Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon.

Insulin-like growth factor I (IGF-I) is known to exert an anabolic effect on tendon fibroblast production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon because they both are produced in fibroblast; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-growth factor (MGF) is upregulated and known to be important for adaptation in loaded muscle. However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and 16 days after surgical removal of synergistic muscle to the plantaris muscle of the rat, thus increasing the load to plantaris muscle and tendon. Nearly a doubling of the tendon mass was observed after 16 days of loading. A rapid rise in tendon procollagen III mRNA was seen after 2 days whereas the increase in procollagen I mRNA was significant from day 8. MGF was expressed and upregulated in loaded tendon tissue with a faster response than IGF-I, which was increased from day 8. Finally, IGFBP-4 mRNA was increased with a time pattern similar to procollagen III, whereas IGFBP-5 decreased at day 8. In conclusion, loading of tendon tissue results in an upregulation of IGF-I, IGFBP-4, and procollagen and is associated with an increase in tendon mass. Also, MGF is expressed with an early upregulation in loaded tendon tissue. We suggest that the IGF-I system could be involved in collagen synthesis in tendon in response to mechanical loading.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

Expression of a biologically active analogue of somatomedin-C/insulin-like growth factor I

A synthetic gene coding for an analogue of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was synthesized by solid support phosphoramidite chemistry and subsequently cloned and expressed in Escherichia coli as a fusion protein. The gene, designed with a threonine codon substituted for a methionine codon at position 59 was expressed fused to an eight-amino acid leader peptide under the direction of the E. coli tryptophan promoter. The fusion protein, termed L0-[Thr59]-Sm-C/IGF-I was purified extensively (greater than 97%) and found to be 60% as active as native Sm-C/IGF-I in a radioimmunoassay and 50% as potent as native Sm-C/IGF-I in a radioreceptor assay. Like native Sm-C/IGF-I it was also mitogenic for Balb/c 3T3 cells. After removal of the eight amino acid leader peptide by cyanogen bromide treatment, the resulting threonine analogue, termed [Thr59]-Sm-C/IGF-I was 80% as potent as native Sm-C/IGF-I in both the RIA and the radioreceptor assays. It was also mitogenic in Balb/c 3T3 cells. These two analogues, therefore, display biological activities similar to human-derived Sm-C/IGF-I.     

Expression, purification and characterization of recombinant human insulin-like growth factor I in yeast

Insulin-like growth factor I (IGF-I) is a 70 amino acid (aa) protein that is structurally similar and functionally related to insulin. We have inserted a synthetic gene coding for human IGF-I into a Saccharomyces cerevisiae expression vector utilizing the MF alpha 1 promoter and pre-pro leader peptide. This vector directs the expression and secretion of native, biologically active growth factor. Cleavage of the pre-pro alpha factor leader sequence in vivo results in the secretion of a 70-aa recombinant IGF-I molecule with the native N-terminal glycine residue. Human IGF-I purified from yeast culture supernatant is equipotent to serum-derived IGF-I in inhibiting [125I]IGF-I binding to type-I IGF receptors and crude human serum-binding proteins. Recombinant IGF-I is also equipotent to human IGF-I in the stimulation of DNA synthesis in rat aortic smooth-muscle cells. In contrast, yeast recombinant IGF-I is less potent than serum-derived IGF-I in binding to type-2 IGF receptors. The ability to produce native, biologically active IGF-I in yeast will allow the elucidation of binding domains through the expression and characterization of specific structural analogs.     

Acromegaly and serum insulin-like growth factor I

Random levels of growth hormone (GH) are usually not helpful in diagnosing either GH deficiency or GH hypersecretion because GH is secreted in a pulsatile fashion. Insulin-like growth factor I (IGF-I), however, is a good indicator of GH secretion and action, particularly at the level of the liver. There is a good correlation between IGF-I and several clinical indices of acromegaly. Measurements of both IGF-I and GH are cornerstones of biochemical diagnosis and follow-up of acromegaly, although in patients treated with pegvisomant, IGF-I levels should be followed rather than GH levels. IGF-I immunoassays differ in assay design, label, intra- and inter-assay precision, and calibrator or standard used, so IGF-I assays may be difficult to compare with one another. Hence, it is essential that the assays used in the laboratory are well validated and adequate normal ranges are available for the levels to be interpreted in a robust manner.     

Exercise and circulating insulin-like growth factor I

Determinations of serum concentrations of total insulin-like growth factor I (tIGF-I) are important in the diagnosis, monitoring of treatment and safety evaluation of patients with growth disorders and/or metabolic disease. It is well established that tIGF-I status varies over time. Changes in tIGF-I levels in relation to an acute bout of exercise or repeated bouts, known as training, are likely to contribute to this variation. Serum tIGF-I has also been found to be of predictive value in growth prediction models employed before the start of growth hormone (GH) treatment. Furthermore, IGF-I generation tests have been suggested to be of value in the assessment of the growth response to GH administration in patients suspected of GH deficiency with or without some degree of GH insensitivity. This is discussed elsewhere in this issue. Recent progress in our understanding of growth hormone-dependent and -independent expression of the IGF1 gene in skeletal muscle and the role of sufficient energy intake during training for muscle and liver generation of IGF-I raises important questions regarding their relative contribution to the circulating pool of IGF-I. The present review is focused on circulating levels of tIGF-I in relation to a single bout of exercise or to a period of training. In addition, the expression of IGF-I locally in muscle in response to these stimuli will be discussed.     

Human insulin-like growth factor I receptor 950tyrosine is required for somatotroph growth factor signal transduction.

Insulin-like growth factor I (IGF-I), a growth hormone (GH)-dependent growth factor exerts feedback regulation of GH by inhibiting GH gene expression. IGF-I inhibition of GH secretion is enhanced 3-5-fold in GC rat pituitary cells overexpressing the wild type 950Tyr human IGF-I receptor which autophosphorylates appropriately. To determine the critical amino acid sequence responsible for IGF-I signaling, insertion, deletion, and site-directed mutants were constructed to substitute for 950Tyr in exon 16 of the human IGF-I receptor beta-subunit transmembrane domain. All mutant transfectants bound IGF-I with a similar Kd to untransfected cells but had markedly increased (7-34-fold) IGF-I-binding sites. GH responsiveness to IGF-I was tested in mutant transfectants. Overexpressed site-directed and insertion mutant IGF-I receptors exhibited a modest suppressive effect on GH in response to the IGF-I ligand, similar to that observed in untransfected cells. Deletion mutant (IG-FIR delta 22) (amino acid 944-965) did not transduce the IGF-I signal to the GH gene. Site-directed and insertion mutants therefore did not enhance the IGF-I response of the endogenous rat receptor, unlike the 950Tyr wild type transfectants which enhanced the IGF-I signal. All mutant transfectants, except the deletion mutant, internalized radioactive ligand similarly to 950Tyr wild type transfectants. 950Tyr of the human IGF-I receptor is therefore required for IGF-I signal transduction in the pituitary somatotroph, but not for IGF-I-mediated internalization.     

Genetic variations of insulin-like growth factor I in Italy

Four Italian populations were examined for a HindIII RFLP associated with the human insulin-like growth factor I gene. No differences were observed among the four samples. The allele frequencies in Italy were: IGF-I HF = 0.834; IGF-I HS = 0.166. The polymorphism appears to be due to a 400-bp sequence insertion-deletion mechanism.     

Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor

Receptor-mediated endocytosis may represent an important mechanism whereby peptide hormones exert their biological effects. The ability of recombinant insulin-like growth factor (IGF)-I to be internalized by cultured cells was evaluated in BRL-3A2 cells, a rat liver-derived cell line which lacks insulin receptors. Since recombinant IGF-I does not bind to the Type II IGF receptor, all specific binding of 125I-IGF-I in BRL-3A2 cells represents binding to the Type I receptor. Exposure of BRL-3A2 cells to IGF-I resulted in a rapid 50% downregulation of Type I IGF receptors. Only one-half of these binding sites were sensitive to treatment with trypsin, a phenomenon which indicates that the peptide and its receptor were internalized after the cells were exposed to IGF-I. In conclusion, these experiments demonstrate that IGF-I can be internalized by cultured cells via the Type I IGF receptor, and suggest that IGF hormone action may be exerted by receptor-mediated endocytosis.     

Distribution of insulin-like growth factor peptides in the developing chick embryo

Growth factors are likely to be of major significance in developmental biology. Here, the distribution of insulin-like growth factor (IGF) peptides is described during development of the chick embryo. IGF was immunolocalised using a polyclonal antibody to human IGF I detected with a modified Vectastain ABC procedure. Under the conditions used, the antibody binds strongly to IGF I and weakly to IGF II; thus the distribution of IGF peptide, rather than the individual factors, is described. Muscle, peripheral nerve and the notochord were labelled whenever present. Muscle label was associated with the myotubes and neural labelling with neurons; Schwann cells were unlabelled. IGF distribution changed during differentiation of connective tissues. Regions of mesenchyme destined to form cartilage labelled weakly or not at all, and cartilage condensations were unlabelled. In the limb, chondrocytes became labelled once cartilage rudiments had formed; however, in later development, label was absent in zones of rounded and flattened chondrocytes and appeared strongly at the onset of hypertrophy. Early osteogenic mesenchyme was also unlabelled, although later bone cells were strongly stained. In the neural tube, label was associated with differentiating neuroblasts and cell bodies and with axons, especially in the developing dorsolateral tracts. These results show a possible correlation between IGF label and cell division in early mesenchyme; cartilage condensations, which have reduced mitotic indices, do not label. In other tissues, notably muscle and nerve but also later connective tissues, label is associated with differentiating, rather than dividing, cells.     

The human gene encoding insulin-like growth factor I is located on chromosome 12

Summary A cDNA probe corresponding to mRNA encoding human somatomedin-C/insulin-like growth factor I (IGF-I) was used for the chromosomal assignment of the IGF-I gene. Southern-blot hybridization analysis of DNA from human-Chinese hamster somatic cell hybrids showed that the IGF-I gene is located on chromosome 12. Comparison of the chromosomal assignments of the IGF-I gene and two other members of the insulin gene family, with three c-ras oncogenes, reveals a remarkable association of the two gene families.     

The insulin-like growth factor I generation test in adults

The insulin-like growth factor I (IGF-I) generation test has the potential to assess the ability of an individual to respond to an acute bolus of growth hormone (GH), in terms of IGF-I, IGF-binding protein 3 and acid-labile subunit responses. This article will discuss something of the history of the IGF-I generation test, and review some of the major studies to date. The IGF-I generation test was first used in adults by Lieberman et al., who studied the effects of ageing and oestrogen administration, and suggested that decreased responsiveness to GH occurs with increasing age and oral oestrogen administration. Our results, however, show that, while activity of the GH/IGF-I axis declines with age, peripheral responsiveness to GH is not affected. As in the Lieberman study, we found that oral oestrogen replacement reduces responses of GH-dependent peptides to GH stimulation in healthy post-menopausal women. Transdermal oestrogen administration also reduced responsiveness to GH, although to a lesser degree than orally administered oestrogen. In addition, utilizing a non-weight-based dose of GH we have demonstrated that obese individuals produce greater increases in IGF-I following an acute bolus of GH. In GH deficiency (GHD), data suggesting enhanced peripheral responsiveness should be interpreted with caution, and with awareness of differences between these groups in terms of age and obesity. The IGF-I generation test may allow a fresh approach to unanswered questions in the field of GHD, but as the IGF-I response to GH is not strictly associated with protein anabolism or clinical benefit, the question remains whether this test will predict the effect of longer-term GH administration.     

Epidermal growth factor enhancement of insulin-like growth factor-I-induced down-regulation of insulin-like growth factor I receptor in cultured placental trophoblastic cells.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) synergistically stimulate placental lactogen (hPL) secretion by placental cells. To understand the mechanism of actions we have investigated a possible heterologous regulatory effect of EGF and IGF-I on each other's receptors. Pretreatment of the cells with IGF-I had no effect on [125I]-EGF binding or the down-regulation of EGF receptor. Pretreatment of the cells with EGF, concomitantly with IGF-I, had no effect on [125I]-IGF-I binding but it augmented the IGF-I down-regulation of IGF-I receptor. The time required to initiate the IGF-I-induced down-regulation of IGF-I receptor was reduced by 4 h in the presence of EGF. IGF-I-down-regulated decreased (P less than 0.05) receptor numbers were further decreased (p less than 0.05) in the presence of EGF. These results suggested that the synergistic effect of EGF and IGF-I seen in hPL secretion by placental cells is not due to direct heterologous hormone-receptor interactive effects. However, the effects seen may be due to a differentiating effect of EGF sensitizing the cells for responsiveness to IGF-I.     

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

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The role of insulin-like growth factor I monitoring in growth hormone-treated children

Growth hormone (GH) therapy has evolved rapidly over the past decade, and continuing research has established a clear role for therapeutic GH in a wide spectrum of disorders, including idiopathic GH deficiency (childhood- and adult-onset), Turner syndrome, Prader-Willi syndrome, small-for-gestational age children with failure of catch-up growth, AIDS-related catabolism, children with chronic renal failure, and idiopathic short stature. Although GH is used therapeutically in a wide variety of conditions, actual guidelines regarding the logistics of GH dosing continue to evolve, with data emerging regarding efficacy and safety. This review proposes a role for insulin-like growth factor I measurement in optimizing GH dosing.     

Study of insulin-like growth factor I in human obesity.

In obesity there is a decrease in basal and stimulated GH secretion. IGF-I, which has negative feedback effects on GH secretion, could be the initial mediator of such alterations. We studied IGF-I levels in obese subjects and their relationship to the obesity level and GH secretion. We determined plasma IGF-I, basal and stimulated GH in 30 normal and 30 obese women and related these variables to obesity indices (body mass index, BMI, and % overweight). Baseline plasma GH values were 1.2 +/- 0.3 and 2.3 +/- 0.6 micrograms/l in obese subjects and controls, respectively (NS). Mean peak GH secretion after stimuli were 11.2 +/- 1.4 and 34.4 +/- 5.6 micrograms/l in obese subjects and controls, respectively (p less than 0.001). Plasma IGF-I were 1.0 +/- 0.1 U/ml and 0.7 +/- 0.1 U/l in obese subjects and controls, respectively (NS). There was a significant negative correlation between plasma IGF-I and age (r = -0.55, p less than 0.001) and a significant negative correlation between mean peak GH secretion and weight (r = -0.60, p less than 0.001), BMI (r = -0.64, p less than 0.001) and percentage of ideal body weight (r = -0.67, p less than 0.001). We did not find any correlation between IGF-I and indices of overweight. These data suggest that the reduced GH secretion found in obesity is not related to a negative feedback inhibition by elevated levels of IGF-I and that adiposity is not associated with a decline in IGF-I levels. We confirm the existence of a negative correlation between GH secretion and obesity indices.     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.