Production, in vitro characterisation, in vivo clearance, and tissue localisation of recombinant barramundi (Lates calcarifer) insulin-like growth factor II.

Recombinant barramundi insulin-like growth-factor-II (bIGF-II) has been produced in Escherichia coli after modification of an expression plasmid that coded for a chicken IGF-II fusion protein. The bIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, and refolded. The protein was then released from the fusion protein by cleavage with subtilisin BPN'. Finally the protein was purified to homogeneity with a number of HPLC steps. In vitro analysis of recombinant bIGF-II demonstrated decreased potency in stimulating protein synthesis when compared to human and barramundi IGF-I (bIGF-I). The in vivo distribution of radiolabeled bIGF-II and bIGF-I in the circulation and tissue uptake of radiolabeled bIGF-II was also compared in juvenile barramundi (Lates calcarifer). Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that bIGF-II was degraded faster than bIGF-I. Moreover, neutral gel chromatography of these samples suggested this difference may be due to reduced affinity of bIGF-II, compared to blGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi circulation. Based on these results, it would appear that elements important in the function of IGFs have been well conserved during vertebrate evolution. However, to clearly define the IGF system in fish it will be necessary to characterise the IGFBPs present and to determine how they influence the biological actions of native IGFs.

IGFs stimulate zebrafish cell proliferation by activating MAP kinase and PI3-kinase-signaling pathways.

Insulin-like growth factor (IGF)-I and -II have been cloned from a number of teleost species, but their cellular actions in fish are poorly defined. In this study, we show that both IGF-I and -II stimulated zebrafish embryonic cell proliferation and DNA synthesis in a concentration-dependent manner, whereas insulin had little mitogenic activity. Affinity cross-linking and immunoblotting studies revealed the presence of IGF receptors with the characteristics of the mammalian type I IGF receptor. Competitive binding assay results indicated that the binding affinities of the zebrafish IGF-I receptors to IGF-I, IGF-II, and insulin are 1.9, 2.6, and >190 nM, indicating that IGF-I and -II bind to the IGF-I receptor(s) with approximately equal high affinity. To further investigate the cellular mechanism of IGF actions, we have studied the effects of IGFs on two major signal transduction pathways: mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase). IGFs activated MAPK in zebrafish embryonic cells in a dose-dependent manner. This activation occurred within 5 min of IGF-I stimulation and disappeared after 1 h. IGF-I also caused a concentration-dependent activation of protein kinase B, a downstream target of PI3 kinase, this activation being sustained for several hours. Inhibition of MAPK activation by the MAPK kinase inhibitor PD-98059 inhibited the IGF-I-stimulated DNA synthesis. Similarly, use of the PI3 kinase inhibitor LY-294002 also inhibited IGF-I-stimulated DNA synthesis. When both the MAPK and PI3 kinase pathways were inhibited using a combination of these compounds, the IGF-I-stimulated DNA synthesis was completely negated. These results indicate that both IGF-I and -II are potent mitogens for zebrafish embryonic cells and that activation of both the MAPK and PI3 kinase-signaling pathways is required for the mitogenic action of IGFs in zebrafish embryonic cells.

Comparison of recombinant barramundi and human insulin-like growth factor (IGF)-I in juvenile barramundi (Lates calcarifer): in vivo metabolic effects, association with circulating IGF-binding proteins, and tissue localisation.

The in vivo actions of human and fish insulin-like growth factor (IGF)-I have been compared to extend the understanding of the metabolism of IGFs in fish and to identify potential differences in their actions. The effects of acute administration of these proteins on the incorporation of glucose into muscle glycogen and leucine into liver protein in juvenile barramundi were investigated. In these in vivo metabolic assays, both baramundi IGF-I (bIGF-I) and human IGF-I (hIGF-I) increase the incorporation of D-[14C]glucose into muscle glycogen and [14C]leucine into liver protein. The distribution of radio-labeled human and barramundi IGF-I in the circulation and their uptake by tissue was also compared in juvenile barramundi (Lates calcarifer). Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that hIGF-I was degraded faster than bIGF-I. Neutral gel chromatography of these samples suggested that this difference is due to reduced affinity of hIGF-I, compared to bIGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi. Tissue uptake of [125I]-labeled hIGF-I and bIGF-I was similar except that [ 125I]bIGF-I uptake by the kidney exceeded that of hIGF-I. It is suggested that while some of the in vivo actions of IGFs in fish are conserved, functional differences between mammalian and teleostean IGFs exist, particularly with respect to their interactions with fish IGFBPs.

Evolution of insulin-like growth factor-I (IGF-I) action: in vitro characterization of vertebrate IGF-I proteins.

While there is considerable structural evidence that IGFs share a long evolutionary history, less is known about the conservation of IGF action. These studies have primarily been hampered by the small amounts of purified IGFs that have been available for testing. More recently, however, we have adopted recombinant strategies to produce milligram quantities of IGFs for biological studies. Thus we have been able to compare the properties of rat, kangaroo, chicken, salmon and barramundi IGF-I, proteins that differ from human IGF-I by 3, 6, 8, 14 and 16 amino acids respectively. While we have found that the IGF-I proteins exhibit similar biological activities and type-I IGF receptor binding affinities, regardless of whether mammalian, avian or piscine cell lines are used, there was a trend suggesting that the fish proteins at least, were most effective in studies using homologous systems. Thus, salmon IGF-I was not as potent as human IGF-I in bioassays in mammalian cells, but was as effective as human IGF-I in piscine cells. As expected, the IGF-I proteins competed poorly for binding to type-2 receptors present on ovine placental membranes. Interestingly however, the two fish IGF-I proteins exhibited greater affinity for this receptor than the other IGF-I proteins, hence reminiscent of the results previously found with recombinant hagfish IGF. Despite these small differences, these results taken together indicate that the IGF-I proteins appear to have been remarkably conserved in both structure and in vitro action during vertebrate radiation.