcript were impaired, we would expect to observe the accumulation of spliced IGF-I mature transcript, not nonspliced pre-mRNA. Our results suggest that fasting causes delayed IGF-I pre-mRNA splicing, which leads to attenuation of the maturation of IGF-I mRNA and a decrease in cytoplasmic IGF-I mRNA abundance. This is consistent with the view that pre-mRNA splicing is the principal rate-limiting step in the posttranscriptional control of gene expression in mammalian cells (53). Our findings do not indicate the mechanism involved in diminished IGF-I pre-mRNA splicing during fasting. The lack of a significant decrease in IGF-I-spliced transcript in liver nuclei of fasted rats is probably a combined effect of diminished IGF-I pre-mRNA splicing and attenuated maturation of spliced IGF-I transcript. Our results also indicate that it is not always possible to predict the level of transcription of a specific gene based on the level of its pre-mRNA. Although abundance of a gene’s pre-mRNA is a better predictor of the rate of the gene’s transcription than is abundance of its mRNA, nuclear run-on transcription assays or transcription chase experiments are much more likely to predict the rate of transcription (51). We believe that care must be exercised when the pre-mRNA level for a specific gene is used to determine its transcription.

We assessed the decay of IGF-I mRNAs in cultured rat hepatocytes after IGF-I gene transcription was arrested by addition of DRB to the culture medium. We observed that reduction of amino acid concentration resulted in more rapid degradation of 7.5-kb IGF-I mRNA. The other two smaller IGF-I mRNAs also exhibited faster decay in 0.2 x medium than in 1 x medium. From these observations in an in vitro system, we speculate that fasting decreases the stability of liver IGF-I mRNAs in vivo. Our results are consistent with previous reports that 7.5-kb IGF-I mRNA is less stable in protein-restricted animals than in control animals (9, 10) and is less stable than the other two smaller IGF-I mRNA species in vivo (56).

Our results lead us to conclude that as IGF-I gene transcription proceeds during fasting, IGF-I pre-mRNA splicing is reduced. This probably attenuates the generation of mature IGF-I mRNA. Also, reduced stability of cytoplasm IGF-I mRNA in cultured hepatocytes by amino acid restriction may indicate a possible decline of the stability of liver IGF-I mRNA in the fasted rat. These events are likely to contribute to the reduction in steady state levels of IGF-I mRNAs in fasting and perhaps in other forms of nutrient restriction. The observation that nutrient restriction has effects on more than one event in the steps immediately following IGF-I gene transcription is not surprising in light of previous observations showing that dietary deficiencies attenuate the GH-IGF system at multiple levels, including reduction of GH receptors (57), production of a postreceptor defect in GH action (12), stalling of the translation of IGF-I mRNA (11), accelerated clearance of IGF-I from serum (13), changes in IGFBPs (58), and resistance to IGF-I action in target tissues (14).

	Acknowledgments

 
We thank Drs. Ali S. Calikoglu, Eyvonne Bruton, Susan Hall, and Katherine Hamil for their expert technical assistance. We are grateful to Drs. A. Joseph D’Ercole and Billie M. Moats-Staats for critical reading of this manuscript and helpful comments.

	Footnotes

 
¹ This work was supported by NIH Grant HD-26871 and an Endocrine Fellowship grant from Eli Lilly & Co. The services (rat hepatocyte primary cultures) provided by the Center for Gastrointestinal Biology and Disease of the University of North Carolina were supported by Grant P30-DK-34987.

Received April 27, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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