Mature Insulin-like Growth Factor I Research Articles

Characterization of a novel alternatively-spliced 5′ exon in the human insulin-like growth factor I (IGF-I) gene, expressed in liver and some cancers

In mammals, the large IGF-I gene comprises 6 exons, which are subject to alternative splicing. All transcripts contain exons 3 and 4, encoding mature IGF-I, but the other exons are included in various combinations, giving at least 6 possible mature mRNAs. At the 5′ end, exons 1 and 2 are spliced alternatively to exon 3, giving different leader/signal sequences. It is shown in this study that in human an additional exon (designated exon 0) is present, upstream of exon 1. This can be spliced directly to exon 3 or, less frequently, into exon 1. Exon 0 is utilized in liver, in about 24% of IGF-I transcripts, to a minor extent in prostate and endometrium (<1% of transcripts), but not in any of 29 other normal human tissues examined. The exon 0 sequence includes an in-frame ATG/AUG, potentially providing a translation start point giving an IGF-I precursor with a very long signal peptide. However, this ATG is very close to the 5′ end, and may not be included in all transcripts; an in-frame ATG in exon 3 could provide an alternative start point. Utilization of exon 0 was detected in other apes, and to a small extent in Old World monkeys, but not in New World monkeys, prosimians or various non-primate mammals. Exon 0 was not expressed in most human tumours, but was utilized in many prostate tumours, at levels much greater than seen in normal prostate, and in liver tumours, at a lower level than in normal liver.

Viral expression of insulin-like growth factor I E-peptides increases skeletal muscle mass but at the expense of strength

Insulin-like growth factor I (IGF-I) is a protein that regulates and promotes growth in skeletal muscle. The IGF-I precursor polypeptide contains a COOH-terminal extension called the E-peptide. Alternative splicing in the rodent produces two isoforms, IA and IB, where the mature IGF-I in both isoforms is identical yet the E-peptides, EA and EB, share less than 50% homology. Recent in vitro studies show that the E-peptides can enhance IGF-I signaling, leading to increased myoblast cell proliferation and migration. To determine the significance of these actions in vivo and to evaluate if they are physiologically beneficial, EA and EB were expressed in murine skeletal muscle via viral vectors. The viral constructs ensured production of E-peptides without the influence of additional IGF-I through an inactivating mutation in mature IGF-I. E-peptide expression altered ERK1/2 and Akt phosphorylation and increased satellite cell proliferation. EB expression resulted in significant muscle hypertrophy that was IGF-I receptor dependent. However, the increased mass was associated with a loss of muscle strength. EA and EB have similar effects in skeletal muscle signaling and on satellite cells, but EB is more potent at increasing muscle mass. Although sustained EB expression may drive hypertrophy, there are significant physiological consequences for muscle.

Persian sturgeon insulin-like growth factor I: molecular cloning and expression during various nutritional conditions

The effects of different periods of starvation (1, 2, 3, and 4weeks) and subsequent re-feeding (over a 4week) on the compensatory growth performance and insulin-like growth factor I (IGF-I) mRNA expression in liver and white muscle were investigated in juvenile Persian sturgeon (Acipenser persicus). First, a fragment of 617 nucleotides coding for IGF-I was cloned from liver, which included an open reading frame of 486 nucleotides, encoding a 162 amino acid preproIGF-I. This is composed of a 45 aa for signal peptide, a 117 aa for the mature peptide comprising the B, C, A, and D domains, and a 47 aa for E domain. The mature Persian sturgeon IGF-I exhibits high sequence identities with other sturgeon species and teleost, ranging between 68 and 95%. The pattern of IGF-I mRNA expression in the liver and white muscle was measured in response to different periods of starvation and subsequent re-feeding. Nutritional status influenced IGF-I mRNA expression pattern in both liver and muscle. IGF-I mRNA expression in the liver increased during starvation, before decreasing after re-feeding. Furthermore, white muscle IGF-I mRNA expression showed better responses to nutritional status and decreased following starvation and increased by re-feeding. However, changes in the expression of IGF-I mRNA were not significantly different between any of the treatments in both tissues. These data suggest that muscle and liver IGF-I mRNA expression do not have a regulatory role for somatic growth induced by compensatory growth in Persain sturgeon.

The Complexity of the IGF1 Gene Splicing, Posttranslational Modification and Bioactivity

The insulinlike growth factor-I (IGF-I) is an important factor which regulates a variety of cellular responses in multiple biological systems. The IGF1 gene comprises a highly conserved sequence and contains six exons, which give rise to heterogeneous mRNA transcripts by a combination of multiple transcription initiation sites and alternative splicing. These multiple transcripts code for different precursor IGF-I polypeptides, namely the IGF-IEa, IGF-IEb and IGF-IEc isoforms in humans, which also undergo posttranslational modifications, such as proteolytic processing and glycosylation. IGF-I actions are mediated through its binding to several cell-membrane receptors and the IGF-I domain responsible for the receptor binding is the bioactive mature IGF-I peptide, which is derived after the posttranslational cleavage of the pro-IGF-I isoforms and the removal of their carboxy-terminal E-peptides (that is, the Ea, Eb and Ec). Interestingly, differential biological activities have been reported for the different IGF-I isoforms, or for their E-peptides, implying that IGF-I peptides other than the IGF-I ligand also possess bioactivity and, thus, both common and unique or complementary pathways exist for the IGF-I isoforms to promote biological effects. The multiple peptides derived from IGF-I and the differential expression of its various transcripts in different conditions and pathologies appear to be compatible with the distinct cellular responses observed to the different IGF-I peptides and with the concept of a complex and possibly isoform-specific IGF-I bioactivity. This concept is discussed in the present review, in the context of the broad range of modifications that this growth factor undergoes which might regulate its mechanism(s) of action.

Mature IGF-I excels in promoting functional muscle recovery from disuse atrophy compared with pro-IGF-IA

Prolonged disuse of skeletal muscle results in atrophy, and once physical activity is resumed, there is increased susceptibility to injury. Insulin-like growth factor-I (IGF-I) is considered a potential therapeutic target to attenuate atrophy during unloading and to enhance rehabilitation upon reloading of skeletal muscles, due to its multipronged actions on satellite cell proliferation, differentiation, and survival, as well as its actions on muscle fibers to boost protein synthesis and inhibit protein degradation. However, the form of IGF-I delivered may alter the success of treatment. Using the hindlimb suspension model of disuse atrophy, we compared the efficacy of two IGF-I forms in protection against atrophy and enhancement of recovery: mature IGF-I (IGF-IS) lacking the COOH-terminal extension, called the E-peptide, and IGF-IA, which is the predominant form retaining the E-peptide. Self-complementary adeno-associated virus harboring the murine Igf1 cDNA constructs were delivered to hindlimbs of adult female C57BL6 mice 3 days prior to hindlimb suspension. Hindlimb muscles were unloaded for 7 days and then reloaded for 3, 7, and 14 days. Loss of muscle mass following suspension was not prevented by either IGF-I construct. However, IGF-IS expression maintained soleus muscle force production. Further, IGF-IS treatment caused rapid recovery of muscle fiber morphology during reloading and maintained muscle strength. Analysis of gene expression revealed that IGF-IS expression accelerated the downregulation of atrophy-related genes compared with untreated or IGF-IA-treated samples. We conclude that mature-IGF-I may be a better option than pro-IGF-IA to promote skeletal muscle recovery following disuse atrophy.

Evidence for the Possible Biological Significance of the igf-1 Gene Alternative Splicing in Prostate Cancer

Insulin-like growth factor-I (IGF-I) has been implicated in the pathogenesis of prostate cancer (PCa), since it plays a key role in cell proliferation, differentiation, and apoptosis. The IGF-I actions are mediated mainly via its binding to the type I IGF receptor (IGF-IR), however IGF-I signaling via insulin receptor (IR) and hybrid IGF-I/IR is also evident. Different IGF-I mRNA splice variants, namely IGF-IEa, IGF-IEb, and IGF-IEc, are expressed in human cells and tissues. These transcripts encode several IGF-I precursor proteins which contain the same bioactive product (mature IGF-I), however, they differ by the length of their signal peptides on the amino-terminal end and the structure of the extension peptides (E-peptides) on the carboxy-terminal end. There is an increasing interest in the possible different role of the IGF-I transcripts and their respective non-(mature)IGF-I products in the regulation of distinct biological activities. Moreover, there is strong evidence of a differential expression profile of the IGF-I splice variants in normal versus PCa tissues and PCa cells, implying that the expression pattern of the various IGF-I transcripts and their respective protein products may possess different functions in cancer biology. Herein, the evidence that the IGF-IEc transcript regulates PCa growth via Ec peptide specific and IGF-IR/IR-independent signaling is discussed.

Insulin-Like Growth Factor-I E-Peptide Activity Is Dependent on the IGF-I Receptor

Insulin-like growth factor-I (IGF-I) is an essential growth factor that regulates the processes necessary for cell proliferation, differentiation, and survival. The Igf1 gene encodes mature IGF-I and a carboxy-terminal extension called the E-peptide. In rodents, alternative splicing and post-translational processing produce two E-peptides (EA and EB). EB has been studied extensively and has been reported to promote cell proliferation and migration independently of IGF-I and its receptor (IGF-IR), but the mechanism by which EB causes these actions has not been identified. Further, the properties of EA have not been evaluated. Therefore, the goals of this study were to determine if EA and EB possessed similar activity and if these actions were IGF-IR independent. We utilized synthetic peptides for EA, EB, and a scrambled control to examine cellular responses. Both E-peptides increased MAPK signaling, which was blocked by pharmacologic IGF-IR inhibition. Although the E-peptides did not directly induce IGF-IR phosphorylation, the presence of either E-peptide increased IGF-IR activation by IGF-I, and this was achieved through enhanced cell surface bioavailability of the receptor. To determine if E-peptide biological actions required the IGF-IR, we took advantage of the murine C2C12 cell line as a platform to examine the key steps of skeletal muscle proliferation, migration and differentiation. EB increased myoblast proliferation and migration while EA delayed differentiation. The proliferation and migration effects were inhibited by MAPK or IGF-IR signaling blockade. Thus, in contrast to previous studies, we find that E-peptide signaling, mitogenic, and motogenic effects are dependent upon IGF-IR. We propose that the E-peptides have little independent activity, but instead affect growth via modulating IGF-I signaling, thereby increasing the complexity of IGF-I biological activity.

Cloning, recombinant expression and purification ofturbot (&lt;I&gt;Scophthalmus maximus&lt;/I&gt;) mature IGF-I

Insulin-like growth factor-I (IGF-I) is a conserved peptide expressed ubiquitously,which shows highly homologous diverse effects on development,growth,and metabolism. With RT-PCR,the fragment encoding the turbot (Scophthalmus maximus) mature IGF-I peptide was amplified. It was predicted that the mature peptide was composed of 70 amino acids including 6 cysteines which may form 3 disulfide bonds. The target fragment was then successfully subcloned into the express vector pGEX-4T-1 and was highly expressed in E.coli BL21(DE3)plysS. The result of SDS-PAGE showed that the fusion protein expressed in the form of inclusion bodies with molecular weight of 34 ku and maximally amounted to 59 % of the whole protein in the E.coli cell 4 hours after being induced with IPTG. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. The inclusion bodies were dissolved in 6 mol/Lguanidine chloride followed by pulse renaturation in refolding buffer containing 0.5 mol/L L-Arginine,1.0 mol/L GSH and 0.2 mol/L GSSG. Then the renatured recombinant protein was purified by GSTrap FF affinity chromatography. The effect of purified GST-IGF-I on turbot kidney cells was analysed,and it indicated that the recombinant GST-IGF-I can stimulate the proliferation of the cells.

The insulin-like growth factor (IGF)-I E-peptides are required for isoform-specific gene expression and muscle hypertrophy after local IGF-I production

Insulin-like growth factor I (IGF-I) coordinates proliferation and differentiation in a wide variety of cell types. The igf1 gene not only produces IGF-I, but also generates multiple carboxy-terminal extensions, the E-peptides, through alternative splicing leading to different isoforms. It is not known if the IGF-I isoforms share a common pathway for their actions, or if there are specific actions of each protein. Viral administration of murine IGF-IA, IGF-IB, and mature IGF, which lacked an E-peptide extension, was utilized to identify IGF-I isoform-specific responsive genes in muscles of young growing mice. Microarray analysis revealed responses that were driven by increased IGF-I regardless of the presence of E-peptide, such as Bcl-XL. In contrast, distinct expression patterns were observed after viral delivery of IGF-IA or IGF-IB, which included matrix metalloproteinase 13 (MMP13). Expression of Bcl-XL was prevented when viral administration of the IGF-I isoforms was performed into muscles of MKR mice, which lack functional IGF-I receptors on the muscle fibers. However, MMP13 expression persisted under the same conditions after viral injection of IGF-IB. At 4 mo after viral delivery, expression of IGF-IA or IGF-IB promoted muscle hypertrophy, but viral delivery of mature IGF-I failed to increase muscle mass. These studies provide evidence that local production of IGF-I requires the E-peptides to drive hypertrophy in growing muscle and that both common and unique pathways exist for the IGF-I isoforms to promote biological effects.

The ABCs of IGF-I isoforms: impact on muscle hypertrophy and implications for repair

Insulin-like growth factor I (IGF-I) plays a critical role in the growth and development of many tissues in the body. It is a key regulator of skeletal muscle development, and continues to enhance the ability for muscle to grow and undergo repair throughout life. Although the focus of research has been on the molecular actions and physiological impact of IGF-I, there has also been a growing undercurrent of studies geared toward the characterization of additional potentially active peptides produced by the igf1 gene. Alternative splicing of the gene results in multiple isoforms that retain the identical sequence for mature IGF-I, but also give rise to divergent C-terminal peptides. The peptides might modulate the actions, stability, or bioavailability of IGF-I, or they might have independent activity. These possibilities have gained the attention of the skeletal muscle field, where novel actions of IGF-I could have significant impact on muscle mass, strength, and repair.

Insulin-like growth factor I of Japanese eel, Anguilla japonica: cDNA cloning, tissue distribution, and expression after treatment with growth hormone and seawater acclimation

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 µg g−1 body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL−1 for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.

Molecular cloning, recombinant expression, and growth–promoting effect of mud carp ( Cirrhinus molitorella) insulin-like growth factor-I

A full-length cDNA encoding insulin-like growth factor-I (IGF-I) was cloned from mud carp ( Cirrhinus molitorella) liver tissue using reverse transcription polymerase-chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The IGF-I precursor cDNA consists of 822 bp in size with a 218 bp 5′-untranslated region and 118 bp 3′-untranslated region. The 486 bp open reading frame encodes a 161 amino acid peptide with a molecular weight of 17.9 kDa. The deduced IGF-I amino acid sequence shared 82.5 ∼ 97% and 82.5 ∼ 84% sequence identity with fish and mammalian counterparts, respectively. The mature IGF-I was overexpressed in Escherichia coli, and the expression level of recombinant mcIGF-I reached to 34.1% of the cell total protein. After purification and refolding of recombinant mcIGF-I, growth–promoting effect of recombinant mcIGF-I was investigated, the results showed that the recombinant mcIGF-I significantly enhanced the growth rate of juvenile tilapia. After 6-week treatment, the growth rates of group1 and 2 were 53 and 67.3% higher than the saline-treated control group. The recombinant mcIGF-I was more effective than recombinant mcGH to enhance the growth rate of juvenile tilapia. The recombinant mcIGF-I-treated fish revealed no significant changes of content of protein, lipid, ash and moisture in muscle.

Viral expression of insulin-like growth factor-I isoforms promotes different responses in skeletal muscle

Insulin-like growth factor I (IGF-I) is a critical protein for skeletal muscle development and regeneration. Its ability to promote skeletal muscle hypertrophy has been demonstrated by several methods. Alternative splicing of the Igf-1 gene does not affect the mature IGF-I protein but does produce different E peptide extensions, which have been reported to modify the potency of IGF-I. Viral-mediated delivery of murine IGF-IA and IGF-IB into skeletal muscle of 2-wk-old and 6-mo-old mice was utilized to compare the effects of the isoforms on muscle mass. In young mice, tissue content of IGF-I protein was significantly higher in rAAV-treated muscles than control muscles at 1, 2, and 4 mo postinjection. Viral injection of IGF-IB produced two- to sevenfold more IGF-I than rAAVIGF-IA. Hypertrophy was observed 2 and 4 mo postinjection, where both rAAVIGF-IA and rAAVIGF-IB were equally effective in increasing muscle mass. These results suggest that there is a threshold of IGF-I production necessary to promote muscle hypertrophy in young growing animals regardless of isoform. In 6-mo-old animals, only rAAVIGF-IA produced significant increases in muscle size, even though increased IGF-I content was observed after injection of both isoforms. Therefore, the ability for IGF-IB to promote muscle hypertrophy is only effective in growing animals, suggesting that the bioavailability of this isoform or its receptor affinity diminishes with age.

Changes in mRNA expression of grouper ( Epinephelus coioides) growth hormone and insulin-like growth factor I in response to nutritional status

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are key links to nutritional condition and growth regulation in teleost. To understand the endocrine mechanism of growth regulation in grouper, we cloned the cDNAs for grouper GH and IGF-I and examined their mRNA expression during different nutritional status. Grouper GH cDNA is 936 base pairs (bp) long excluding the poly-A tail. It contained untranslated regions of 85 and 231 bp in the 5′- and 3′-ends, respectively. It has an open reading frame of 612 bp coding for a signal peptide of 17 amino acids (aa) and a mature hormone of 187 aa residues. Based on the aa sequence of the mature hormone, grouper GH shows higher sequence identity (>76%) to GHs of perciforms than to GHs of cyprinids and salmonids (53–69%). Grouper preproIGF-I cDNA consisted of 558 bp, which codes for 186 aa. This is composed of 44 aa for the signal peptide, 68 aa for the mature peptide comprising B, C, A, and D domains, and 74 aa for the E domain. Mature grouper IGF-I shows very high sequence identity to IGF-I of teleost fishes (84–97%) compared to advanced groups of vertebrates such as chicken, pig, and human (⩽80%). Using DNA primers specific for grouper GH and IGF-I, the changes in mRNA levels of pituitary GH and hepatic IGF-I in response to starvation and refeeding were examined by a semi-quantitative RT-PCR. Significant elevation of GH mRNA level was observed after 2 weeks of food deprivation, and increased further after 3 and 4 weeks of starvation. GH mRNA level in fed-controls did not change significantly during the same period. Hepatic IGF-I mRNA level decreased significantly starting after 1 week of starvation until the 4th week. There was no significant change in IGF-I mRNA levels in fed-controls. One week of refeeding can restore the GH and IGF-I mRNA back to its normal levels. Deprivation of food for 1–4 weeks also resulted in cessation of growth and decrease in condition factor.

Cyclic Adenosine 3',5'-Monophosphate Inhibits Insulin-Like Growth Factor I Gene Expression in Rat Glioma Cell Lines: Evidence for Regulation of Transcription and Messenger Ribonucleic Acid Stability

cAMP inhibits growth and stimulates differentiation in glioma cells. We examined the effect of cAMP on insulin-like growth factor I (IGF-I) gene expression in the C6 cell line, a rat glioma cell line previously reported to grow in response to autocrine IGF-I. cAMP potently inhibited IGF-I messenger RNA (mRNA) and peptide secretion in C6 cells, associated with an attenuation of DNA synthesis. Exogenous IGF-I peptide at least partially prevented the inhibition of DNA synthesis, suggesting that the reduction in IGF-I biosynthesis may contribute to the inhibitory effect of cAMP on C6 cell growth. cAMP also inhibited IGF-I mRNA in rat RG2 glioma cells, but not in three other nonglioma tumor cell lines. The nuclear IGF-I pre-mRNA level and the half-life of mature IGF-I mRNA were both reduced by cAMP in C6 cells, suggesting effects on gene transcription and mRNA stability. However, cAMP had no effect on the activities of IGF-I exon 1 promoter-luciferase constructs. Protein synthesis inhibition partially reduced the inhibition of IGF-I mRNA by cAMP. Inhibition of cAMP-activated protein kinase A activity by H89 did not alter the inhibition of IGF-I gene expression in response to cAMP, suggesting that protein kinase A does not mediate the cAMP inhibitory effect on IGF-I gene expression.

Cyclic adenosine 3',5'-monophosphate inhibits insulin-like growth factor I gene expression in rat glioma cell lines: evidence for regulation of transcription and messenger ribonucleic acid stability.

cAMP inhibits growth and stimulates differentiation in glioma cells. We examined the effect of cAMP on insulin-like growth factor I (IGF-I) gene expression in the C6 cell line, a rat glioma cell line previously reported to grow in response to autocrine IGF-I. cAMP potently inhibited IGF-I messenger RNA (mRNA) and peptide secretion in C6 cells, associated with an attenuation of DNA synthesis. Exogenous IGF-I peptide at least partially prevented the inhibition of DNA synthesis, suggesting that the reduction in IGF-I biosynthesis may contribute to the inhibitory effect of cAMP on C6 cell growth. cAMP also inhibited IGF-I mRNA in rat RG2 glioma cells, but not in three other nonglioma tumor cell lines. The nuclear IGF-I pre-mRNA level and the half-life of mature IGF-I mRNA were both reduced by cAMP in C6 cells, suggesting effects on gene transcription and mRNA stability. However, cAMP had no effect on the activities of IGF-I exon 1 promoter-luciferase constructs. Protein synthesis inhibition partially reduced the inhibition of IGF-I mRNA by cAMP. Inhibition of cAMP-activated protein kinase A activity by H89 did not alter the inhibition of IGF-I gene expression in response to cAMP, suggesting that protein kinase A does not mediate the cAMP inhibitory effect on IGF-I gene expression.

Monoclonal antibodies to the carboxy-terminal Ea sequence of pro-insulin-like growth factor-IA (proIGF-IA) recognize proIGF-IA secreted by IM9 B-lymphocytes.

Insulin-like growth factor-I (IGF-I) circulates in human serum as a 7 kDa peptide but analysis of IGF-I cDNAs predicts two pro-hormone precursors (proIGF-IA and proIGF-IB) with distinct C-terminal E domains. The function of these precursors, and the E peptides generated on cleavage to mature IGF-I, is unknown, largely because of a lack of tools for distinguishing precursors from constituent peptides. We used a synthetic Ea peptide to develop monoclonal antibodies (MAbs) which can recognize the carboxy-terminal sequence of proIGF-IA. These were characterized using proIGF-IA generated by transfected HEK293 cells. The anti-proIGF-IA MAbs immunoprecipitated two peptides (19--21 and 14 kDa) which were also recognized by MAbs to mature IGF-I. The proIGF-IA MAbs could also detect peptides of 9 and 4 kDa predicted to be Ea peptides. Treatment with N -glycosidase proved the 19--21 kDa and 9 kDa bands to be glycosylated proIGF-IA and Ea peptide respectively. Using these antibodies, we have identified proIGF-IA secreted from the IM9 B-lymphocyte cell line. This work paves the way for studies on proIGF-IA and Ea peptide regulation and function.

Primary nucleotide structure of predominant and alternate splice forms of equine insulin-like growth factor I and their gene expression patterns in tissues

Abstract Objective To isolate, clone, and determine primary nucleotide sequence of equine insulin-like growth factor I (IGF-I) and to examine IGF-I gene expression in tissues and cartilage from horses. Animals Horses of various ages. Methods Total RNA was isolated from tissues and purified. Complementary DNA (cDNA) was derived by reverse transcription and polymerase chain reaction (PCR) amplification and subcloned to plasmid vectors for sequencing and comparison with other species. Total RNA from various tissues was probed with radiolabeled cDNA or complimentary RNA constructs by use of northern blotting, tube hybridization, or PCR procedures to determine IGF-I expression patterns. Results Nucleotide sequence of equine IGF-I was 90% homologous to that of cows, 88% homologous to that of humans and sheep, and 77% homologous to that of rats. Amino acid sequence was identical to that of humans, cows, dogs, and pigs. A larger PCR product (IGF-IB) was consistent with alternate splicing with retention of IGF-I exon 4 sequence, similar to rats and mice. Northern blot analysis revealed multiple IGF-I transcripts; predominant sizes were 1.6 and 4.5 kb. The IGF-I message was commonly detected in liver, kidney, and cartilage from young foals and was diminished in cartilage from a 12-month-old horse. Conclusions Nucleotide sequences of equine pre-propeptides were different from those of other species, but the sequence coding the mature IGF-I peptide was more closely homologous. The larger IGF-IB form differed substantially in the carboxy-terminal. The biological action of the cleaved terminal was speculated to be autocrine feedback. Expression of IGF-I was apparent in many tissues, including cartilage, and was greater in immature horses. (Am J Vet Res 1999;60:1234–1241)

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 mRNA

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 β-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 β-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.

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 mRNA.

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 beta-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 beta-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.