Significant levels of IGF-I are found in wound fluid. The contribution that systemic IGF-I makes to the total IGF-I pool in wounds and the influence of IGF binding proteins (IGFBPs) on the delivery of systemic IGF-I to these wound sites has not been established. In the present series of experiments, we have shown that IGF-I flux across a model endothelial cell barrier is decreased in the presence of IGFBPs, whereas flux of Long R(3)IGF-I (LR(3)IGF-I, an IGF-I analogue with low affinity for IGFBPs) is unaffected. On the basis of these findings, the transport of IGF-I and LR(3)IGF-I from blood to extracellular wound fluid was assessed. Wound chambers were implanted subcutaneously in the backs of adult male rats and left in place for 14 days. A single i.v. bolus of either (125)I-IGF-I or (125)I-LR(3)IGF-I (10x10(6) c.p.m.) was administered via a jugular catheter and wound fluid and plasma samples taken at sequential time points between 5 and 240 min. (125)I-LR(3)IGF-I was removed from the circulation more rapidly than (125)I-IGF-I in both sham control and chamber implanted rats. Although implantation of the chambers did not alter the pharmacokinetic parameters of (125)I-IGF-I, significant increases in the steady state volume of distribution, clearance rate and half-life were recorded for (125)I-LR(3)IGF-I. In addition, significantly more intact (125)I-LR(3)IGF-I was recovered in wound fluid than (125)I-IGF-I at each time point, although only 0.08% of administered (125)I-LR(3)IGF-I was recovered per ml of wound fluid at 240 min. Compared with plasma, a greater proportion of wound fluid IGF-I radioactivity had distributed to the lower molecular weight IGFBPs or existed as free peptide. However, a small amount of wound fluid (125)I-IGF-I was detected in the 150 kDa region 30 min after injection. A greater proportion of (125)I-LR(3)IGF-I was associated with the lower molecular weight IGFBPs or existed as free peptide in both wound fluid and plasma. These data point to the importance of IGFBPs in determining the pharmacokinetic parameters of IGF-I in an extracellular fluid-expanded state. They also suggest only a minor role for endocrine IGF-I in surface wound repair.
SE Bastian, PE Walton and DA Belford
SE Bastian, PE Walton, FJ Ballard and DA Belford
Epithelial cells line the lumens of organs including the gastrointestinal tract, kidney tubules and respiratory airways, where they regulate the transport of electrolytes and the movement of macromolecules. The current study aimed to investigate the transport of IGF-I across epithelial cell barriers. Epithelial cell lines derived from gut (IEC-6), kidney (MDBK) and lung (Mv1Lu) were shown to possess high-affinity, functional receptors for IGF-I and formed tight junctions in monolayer culture. To investigate the transport of IGF-I, the three cell lines were grown on microporous filters in a bi-chamber system. In comparison with filters without cells, IEC-6 and Mv1Lu epithelial cell monolayers restricted the passage of (125)I-IGF-I and [(3)H]inulin, whereas the MDBK cells virtually occluded any passage of these molecules. Transport of (125)I-IGF-I across the epithelial cell monolayers was significantly less than that of [(3)H]inulin, suggesting that the binding of (125)I-IGF-I to high-affinity IGF receptors or IGF-binding proteins retarded its transport. Moreover, (125)I-IGF-I transport was not inhibited by the presence of excess unlabelled IGF-I. Our findings provide evidence for the restricted diffusion of intact, free IGF-I across gut, kidney and lung epithelial cell monolayers via a paracellular or low-affinity transcellular pathway.
V Dunaiski, FR Dunshea, PE Walton and C Goddard
Growth hormone (GH) improves growth performance in the pig. Analogues of insulin-like growth factor-I (IGF-I) that bind poorly to IGF binding proteins (IGFBP) stimulate growth in the rat but, in contrast, inhibit growth in the pig. This study was designed to determine the effect of IGF peptides alone or in combination with porcine GH (pGH) on growth characteristics and plasma hormone concentrations in finisher pigs. A four-day infusion of Long [R3] IGF-I (LR3IGF-I; 180 micrograms/kg/day) decreased the average daily gain, food intake, and plasma IGFBP-3, IGF-I and insulin concentrations. The mean plasma GH concentration was decreased by 23% and the area under the GH peaks was reduced by 60%. Co-administration of pGH (30 micrograms/kg/day) with LR3IGF-I had no interactive effect on growth performance, and plasma insulin, IGFBP-3 and IGF-I concentrations remained suppressed. The area under the GH peaks was not restored with this combination treatment although mean plasma GH concentrations were elevated in all animals receiving pGH. Infusion of IGF-I (180 micrograms/kg/day) decreased plasma insulin and mean GH concentrations but had no significant effect on IGFBP-3 concentrations. Average daily gain and feed intake were not changed by IGF-I treatment. A combination of IGF-I and pGH injection (30 micrograms/kg/day) increased plasma IGFBP-3 concentrations but plasma insulin levels remained suppressed. Plasma glucose levels were unaffected by any treatment. The study demonstrates that both IGF-I and LR3IGF-I suppress plasma GH concentrations in finisher pigs. This, in turn, may be responsible for the reduction in the plasma concentration of IGF-I, IGFBP-3 and insulin seen in LR3IGF-I-treated animals. The decrease in these parameters may contribute to the inhibitory effect of LR3IGF-I on growth performance in the pig.
FM Tomas, PE Walton, FR Dunshea and FJ Ballard
The relative acute hypoglycaemic potencies of IGF-I and several variants of IGF-I which bind poorly to the IGF-I binding proteins (IGFBPs) have been examined in marmosets (Callithrix jacchus) and the pig. In the marmoset study, IGF-I and des(1-3)IGF-I were compared in anaesthetised and conscious animals in a range of bolus doses from 42 to 270 micrograms/kg body weight. In the pig study, IGF-I was compared with four variants, des(1-3)IGF-I long-IGF-I, R3IGF-I and long-R3IGF-I (LR3IGF-I), which show reduced affinity for the IGFBPs as well as with insulin. Doses in the pig were 20 and 50 micrograms/kg body weight for the IGFs and 3 micrograms/kg for insulin. In each study serial blood samples were taken from 30 min before to 4 h after the bolus injection. Plasma glucose levels were decreased in a dose-responsive manner with the pig more sensitive than either the conscious or anaesthetised marmoset (maximum lowering 4.8, 3.7 and 2.5 mmol/l respectively). The IGF variants were consistently 2- to 3-fold more potent than IGF-I in each animal for lowering of plasma glucose to the nadir, with the potency reflecting the relative affinities for binding to the IGFBPs and the IGF-I receptors. Thus, hypoglycaemic potency was in the order IGF-I < long-IGF-I < R3IGF-I approximately LR3IGF-I < des (1-3)IGF-I. Notably the variants suppressed plasma glucose levels over a much longer period than did IGF-I, the cumulative suppression over four hours showing an approximately 4- to 8-fold increase in the extent of hypoglycaemia. The prolonged suppression was not simply proportional to the hypoglycaemic nadir; at doses equipotent for glucose lowering, the cumulative hypoglycaemic effect for the variants in either species was about 2-fold that for IGF-I. The differential effect of the variants in the marmoset could not be accounted for by correlated changes in plasma insulin, IGF-I or IGFBP levels in plasma. Indirect effects via inhibition of glucagon, or direct effects via hepatic insulin receptors are postulated to account for the results. There was a dose-related reduction in plasma amino acids in the pig but, unlike the case for plasma glucose, only one analogue, LR3IGF-I was more potent than IGF-I. The response to LR3IGF-I was accentuated at the high dosage but on the basis of the other variants tested this effect could not be ascribed to either of the incorporated molecular variations. Despite their more rapid clearance from the circulation, variants of IGF-I which show lower affinity for binding to IGFBPs show proportionately superior potency for sustained hypoglycaemic action. Since our data were obtained in animal models of accepted relevance to humans these results point to the possible superior efficacy of the variants, especially des(1-3)IGF-I, over IGF-I for use as an adjunct to insulin treatment of hyperglycaemic conditions.
KL Gatford, KJ Quinn, PE Walton, PA Grant, BJ Hosking, AR Egan and PC Owens
The ontogeny of the IGF endocrine system was investigated in 15 young lambs before and after weaning at 62 days of age. Before weaning, plasma IGF-I concentrations were higher in rams than ewes, and plasma concentrations of IGF-II and IGF-binding protein-3 (IGFBP-3) also tended to be higher in rams than in ewes. Feed intake of ewes and rams was restricted after weaning to remove sex differences in feed intake. Plasma concentrations of IGF-I and IGFBP-3 did not differ between rams and ewes at 100 days of age, but plasma IGF-II was higher in rams than in ewes at this time. Since circulating concentrations of GH were higher in rams than in ewes at 100 days of age, this implies that the restricted feed intake blocked the IGF-I and IGFBP-3 responses to GH. We conclude that sex differences in circulating IGF-I and IGFBP-3 concentrations in the growing lamb alter with age, and are not present when nutrition is restricted.
RC Baxter, M Svejkar, MJ Khosravi, GL Bennett, KV Hardman, A Senese, J Mistry, PE Walton and V Quarmby
The acid-labile subunit (ALS) of the high molecular weight insulin-like growth factor binding protein complex is a liver-derived glycoprotein which is regulated by growth hormone and serves as a serum marker of growth hormone action. We have compared the measurement of ALS by four immunoassay methods (two RIAs, two ELISAs) utilizing various polyclonal and monoclonal antibodies raised against natural or recombinant human ALS, or synthetic ALS peptides. Despite the variety of methodologies and reagents, results obtained by the four methods were highly correlated for 125 sera from various patient groups, and when compared for individual groups of sera from healthy children and adults, growth hormone-deficient children and adults, and subjects with acromegaly. Some weaker correlations among methods were seen when measuring ALS levels in groups of sera from pregnant subjects and subjects with chronic renal failure. An assay using antibodies raised against recombinant ALS yielded lower apparent values than the other methods in patient sera, the discrepancy probably being attributable to a difference in standardization. We conclude that a variety of assay formats and reagents can yield serum ALS values of potential clinical utility.