P E Walton

In vivo actions of IGF analogues with poor affinities for IGFBPs: Metabolic and growth effects in pigs of different ages and GH responsiveness

IGF-I analogues that bind poorly to IGFBPs are substantially more potent than IGF-I at stimulating growth in rats. However, rodents differ from other mammals because they contain only minimal circulating levels of IGF-II and they are poorly responsive to GH. In this report we review a series of experiments carried out in pigs, a species that is both GH responsive and has high blood concentrations of IGF-II. Intravenous bolus administration of IGFs to 55 kg pigs depressed blood glucose with the potency greatest for analogues such as des (1-3) IGF-I, R3IGF-I and Long R3IGF-I that showed the weakest binding to pig IGFBP-3, a similar efficacy pattern to that reported in the rat. Chronic subcutaneous administration of Long R3IGF-I, however, reduced growth rates, led to a depression in food intake and lowered concentrations of IGF-I, IGF-II and IGFBP-3. IGF-I itself depressed IGF-II concentrations and did not stimulate growth. Subcutaneous infusion of IGFs over a 3-day period, also in 55 kg pigs, demonstrated that analogues that bound least well to IGFBP-3 were the most effective at reducing the concentration of this binding protein, suggesting that the inhibition of growth was related to the depression of IGFBP-3. On the other hand, IGF-I and Long R3IGF-I increased growth rats in neonatal pigs, especially under conditions of reduced food intake. As these anabolic effects occur at a developmental stage where the animals are insensitive to GH in a manner analogous to the situation in rats, it is plausible that the feed-back inhibition of GH secretion explains the catabolic response to IGFs in older pigs.

Paracellular transport of insulin‐like growth factor‐I (IGF‐I) across human umbilical vein endothelial cell monolayers

Insulin‐like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF‐IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF‐I and IGF‐II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio‐receptor assays using both IGF‐I and IGF‐II and (ii) affinity label cross‐linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP‐2, ‐3, and ‐4 are secreted into serum‐free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF‐I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of 125I‐IGF‐I and [3H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of 125I‐IGF‐I across HUVE cell monolayers was not significantly different to that of [3H]inulin, a paracellular probe. Moreover, 125I‐IGF‐I transport was not inhibited by either excess unlabelled IGF‐I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit 125I‐IGF‐I binding to HUVE cell monolayers. Our findings show that the movement of free IGF‐I across HUVE cell monolayers occurs via a paracellular route and not by a receptor‐mediated, transcellular pathway. J. Cell. Physiol. 170:290–298, 1997.

Growth hormone but not insulin-like growth factor-I improves wound strength in pigs.

Systemic growth hormone and locally administered insulin‐like growth factor‐I have been shown in a number of studies to improve the breaking strength of incisional wounds, especially in compromised animals. The objective of the present study was to compare these two agents when administered subcutaneously distant from an incisional wound site in pigs, as well as to examine effects of a combination growth hormone/insulin‐like growth factor treatment. Growth hormone was shown to increase wound breaking strength in two experiments, whereas insulin‐like growth factor‐I or a more potent analog had no effect. Moreover, breaking strength was only minimally improved above the vehicle groups by the combination of growth hormone and insulin‐like growth factor‐I. These effects could not be explained by changes in plasma insulin‐like growth factor‐I concentrations which were highest in the combination groups, nor by plasma insulin‐like growth factor binding protein‐3 which was raised equally whenever growth hormone was administered. We conclude that systemic growth hormone but not insulin‐like growth factor‐I improves wound strength in normal pigs, whereas insulin‐like growth factor‐I reduces the magnitude of the growth hormone effect by an unknown mechanism.

Development of Unique Growth Factors for Serum-Free Cell Culture using Fusion Partner Technology

Using fusion partner technology in an E. coli expression system designed to give selective attributes, we have produced novel recombinant growth factor analogs specifically for serum-free culture. Long EGF, a human EGF with a 53 amino acid N-terminal fusion partner, and LR3IGF-I, a human IGF-I with an arginine in place of glutamate at position 3 and a 13-residue N-terminal fusion partner, have been developed and tested in serum-free cell culture. Advantages of this approach are a substantially higher yield and improved economy of recombinant peptide production compared to making the native growth factors, and significantly higher potency in cell culture, particularly in the case of LR3IGF-I. Combinations of these growth factor analogs are potently synergistic and provide an important tool for formulating serum-free media.