The Babraham Institute, Cambridge CB2 4AT, United Kingdom
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ABSTRACT |
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This study investigated mechanisms regulating hepatic insulin-like growth factor (IGF)-I class 1 and 2 mRNA levels. Lambs were treated with growth hormone (GH) either as an acute, single dose or over a longer term. Total hepatic unspliced, pre-mRNA levels increased after the single dose of GH but were attenuated after 8 days of GH, with exon 1- and 2-derived pre-mRNA levels displaying coordinate responses. Surprisingly, changes in total spliced, mature mRNA levels did not reflect those for pre-mRNA, instead being augmented after 8 days of GH. GH also induced a differential increase in the ratio of mature class 2-to-class 1 IGF-I mRNA; therefore, this must be predominantly via posttranscriptional mechanisms. Increases in the ratio of class 2-to-class 1 mRNA were observed in polysomal vs. total RNA preparations derived from GH-treated but not control lambs, indicating an increased proportion of class 2 transcripts engaged in translation. Our findings indicate that GH may stabilize mature class 2 transcripts or destabilize mature class 1 transcripts and that class 2 mRNA may have a greater translational potential. The following two main functions of hepatic class 2 IGF-I mRNA are suggested: an efficient "monitor" of GH status via providing a rapid negative feedback mechanism and a coordinator of endocrine-regulated tissue growth.
liver; transcription; alternative splicing; sheep; growth
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INTRODUCTION |
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INSULIN-LIKE GROWTH FACTOR I (IGF-I) regulates cell function, including proliferation, survival, and differentiation, and is therefore essential for normal development and growth (28, 38). Postnatally, IGF-I is found in large amounts in the circulation, most being maintained as a ternary complex that consists of IGF-I, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS), thus providing a reservoir of IGF-I peptide (39). Even though the importance of circulating IGF-I for growth regulation has been challenged (26, 27, 46), recent studies in which both hepatic IGF-I and ALS synthesis have been abolished using gene targeting have confirmed that circulating IGF-I is crucial for normal growth, that the ablated hepatic IGF-I synthesis is apparently not compensated by increased IGF-I synthesis by other tissues, and that the molecular form of circulating IGF-I is important (47). The liver therefore has a central role in the maintenance of circulating IGF-I and in directing the complex molecular interactions of IGF-I with its direct and indirect binding partners. Furthermore, the establishment of a growth hormone (GH)-responsive endocrine IGF network is crucial for successful adaptation from fetal to postnatal life (24) and coordinating tissue growth and development, with hepatic IGF-I being in large part responsible for this endocrine role. However, the mechanisms by which the liver achieves these unique functions are uncharacterized.
The IGF-I gene is complex: mature peptide is encoded by only exons 3 and 4, with alternative splicing occurring at both the 5' (exons 1 and 2)- and 3' (exons 5 and 6)-ends of the gene, generating multiple mRNA species (30). Exons 1 and 2 are each associated with unique promoters and have disperse vs. discrete start sites, generating class 1 and class 2 mRNAs, respectively; interestingly, the liver is the major site of promoter 2 activity, with promoter 1 only being active in most tissues (41). Our previous studies and those of others have demonstrated a preferential increase in hepatic IGF-I derived from exon 2 in animals treated chronically with GH (29, 36), with changed nutritional status (36), and during development (1, 25). However, the mechanism accounting for differential increases in class 2 mRNA are not yet identified; differential transcriptional regulation has not been demonstrated to date (e.g., see Ref. 3). Thus, despite sequence conservation across mammalian species, the functional significance of IGF-I promoter 2 and its mRNA is not established.
It is intriguing that the liver is responsible for circulating IGF-I and is also the major tissue for IGF-I promoter 2 activity; correlation between class 2 mRNA and circulating IGF-I concentrations provides compelling evidence for a functional relationship (36). GH is a key regulator of postnatal circulating IGF-I concentrations, and therefore the aim of this study was to investigate mechanisms by which GH regulates hepatic IGF-I class 1 and class 2 mRNA levels. Lambs, rather than rodents, were selected as the most appropriate model, because the GH-IGF-I axis is far more closely related between humans and sheep than between humans and rodents, and therefore our findings could have significance for clinical application. Because the mature, secreted IGF-I peptides derived from exons 1 and 2 are identical, our studies focused on 1) comparing transcriptional vs. nontranscriptional regulation of IGF-I exons 1 and 2 and 2) the potential translatability of class 1 and class 2 mRNAs.
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MATERIALS AND METHODS |
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Animals.
Temporal effects of GH on pre- and mature IGF-I nascent and mature
class 1 and class 2 transcript abundance were investigated by treatment
of lambs with a single dose or 8 days of GH. Twenty 20-wk-old lambs
were randomly allocated to the following four treatment groups:
groups 1 and 2 were injected subcutaneously with
vehicle control and groups 3 and 4 with bovine GH
(0.2 mg · kg1 · day
1 in
carbonate-buffered saline: 150 mM NaCl and 25 mM
Na2CO3, pH 9.4). Groups 1 and
3 were killed [via a lethal dose of pentobarbital sodium
(0.7 mg/kg), followed by exsanguination] exactly 3 h after a
single injection of GH or vehicle, and groups 2 and
4 were killed after 8 days of treatment with GH or vehicle,
again exactly 3 h after the final dose. Samples of liver were
rapidly dissected and frozen in liquid nitrogen. On the final day of
treatment, jugular blood samples (10 ml) were taken every 30 min from
2 h before vehicle or GH administration until the animals were killed.
Extraction of total RNA. Total RNA was extracted from 1-g portions of frozen liver using the guanidinium isothyocyanate method (6). In addition, glycogen was removed from the final RNA pellet preparation by dissolving the precipitated RNA pellet in RNase-free water and incubation overnight at 4°C with the addition of sodium acetate to 3 M (pH 7.0). The RNA pellet was washed with 75% ethanol. Total RNA was quantified by absorbance at 260 nm (1.0 A = 40 µg/ml). RNA integrity was checked by size separation on a denaturing 1.4% agarose gel and visualization with ethidium bromide. To ensure that mRNA concentrations were also consistent, total RNA was hybridized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Northern analysis (described below).
Preparation of polysomal RNA. Polysomal RNA was extracted from fresh liver as described by Foyt et al. (13) with the adaptations incorporated (12) using a discontinuous sucrose gradient for polysomal isolation.
Template-specific PCR to measure nascent and mature mRNA levels.
Nascent (i.e., pre-mRNA) and mature class 1 and class 2 transcripts
were quantified using a modified version of competitive RT-PCR termed
competitive RS-PCR (RNA template-specific PCR; see Ref.
42). This method has the sensitivity of conventional
RT-PCR with the advantage of a reduction in false positives caused by contamination with minute quantities of genomic DNA. Briefly, RNA was
isolated from sheep liver and used as a template for reverse transcription to cDNA using 47-mer primers, of which only the first 17 nucleotides, at the 3' end, hybridize to the target. The remaining
30-nucleotide tag, being of unique sequence (termed T30), was then used
as the downstream primer in all subsequent PCR reactions (35 cycles).
The pairs and sequences of all primers are given in Fig.
1.
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Reverse transcription.
cRNA at decreasing concentrations [doubling dilutions of cRNA
starting with exon 1 (2.49 × 1017 mol), exon 2 (2.77 × 10
17 mol), intron 1 (9.65 × 10
20 mol), and intron 2 (1.60 × 10
19
mol)] was added to eight reactions, each containing 0.1 µg total hepatic RNA, 0.5 mM dNTPs, 1.0 unit RNase inhibitor, 1.0 µM 47-mer downstream primer, and 10.0 units Moloney murine leukemia virus reverse
transcriptase (Promega, Southampton, Hants, UK).
PCR. Each reverse transcription reaction (10% portion) was used in the subsequent PCR reactions, each consisting of 1.5 mM MgCl2, 200 µM dNTPs, 0.3 µM of each primer, and 2.0 units Taq Start polymerase (Sigma Chemical, Poole, Dorset, UK). PCR reactions were electrophoresed on a 1.4% agarose gel.
Quantification of products. The relative amounts of the two PCR products were quantified by ethidium bromide staining using a Gel Doc 1000 (Bio-Rad, Hercules, CA). The ratio of log target product to the log cRNA product was plotted against the log number of moles of cRNA added to the PCR mixture; the number of moles of target product was equivalent to the moles of cRNA (on the abscissa) when the log ratio was 0.0. Amounts of mRNA associated with exon 1, intron 1, and exon 2 could be obtained directly from these calculations; however, nascent transcripts from intron 2 will contain nascent message derived from the promoters associated with both exons 1 and 2, and therefore the true value for class 2 nascent transcripts was obtained after subtraction of class 1 nascent levels. Relative amounts of pre-mRNA and mature mRNA levels derived from both exon 1 and exon 2 were confirmed in selected samples by RNase protection assay using single-exon riboprobes (which contain intronic sequence); similar changes in message abundance were observed from both techniques (data not shown).
Northern analyses. Total RNA was electrophoresed on formaldehyde 1.4% agarose gels, blotted on an MSI nylon transfer membrane (Micron Separations, Westbrook, MA), and ultraviolet cross-linked using a Stratalinker (Stratagene, La Jolla, CA). Asymmetric PCR was used for IGF-I probe synthesis. Initially, double-stranded DNA templates were generated by standard PCR for exons 1, 2, and 4 (primers: exon 1, upstream 5'-TGT GAC ATT GCT CTC AA-3', downstream 5'-CTT CAA GAA ATC ACA AAA G-3'; exon 2, upstream 5'-TAA TAC CCA CCC TGA CC-3', downstream 5'-TGT AGG TGT AAC CAT TT-3'; exon 4, upstream 5'-GGC TCG AGC AGT CGG AG-3', downstream 5'-AGC CTT GGG CAT GTC GG-3') and GAPDH (upstream primer: 5'-AAT ACT GGC AAA GTG GAC-3'; downstream primer 5'-AGA TGG TGA TGG CCT TTC-3'), which was followed by the asymmetric PCR using the downstream primer for each exon and 32P-labeled dCTP as part of the nucleotide mix. Hybridization and washing of blots was performed using Church and Gilbert's (7) reagents.
RNase protection assays. RNase protection assays were used to quantify IGF-I transcripts derived from the leader exons 1 and 2 and the common coding exons 3 and 4 of the IGF-I gene. Antisense riboprobes were generated from cDNA templates specific for exons 1-3-4 or exons 2-3-4, yielding two protected bands after an RNase protection assay, with the full-length band (404 or 356 bases) corresponding to the class 1 or 2 transcripts, respectively, and the smaller band (341 bases) being equivalent to mRNA protected by the common coding exons 3 and 4 (assumed to represent mRNA derived from the reciprocal leader exon). Intensities of protected bands were visualized on X-ray film and quantified using an image analyzer (Ceescan, Cambridge, UK); the intensity of each protected band was corrected for the number of U nucleotides. Total IGF-I mRNA levels could be calculated from the sum of each of the two protected bands. All procedures were carried out exactly as described previously (36).
Measurement of plasma IGF-I concentrations.
Plasma IGF-I concentrations were determined in acid-ethanol-extracted
samples with an additional overnight precipitation of IGFBPs at
20°C after initial incubation and centrifugation of protein in
acid-ethanol as previously described (36). The anti-IGF-I antiserum was raised against human IGF-I in rabbits at the Babraham Institute and had a titer of 1:50,000.
Statistical analysis.
Where appropriate, data were analyzed using two-way ANOVA, with
treatment time (single dose vs. 8 days) and GH as main effects. ANOVA
was followed by standard post hoc t-tests using the pooled SE. However, for Figs. 2-4, individual group errors (as SE) are also presented to show the variation associated with each treatment group.
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RESULTS |
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Differential effects of acute and short-term GH administration on
total hepatic pre- and mature IGF-I mRNA levels.
The first aim of the current study was to determine whether pre- and
mature mRNA levels exhibited similar changes in response to GH,
inferring transcriptional vs. posttranscriptional regulation of IGF-I
mRNA abundance, respectively (3, 16, 21). Total hepatic
pre-IGF-I mRNA levels are presented in Fig.
2A and were about two orders
of magnitude lower than mature levels (Fig. 2B). As
expected, a significant increase in pre-mRNA levels (6-fold; P < 0.001) was observed in response to the single dose
of GH. Surprisingly, after GH treatment for 8 days, total pre-mRNA
levels were attenuated, decreasing significantly (P < 0.02) to a value only fourfold above mean control levels. Total hepatic
mature IGF-I mRNA levels were ~32 × 1019 mol
mRNA/µg total RNA in the saline control groups and increased by
~2.5-fold 3 h after the single dose of GH (P < 0.001). In contrast to pre-mRNA, total IGF-I message levels did not
decrease after 8 days of GH treatment but were augmented further to
3.5-fold above mean control values (P < 0.002 compared
with the single dose of GH), therefore demonstrating temporal
regulation of IGF-I mRNA levels by GH.
Circulating IGF-I concentrations. Mean circulating IGF-I concentrations were on average 207.4 ng/ml in saline-treated lambs and exhibited no change after only 3 h of GH treatment (i.e., the single dose). However, concentrations increased significantly (P < 0.001) by 2.4-fold after 8 days of GH [single-dose saline, 201.4; 8 days of saline, 213.4; single-dose GH, 214.6; 8 days of GH, 506.9; pooled SED (n = 5), 23.3 ng/ml]. Thus, even though pre- and mature IGF-I mRNA levels respond after 3 h of GH treatment, circulating levels of IGF-I do not increase after such a short time period.
Promoter 1- and 2-derived IGF-I mRNA: coordinate regulation of
pre-mRNA but differential regulation of mature mRNA.
We determined the contributions of mRNA derived from the individual
promoters 1 and 2 to the changes in total pre- and mature hepatic IGF-I
mRNA (represented in Fig. 3 as a degree
of increase above mean saline-treatment levels). Promoter 1- and
2-derived pre-mRNA levels were equivalent in saline-treated control
animals (intron 1, 0.072; intron 2, 0.080 × 1019
mol mRNA/µg total RNA; means ± SE, n = 10).
After the single dose of GH, both increased, with the increase for
intron 2 exceeding that for intron 1 (5.3- vs. 7.1-fold), although not
significantly. After 8 days of GH treatment, the increases in both
promoter 1- and 2-associated pre-mRNA levels were attenuated; the
decrease in promoter 2 pre-mRNA but not in promoter 1 pre-mRNA was
significant (P < 0.02). Thus, in overall terms, the
responses of promoter 1- and 2-derived IGF-I pre-mRNA appeared
qualitatively coordinate, but the magnitude of the changes observed for
promoter 2-derived mRNA exceeded those for promoter 1-derived message.
Differential polysomal association of class 1 and 2 IGF-I mRNA. We next examined whether polysomal association, as an indicator of mRNA translatability (12, 13), was equivalent between class 1 and 2 mRNAs. Initially, Northern analysis was performed on total and polysomal RNA by using a specific exon 4 cDNA probe, which would detect all classes of IGF-I mRNA (Fig. 4A). For animals treated with vehicle or GH, the most abundant transcript in total RNA preparations (Fig. 4A, left) was detected at 7.5 kb, in agreement with previous studies (30), with weaker hybridization to 0.8- to 1.2-kb transcripts; after 8 days of GH treatment, the intensities of all these transcripts were increased. In marked contrast, no hybridization to the 7.5-kb transcript could be detected in polysomal preparations (Fig. 4A, right), even though more intense bands were detected at 0.8 to 1.2 kb in animals treated with GH for 8 days. Similar patterns of hybridization were also observed using exon 1-only or exon 2-only cDNA probes, i.e., no 7.5-kb transcript in polysomal preparations (data not shown); also, it appeared that the exon 2 probe hybridized more strongly to the 0.8- to 1.2-kb transcripts in the 8-day GH-treated animals. Consistency for RNA preparations was confirmed using GAPDH Northern blots (data not shown); however, Northern analysis yielded blots that were difficult to quantify reliably for IGF-I mRNA, and therefore RNase protection assays were used to further confirm relative abundances of class 1 and 2 mRNA in total and polysomal preparations.
Representative RNase protection assays to show class 1 and class 2 mRNA levels in the total mRNA population and associated with polysomes are shown in Fig. 4B. Quantified data derived from all samples are shown in Fig. 4C as the ratio of the abundance of class 2 to class 1 in total and polysomal preparations. In vehicle-treated animals, the ratio of class 2 to class 1 mRNA in total and polysomal RNA preparations was ~0.6 in both total and polysomal preparations. In animals given a single dose of GH, this ratio increased significantly to 0.69 in total RNA (P < 0.05) and further (P < 0.01) to 0.84 in polysomal RNA. These changes were augmented further in animals treated with GH for 8 days, with polysomal class 2 to class 1 mRNA significantly exceeding that for total RNA (P < 0.05). Overall, therefore, GH induced a mean 13% increase in the ratio of class 2 to class 1 mRNA derived from polysomes vs. total RNA in liver, indicating a modest preferential association of class 2 with protein synthetic machinery. ![]() |
DISCUSSION |
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The two IGF-I gene promoters are independently regulated and encode unique mRNAs that are conserved across different species, implying differential functional importance; however, none has yet been identified unequivocally. We have made the following novel observations on GH regulation of IGF-I gene expression: 1) the preferential increase in class 2 IGF-I mature mRNA that occurs after several days of treatment is the result of posttranscriptional rather than transcriptional mechanisms, whereas after acute treatment regulation is largely transcriptional; and 2) class 2 mature mRNA has a greater overall translational potential than class 1 mRNA. These observations are of significance in terms of our understanding of IGF-I gene expression and also for clinical treatment with GH.
Determination of an optimal therapeutic dose of GH for deficient patients to maximize potential clinical benefit while minimizing risks associated with excessive GH exposure has proved difficult; responsiveness to GH varies over time, and endogenous GH and IGF-I secretion and sensitivity vary with age in normal patients (11). Reproduction of normal physiological patterns of pulsatile GH secretion is not possible, although a single daily subcutaneous injection of GH is optimal for improved growth rate in children (19, 37), as has been used in the current study. Serum IGF-I concentrations have been used as a marker for excess GH, particularly after the observations that the risk of developing some forms of carcinoma is correlated with circulating IGF-I levels (5, 15). Of potential concern, serum IGF-I concentrations have continued to increase gradually when patients are given a constant dose of GH (9) or have remained elevated in patients even after a reduction in GH dose (45). Our study may provide a mechanism to account for sustained IGF-I synthesis, since hepatic mature mRNA levels "accumulated" after several days of GH. In support of this hypothesis, we observed a continual increase in circulating IGF-I concentrations in lambs treated with a constant dose of GH for 10 wk from 200 to >1,000 ng/ml, and GH binding to hepatic receptors also increased (36).
A key finding from this study is the modest increase in class 1 IGF-I mature transcripts vs. the larger increase in class 2 mature transcripts in response to GH, despite coordinate changes in their respective pre-mRNA levels. Pre-mRNA is processed, migrating from the nucleus very soon after such maturation; thus, usually only fully spliced RNA is exported from the nucleus (10, 20, 23, 32). Therefore, the unspliced pre-mRNA levels measured in this study represent the overall IGF-I transcription rate; this is supported by the excellent correlation of IGF-I transcription, quantified independently using nuclear run-on assays, with measurements of IGF-I pre-mRNA during development (21), fasting (16), and GH treatment (3). We therefore conclude that GH regulates transcription from promoters 1 and 2 of the ovine IGF-I gene in a coordinate manner, as originally suggested by Bichell et al. (3). Thus the differential levels of mature class 1 and 2 mRNAs suggest posttranscriptional regulation.
GH therefore could change the relative rates of 1) splicing of promoter 1- and 2-derived mRNA, 2) transport of mature class 1 and 2 mRNAs from the nucleus, or 3) mature class 1 or 2 mRNA degradation. The current experiment has not addressed pre-mRNA splicing or mRNA export, although splicing has been suggested as a regulatory mechanism controlling IGF-I mRNA abundance in fasted rats (49). The data described here are also consistent with the hypothesis that IGF-I class 2 mRNA may be more stable than class 1 mRNA or that exon 1-derived mRNA is specifically destabilized. mRNA degradation is certainly established as important in the regulation of cytoplasmic mRNA levels (8, 44). Sequences at both the 5' and 3' ends of mRNA, the secondary structure, and association with RNA-binding proteins may all determine mRNA stability (33) by inhibiting or accelerating mRNA degradation. The size heterogeneity of IGF-I mRNA species observed on Northern blots is because of the use of different polyadenylation sites containing potentially destabilizing AU-rich regions within the 3'-untranslated region (3'-UTR) encoded by exon 6 (17). Specific stabilization of IGF-I mRNA by insulin, without changes in transcription rate, has been reported recently (14). However, at present, there is no evidence to support the hypothesis that the choice of 5' splicing determines the length of the 3'-UTR (30), and therefore it is not possible to associate GH regulation of class 1 and 2 mRNA with current mechanisms suggested for IGF-I mRNA degradation.
Polysomal association of IGF-I mRNA was determined to establish the number of transcripts potentially engaged in translation. Northern analysis revealed that only the smaller-size transcripts (0.8-1.2 kb) were associated with polysomal fractions, and these appeared to be enriched in GH-treated animals. These findings are in contrast with those of Thissen and Underwood (43), who detected the 7.5-kb IGF-I mRNA in polysomal fractions, but are in agreement with Foyt et al. (13). It is possible that the polyadenylation, which is in large part responsible for the size difference between the 0.8- to 1.2-kb and 7.5-kb mRNAs, in some manner inhibits the initiation of translation. Quantitative aspects of class 1 and class 2 IGF-I mRNAs associated with polysomes indicated that GH treatment enriched the proportion of class 2-to-class 1 transcripts by a modest but significant amount, suggesting that the proportion of the two mRNAs may have a role in determining overall IGF-I translational efficiency. In agreement with our findings for control animals, Foyt et al. (13) observed no enrichment of polysomes with class 2 mRNA. However, despite observing a small increase, Foyt et al. (12) later concluded that the relative proportions of exon 2 transcripts between total and polysomal populations were not statistically significantly different in response to changed GH status. Nielsen et al. (34) have reported the existence of specific mRNA-binding proteins for IGF-II that interact with 5' regions in a leader exon-specific manner; even though this would provide a mechanism for differential translatability of IGF-I class 1 and 2 mRNA, no such RNA-binding proteins have been reported for IGF-I to date.
Yang et al. (48) measured IGF-I translation directly in vitro using synthetic IGF-I RNAs; class 2 and class 1 start site 4 mRNAs were translated severalfold more efficiently than mRNAs derived from upstream, longer class 1 start sites. Because start site 4 usage is extremely low (13, 36, 41), it would be expected that class 2 transcripts are the most efficiently translated in vivo. RNA secondary structure is a major determinant of translation initiation (22), and molecular modeling (M-fold software; Babraham Bioinformatics Department) of class 1 and 2 IGF-I 5'-UTR implied extensive opportunity for a secondary structure in class 1 but not class 2 5'-UTR.
Ovine class 1 and 2 transcripts encode pre-IGF-I peptides containing 49 and 33 residues of the NH2-terminal signal sequence, respectively, of which the COOH-terminal 28 residues are encoded by the
common exon 3. Usually, signal peptides are highly variable and share
little in common except a hydrophobic core of ~7-15 amino acids,
the cleavage sequence of two alanines at 1 and
3, and a positively
charged NH2-terminal (2, 40). It is therefore surprising that the IGF-I class 1 and 2 signal peptide sequences display a high degree of conservation across species. The signal peptide hydrophobic core appears to be of key importance
(31), and in the case of IGF-I this is derived from the
common coding exon 3. The splicing of exon 2 to exon 3 only adds
another five residues, which are not likely to be structurally
significant, whereas exon 1 splicing adds another 21 residues.
Molecular modeling (Insight-II Software; MSI, San Diego, CA) suggested
that the additional residues derived from exon 1 generate another
hydrophobic patch that is surrounded by charged residues and that the
presence of a proline residue in the class 1 signal peptide introduces
additional structural complexity. This initially translated sequence
may impede secretory efficiency and therefore act as another level of
controlling hepatic IGF-I synthesis.
The physiological function of hepatic IGF-I has been subject to controversy. In the absence of hepatic IGF-I synthesis (26, 27, 46), circulating IGF-I concentrations are markedly reduced, confirming the liver as principally responsible for endocrine IGF-I. Furthermore, increased GH concentrations were observed, supporting a role for either circulating or hepatic IGF-I in negative feedback mechanisms for GH synthesis. Surprisingly, however, mice lacking hepatic IGF-I synthesis grew at normal rates, suggesting that liver-derived endocrine IGF-I was not relevant for growth regulation and casting doubt on the original "somatomedin hypothesis." However, in important recent studies, the ALS has also been ablated, generating growth-retarded mice (47). Because the ALS is responsible for stabilizing the circulating IGF-I pool, these studies support a role for circulating endocrine IGF-I in growth regulation but crucially also suggest that the molecular form of IGF, i.e., association with IGFBPs and formation of ternary complex, is important. Hepatic class 2 IGF-I mRNA is important for the GH stimulation of circulating IGF-I concentrations, but the mechanism by which this occurs has not been identified to date. Our findings suggest that GH stimulation of hepatic IGF-I class 2 mRNA is temporally regulated via posttranscriptional rather than transcriptional mechanisms and that class 2 IGF-I mRNA may be translated and secreted with greater efficiency than exon 1-derived IGF-I. We therefore propose the hypothesis that exon 2-derived IGF-I represents a form of IGF-I that can be synthesized and secreted rapidly in response to GH with the function of coordinating postnatal growth and providing an efficient GH feedback mechanism.
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ACKNOWLEDGEMENTS |
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We thank Anthony Jones and staff for expert care of the lambs and John Coadwell (Babraham Bioinformatics) for the molecular modeling.
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FOOTNOTES |
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This work was funded as part of a Competitive Strategic Grant to The Babraham Institute from the Biotechnology and Biological Sciences Research Council.
Address for reprint requests and other correspondence: J. M. Pell, The Babraham Institute, Cambridge, CB2 4AT UK (E-mail: jenny.pell{at}bbsrc.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 27, 2002;10.1152/ajpendo.00016.2002
Received 15 January 2002; accepted in final form 8 March 2002.
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