Abstract

The development of a homologous radioimmunoassay (RIA) for chicken insulin-like growth factor-I (cIGF-I) and its use to investigate the developmental changes in IGF-I in the chicken and turkey is described. A doubleantibody RIA has been developed using recombinantly derived cIGF-I as antigen, radiolabelled tracer and standard. The resulting immunoassay has a minimum detection limit of 0·035 ng and effective dose of 2·5 ng. Dose–response curves of chicken and turkey plasma and tissue extracts were parallel with cIGF-I standard. The antiserum is specific for IGF-I as no cross-reactivity with chicken IGF-II, insulin, glucagon, gastrin or avian pancreatic polypeptide was observed. We have also established that acid/ethanol extraction of chicken and turkey plasma reduced possible interference of IGF-binding proteins (IGFBPs) in the RIA. Comparison of IGF-I immunoactivity in unextracted and acid/ethanol-extracted samples following gel filtration under acidic and neutral conditions indicates that the cIGFBPs may be acid-labile. Analyses of samples from growing chickens and turkeys using the homologous avian reagents revealed higher IGF-I concentrations than if the IGF were quantified using heterologous mammalian-derived reagents. A similar pattern was observed when tissue extracts were assayed for IGF-I content. The application of the homologous RIA to monitor blood and tissue IGF-I levels during embryonic development and posthatch growth in avian species will provide more accurate comparisons of results from studies on the role of IGF-I in growth and metabolism of domestic birds.

Journal of Endocrinology (1994) 142, 225–234

Abstract

While numerous researchers have used rat models to investigate the in vivo actions of IGF-I, interpretation of the results in terms of true concentrations of rat IGF-I (rIGF-I) in plasma has been hampered by the absence of homologous reference standards. In order to overcome this we have produced recombinant rIGF-I (rrIGF-I) from Escherichia coli using procedures similar to those we have previously described for the production of other recombinant IGFs. The rrIGF-I is indistinguishable from serum-derived rIGF-I when characterized in a number of in vitro assays including ability to stimulate protein synthesis and inhibit protein degradation in cultured rat cells, as well as in interactions with the rat type-1 IGF receptor and with rat IGF-binding proteins. Moreover, both the serum-derived and the recombinant rat proteins are similar to recombinant human IGF-I (rhIGF-I) in these assays. However, differences between the human and rat IGFs are apparent when tested in immunoassays using some antibodies raised against rhIGF-I. Furthermore, the differences between rhIGF-I and rrIGF-I are even greater when rhIGF-I is used as the competing radiolabel in these assays, a situation that can lead to a two- to threefold underestimation of the actual concentration of IGF-I in rat plasma. These results indicate that, while immunoassays employing antibodies raised against rhIGF-I and rhIGF-I reference standards reliably indicate trends in IGF-I concentrations in rat plasma, the true amounts of rIGF-I present can only be assured in an assay using homologous tracer and reference peptides.

Journal of Endocrinology (1996) 149, 379–387

ABSTRACT

Insulin-like growth factor-I (IGF-I) and IGF-II have been purified to homogeneity from chicken serum as a step towards the characterization of the roles for these peptides in the growth process. Chicken IGF-I had about half the efficacy of bovine/human IGF-I in a bioassay and in radioimmunoassays with bovine IGF-I as radioligand. Chicken IGF-II competed for the binding of bovine IGF-II to cell receptors while chicken IGF-I reacted minimally in this IGF-II radioreceptor assay. Further evidence of homology was obtained by N-terminal sequence analysis of the first 31 and 35 amino acids of chicken IGF-I and IGF-II respectively. Chicken IGF-I had the same N-terminal as human IGF-I, with the exception of the substitution of serine for asparagine at residue 26. Chicken IGF-II had a unique N-terminal tetrapeptide Tyr-Gly-Thr-Ala, but from residues 5–30 the sequence was identical to that reported for residues 6–31 of human IGF-II. Substitutions also occurred corresponding to residues 32, 33, 35 and 36 of human IGF-II. A variant form of chicken IGF-II that had the same N-terminal pentapeptide as human IGF-II was also detected.

J. Endocr. (1988) 117,173–181

ABSTRACT

The clearance of labelled insulin-like growth factor-I (IGF-I) has been measured in lambs following acid gel-permeation chromatography and immunoprecipitation of plasma samples. The half-lives obtained in three experiments were between 5 and 7 h. Chromatography at neutral pH on a Fractogel HW55(S) column demonstrated that all the radioactivity associated with undegraded peptide in plasma was bound to a carrier protein. Similar studies with IGF-I that had been reduced by prior dithiothreitol treatment showed that two-thirds of the initial radioactivity in plasma decayed with a much shorter half-life and represented material that did not bind to carrier proteins in plasma. The remaining radioactivity was both associated with a binding protein and exhibited the characteristically long half-life of the native growth factor. Analysis of plasma samples using reversed-phase chromatography demonstrated that the radioactive component with a long half-life was IGF-I while that with a short half-life had been reoxidized to an incorrect form of the growth factor. When reoxidation of reduced IGF-I was blocked by S-carboxymethylation before injection of the radioactive peptide into lambs, it remained unbound in plasma and had a 0·8–0·9 h half-life. We suggest that reduced IGF-I only associates with the binding protein upon oxidation and correct folding and that this association is necessary in order for IGF-I to have a relatively long half-life.

J. Endocr. (1988) 117, 183–189

ABSTRACT

We have investigated the clearance of ¹²⁵I-labelled chicken and recombinant human insulin-like growth factor-I (IGF-I) from the circulation of chickens as well as the role that IGF-binding proteins play in this process. Analysis of plasma samples by high-performance liquid chromatography (HPLC) neutral gel permeation on a TSK G3000SW column indicated that the i.v. injected radioactivity was rapidly partitioned between at least three pools. Most of the radioactivity occurred in a complex with binding protein(s), while smaller amounts of radioactivity chromatographed in the free IGF-I peak or appeared as low molecular weight degradation products. The labelled chicken and human IGF-I were rapidly cleared during the first 90 min. The calculated half-life for total labelled IGF-I during this period was 54 min for the chicken tracer and 33 min for the human tracer. The clearance was monitored for 10 h during which the human tracer continued to be cleared more rapidly than the chicken tracer. The proportion of radioactivity appearing as low molecular weight degradation products increased with time. Acid gel permeation and reverse-phase HPLC of the binding protein-associated radioactivity demonstrated that the labelled IGF-I bound was intact IGF-I. Sephadex G-200 gel permeation chromatography of chicken plasma samples at pH 7·4 showed that the binding protein complex labelled in vivo with chicken IGF-I tracer had a molecular mass of 55 kDa. Furthermore, the tracer associated with the binding protein coeluted with the major peak of endogenous IGF-I, suggesting that the tracer was bound to the physiologically relevant binding protein. Similar results were obtained with Superose 12 chromatography. Human plasma chromatographed as expected with much of the immunoreactivity and the labelled IGF-I eluting with a mass of 150 kDa on both Sephadex G-200 and Superose 12 columns. Ligand blotting of plasma binding proteins with ¹²⁵I-labelled IGF-I after first subjecting the samples to polyacrylamide gel electrophoresis and transfer to nitrocellulose confirmed that the predominant binding protein in chicken plasma differed from the major forms in human plasma.

Journal of Endocrinology (1990) 124, 361–370

ABSTRACT

Chicken insulin-like growth factor-II (cIGF-II) has been characterized by amino acid sequencing, by its receptor and binding protein interactions and by its biological activities in cultured cells in order to help define the significance of the peptide in the growth process. Chicken IGF-II has an N-terminal region and several amino acid substitutions in the mid-peptide region that differ from the mammalian growth factor. Nevertheless, cIGF-II was indistinguishable from ovine IGF-II in all assay systems, including those involving chicken receptors and chicken binding proteins. Thus the amino acid substitutions did not modify the biological activities. In chick embryo fibroblasts, labelled bovine IGF-II or cIGF-II bound to a receptor with size and specificity properties expected for a type 1 IGF receptor, except that IGF-I competition for binding was less than IGF-II competition. No evidence for a type 2 receptor was obtained. The relatively lower biological activity of IGF-I compared with IGF-II in chick embryo fibroblasts contrasts with the much higher potency of IGF-I in rat L6 myoblasts. This difference can be explained by a combination of an inhibitory, IGF-II-specific binding protein produced only by the rat cells as well as the unusual receptor specificity of the chicken cells.

Journal of Endocrinology (1990) 124, 89–97

ABSTRACT

Porcine insulin-like growth factor-I (IGF-I) and IGF-II have been characterized to help define the roles of these peptides in the growth process. The amino acid sequence of porcine IGF-I was found to be identical to the human and bovine peptides. Porcine IGF-II was more similar to human IGF-II than to forms of this growth factor in other mammalian species, differing only in the replacement of asparagine for serine at residue 36. In a biological assay that measures the stimulation of protein synthesis in rat L6 myoblasts, porcine IGF-I was approximately ninefold more potent than porcine IGF-II or bovine IGF-II, while recombinant human IGF-I and IGF-II had half the potency of the respective natural peptides. Porcine and recombinant human IGF-I showed essentially equal competition for binding in a human IGF-I radioimmunoassay while between 0·6 and 1·5% cross-reactivity was observed with human, bovine or porcine IGF-II. A receptor assay for IGF-II demonstrated similar potencies for the three IGF-II peptides, while the cross-reactivity of recombinant human IGF-I was only 0·05%. Porcine IGF-I exhibited a higher cross-reactivity, presumably due to very slight contamination with IGF-II.

Journal of Endocrinology (1989) 122, 681–687

Abstract

Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80–100 kDa. In contrast, acid gelfiltration chromatography resolved two peaks of cell growth activity. A peak at 15–25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bioactivity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15–25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor β (TGF-β), and all inhibitory activity for Mv1Lu cells was immuno-neutralised by an antibody against TGF-β. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-β and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-β; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-β account for the 15–25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk.

Journal of Endocrinology (1997) 154, 45–55

Abstract

The effects of continuous 14 day infusion of recombinant human IGF-I (104 or 260 μg/day) or IGF-II (104, 260 or 650 μg/day) via s.c. implanted osmotic pumps were compared in young female rats in order to establish the relative efficacies of these two growth factors.

Significant increases in body weight gain and feed conversion efficiency were achieved by 260 of IGF-I or 650 μg/day of IGF-II. These treatments were associated with increased nitrogen retention and increases in the fractional weights of kidneys, spleen, total gut and individual gut regions. There was an increase in the size of villi and muscularis lining the jejunum, suggesting an increased absorptive capacity of the gut. However there was no significant change in the amount of faecal nitrogen excretion when expressed as a percentage of nitrogen intake. Interestingly, IGF-II was at least as potent as IGF-I in increasing the depth of jejunal crypts. Infusion of equivalent doses of either IGF-I or IGF-II resulted in similar increases in circulating concentrations of the respective peptides, though IGF-II infusion dosedependently decreased plasma IGF-I concentrations from those of the controls. Plasma IGF-binding protein levels were increased by both IGF-I and IGF-II treatments, though IGF-I elicited greater responses.

In summary, IGF-II can promote the growth of young female rats, although generally less potently than IGF-I.

Journal of Endocrinology (1995) 144, 91–98

Abstract

The metabolic clearance of chicken IGF-I (cIGF-I), cIGF-II, human IGF-I (hIGF-I), and hIGF-II was examined in the chicken using ¹²⁵I-labelled growth factors. Superose-12 chromatography of plasma collected at 7·5 min post-infusion revealed peaks of radioactivity corresponding to 150 and 43 kDa and unbound tracer. Statistical analysis of trichloroacetic acid (TCA)-precipitable radioactivity in sequential plasma samples as well as following chromatography of the same samples revealed that clearance of the radiolabelled peptides followed an apparent triphasic pattern. The close similarity of the individual chromatographically defined pools in their clearance rate compared with the three components described by TCA precipitation strongly suggested their identity. Both free ¹²⁵I-labelled cIGF-II (3·11 min) and hIGF-II (3·01 min) were cleared at a greater rate than their IGF-I counterparts. Unbound hIGF-I was cleared at a greater rate than cIGF-I (4·45 vs 5·66 min respectively). A similar pattern for clearance was evident in the radiolabelled growth factors associated with the 43 kDa component, although at a longer half-life. There was no difference in the apparent clearance of the radiolabelled growth factors associated with the 150 kDa component between IGF-I or -II or between species. Analysis of the chromatographic profiles of radioactive IGF-I peptides complexed to serum proteins versus those bound to labelled IGF-II peptides revealed the presence of a large molecular mass binding protein in vivo. Ligand blotting of chicken serum determined that a binding protein with a mass of 70 kDa was detectable with ¹²⁵I-IGF-II probes only, and was not present in pig serum. In addition, tissue uptake of ¹²⁵I-cIGF-I and -II was evaluated. Similar patterns of tissue distribution and uptake were observed for ¹²⁵I-cIGF-I and -II, except that cIGF-II uptake by the liver exceeded that of ¹²⁵I-cIGF-I at 15 min post-infusion. The rank order of tissue distribution was as follows: kidney > testis > heart > liver > pancreas > small intestine> cartilage > bursa > gizzard > leg muscle > breast muscle > brain. We conclude from these studies that the clearance of IGFs from the compartments identified in blood and the potential target tissues is dependent on their interactions with IGF-binding proteins and receptors.

Journal of Endocrinology (1996) 150, 149–160