Short-term infusion of LongR(3) insulin-like growth factor (IGF)-I decreases hepatic IGF-I mRNA but not IGF binding protein-3 mRNA expression in pigs.

Infusion of pigs with an insulin-like growth factor-I (IGF-I) analogue (LongR(3)IGF-I) that does not bind to IGF-binding proteins decreases growth rate and the plasma concentration of growth hormone (GH), IGF-I, IGFBP-3, and insulin. This study was designed to determine whether the decrease is due to changes in IGF-I and IGFBP-3 gene expression. IGF-I or LongR(3)IGF-I (180 microg/kg/day) was infused into 55-kg finisher pigs for 4 days using Travenol infuser pumps. Plasma IGF-I concentration was measured by radioimmunoassay and plasma IGFBP-3 and IGFBP-2 were estimated by Western ligand blotting. Steady-state levels of IGF-I and IGFBP-3 mRNA were measured by RNase protection assay. Neither IGF-I nor LongR(3)IGF-I had a significant effect on hepatic IGF-I class 1 mRNA expression, whereas hepatic IGF-I class 2 mRNA expression was significantly reduced by both peptides. Plasma IGFBP-3 levels were unaffected by IGF-I treatment but were reduced by LongR(3)IGF-I treatment. The decrease in IGFBP-3 was not due to decreased gene expression in porcine liver or kidney, since neither IGF-I nor LongR(3)IGF-I treatment altered IGFBP-3 mRNA. This study infers a direct effect of the IGF analogue LongR(3)IGF-I on GH through its inhibition of plasma IGF-I concentration and class 2 IGF-I mRNA. The decrease in plasma IGFBP-3 was not accompanied by a decrease in hepatic or renal IGFBP-3 mRNA, suggesting that in this case, plasma IGFBP-3 protein levels are posttranslationally regulated or are derived from tissues other than liver or kidney.

Measurement of the acid-labile subunit of the insulin-like growth factor binding protein complex in human serum: a comparison of four immunoassays.

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.

Transport of circulating IGF-I and LR3IGF-I from blood to extracellular wound fluid sites in rats.

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.

Transport of IGF-I across epithelial cell monolayers.

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.

The relationship between endogenous insulin-like growth factors and growth in pigs.

Previous studies have reported conflicting data on gender differences in plasma IGF-I in postnatal pigs. There is also debate over the role of IGF-II in regulation of postnatal growth. We have, therefore, determined the concentrations of plasma IGF-I, IGF-II, and IGF-binding protein-3 (IGFBP-3) in boars, barrows, and gilts and related these to postnatal growth characteristics. Plasma concentrations of IGF-I were higher in boars than in gilts or barrows from 13 wk. of age, and plasma IGF-II levels were generally higher in barrows than in boars or gilts. Plasma IGFBP-3 levels were higher in boars than in gilts or barrows at most ages. Between 15 and 23 wk. of age, IGF-I and IGFBP-3, but not IGF-II, were positively associated with growth rate, voluntary feed intake, and gain:feed ratio. Plasma IGF-II, but not IGF-I or IGFBP-3, was positively associated with backfat depth during this period. These results support the hypothesis that circulating IGF-I and IGF-II are regulators of lean and adipose tissue growth, respectively.

Effect of growth hormone administration on IGF binding protein-3 mRNA levels in porcine tissues.

The effect of short-term GH treatment on steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA levels in liver, kidney, longissimus dorsi muscle, stomach and jejunum was examined in pigs. Ten female crossbred pigs were allocated to either saline or GH (70 microg/kg/day) treatment by subcutaneous injection for 4 days. They were allowed to feed ad libitum, and were weighed daily. At the end of the treatment period, the pigs were slaughtered and samples of liver, kidney, skeletal muscle, stomach and jejunum were collected and total RNA was extracted. Steady-state levels of IGFBP-3 mRNA were quantified by RNase protection assay and were compared with the level of IGF-I class 1 and class 2 transcripts. IGFBP-3 mRNA increased in response to GH in both liver and kidney, but not in the other tissues sampled. Hepatic IGF-I mRNA responded to short-term GH treatment with a fourfold increase in IGF-I class 1 mRNA and an eightfold increase in IGF-I class 2 mRNA, which was liver specific. IGF-I class 1 mRNA was not responsive to GH treatment in other tissues. The short-term nature of this treatment suggests that the increase in hepatic IGFBP-3 and IGF-I transcripts is a relatively early response to treatment with GH, and that the increase in plasma concentrations of IGFBP-3 in response to GH are derived from the liver, the kidney, or both.

Moderate doses of porcine somatotropin do not increase plasma insulin-like growth factor-I (IGF-I) or IGF binding protein-3.

The growth rate of the young pig is generally much less than its potential and may be constrained by endocrine status as well as by nutrient intake. The aim of this study was to determine whether porcine somatotropin (pST) could increase growth in the nursing pig. Fourteen sows nursing litters of 6 (n = 7) or 12 (n = 7) piglets were utilized to establish a high and low plane of nutrition for sucking pigs. On Day 4 of lactation, the median two male pigs from each litter were randomly allocated to one of two doses of pST (0 or 60 micrograms/kg/d) until weaning on Day 31. Pigs were bled on Days 4, 13, 22, and 31 of lactation and the plasma was analyzed for insulin-like growth factor (IGF)-I, IGF-II, and IGF binding protein-3 (IGFBP-3). Pigs were weaned into conventional accommodation and further weighed on Days 63, 91, and 119. Pigs from litters of 6 grew more quickly and weighed 2.2 kg (P = 0.01) and 3.5 kg (P = 0.04) more than pigs from litters of 12 at 31 and 63 d of age, respectively. There was no effect of pST on preweaning growth of sucking pigs (261 vs. 258 g/d, P = 0.68), although growth rate increased in the final 3 d before weaning at 31 d (241 vs. 294 g/d, P = 0.01). IGFBP-3 was greater (1.09 vs. 0.78 micrograms/ml, P < 0.001), whereas IGF-I tended to be greater (206 vs. 176 ng/ml, P = 0.14), in pigs from the small litters. There was no effect of pST on plasma IGF-I (182 vs. 195 ng/ml, P = 0.454) or IGFBP-3 (0.93 vs. 0.94 microgram/ml, P = 0.85) concentrations. Plasma IGF-I and IGFBP-3 were highly correlated with the growth rate of nursing pigs (R = 0.638 and 0.756, respectively). There were no effects of pST (340 vs. 328 ng/ml, P = 0.48) or litter size (336 vs. 333 ng/ml, P = 0.88) on IGF-II. In conclusion, pST had no little or no effect on growth performance or plasma IGF-I, IGF-II, or IGFBP-3 in sucking pigs on either a high or low plane of nutrition.

Ontogenic and nutritional changes in circulating insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins in growing ewe and ram lambs.

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.

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.

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.

Long [R3] insulin-like growth factor-I reduces growth, plasma growth hormone, IGF binding protein-3 and endogenous IGF-I concentrations in pigs.

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.

IGF-I variants which bind poorly to IGF-binding proteins show more potent and prolonged hypoglycaemic action than native IGF-I in pigs and marmoset monkeys.

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.

Effects of chronic renal failure on plasma clearance of insulin-like growth factor I, des-(1-3)IGF-I, and LR3IGF-I.

Using a rat model of chronic renal failure (CRF), we examined insulin-like growth factor I (IGF-I) clearance, degradation, organ distribution, and IGF binding profiles in plasma. The effects of IGF-binding proteins (IGFBP) on IGF clearance and degradation in CRF were studied using the IGF-I analogues des-(1-3)IGF-I and LR3IGF-I, which bind poorly to IGFBP. Although total clearance of IGF-I was not significantly altered in CRF, half-life and area under the curve were increased in the rapid distribution phase and were reduced in the slow elimination phase. Total clearance of LR3IGF-I was significantly increased. Reduced binding of IGF-I in the 150-kDa complex and increased binding to smaller-molecular-weight IGFBP were observed in CRF. Increased degradation of both IGF-I and LR3IGF-I was associated with reduced IGF binding in the 150-kDa complex. The results suggest that the accumulation of lower-molecular-weight IGFBP with reduced IGF binding in the 150-kDa complex, associated with increased degradation of peptide, may explain, at least in part, the reduced bioactivity of IGF-I observed in CRF.

Plasma clearance and tissue distribution of labelled chicken and human IGF-I and IGF-II in the chicken.

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 radio-labelled 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.

Sexual dimorphism of circulating somatotropin, insulin-like growth factor I and II, insulin-like growth factor binding proteins, and insulin: relationships to growth rate and carcass characteristics in growing lambs.

The effects of sex and age on patterns of circulating somatotropin (ST) concentration and plasma IGF-I, IGF-II, insulin, and IGF binding protein-3 (IGFBP-3) were studied in ram, wether, and ewe lambs (n = 7 or 8) sampled at mean ages of 81 (I1) and 158 d (I2). Between 81 and 158 d of age, rams grew more rapidly than wethers (P < .01), and wethers grew more rapidly than ewes (P < .01). The sex differences in growth were reflected in empty body weight at slaughter: rams > wethers > ewes (P < .05). Mean plasma ST concentrations, ST pulse amplitude, and integrated plasma ST concentrations were greater (P < .05) in rams than in ewes at I1 and I2. Characteristics of the ST plasma profile in wethers were generally intermediate between those of rams and ewes. The interpulse interval was greater in ewes than in wethers at I2. The IGF-I and IGFBP-3 concentrations were greater in rams than in ewes at both sampling times. Plasma IGF-II was greater in ewes than in rams at I2. Mean plasma ST was approximately two thirds less at I2 than at I1 regardless of sex. Mean plasma ST and IGF-I at both ages were positively correlated with growth. Mean plasma ST at I2 was negatively correlated with fatness at slaughter. Sex and age significantly affected patterns of circulating ST and concentrations of IGF-I and IGFBP-3 in prepubertal growing lambs, under conditions for which growth rates and composition were also sexually dimorphic.

Pretreatment with bovine growth hormone is as effective as treatment during metabolic stress to reduce catabolism in fasted lambs.

The effects of recombinant bovine GH (rbGH) treatment on the insulin-like growth factor (IGF) axis and protein metabolism during fasting induced metabolic stress were evaluated in young lambs. To explore whether rbGH pretreatment alone might offer a degree of protection against nutritional stress, we compared the effects of rbGH given only before or during the fasting-induced metabolic stress with that given over the whole period. The animals were fed ad libitum for 5 days (well fed phase) and then fasted for 70 h (fasted phase). The rbGH was administered during either the well fed and the fasted phase (G-G), only during the well fed phase (G-S), or only during the fasted phase (S-G), and the effects were compared with those of saline treatment throughout both phases (S-S; n = 7/group). The rate of net protein catabolism, analyzed on the final day of the study, was reduced (P < 0.001) to a similar degree in all rbGH-treated groups compared with that in the S-S group. rbGH pretreatment was as effective as rbGH administered during the catabolic phase. Plasma IGF-I was increased (P < 0.001) in the well fed phase by rbGH treatment and decreased in the fasted phase in all groups. The rbGH treatment during the fasted phase resulted in a smaller fall in plasma IGF-I levels than saline treatment (P < 0.05, G-G vs G-S and S-G vs. S-S), but no difference was observed in the specific binding of [125I]ovine GH to the hepatic membranes from animals of the different groups. There was a negative correlation between net protein catabolism and plasma IGF-I levels (r = -0.48; P < 0.01) and specific binding of [125I]ovine GH to hepatic membranes (r = -0.56; P < 0.001). Plasma IGF-II levels were decreased by rbGH treatment during the well fed phase, but the responses to treatment during the fasted phase were variable, suggesting that plasma IGF-II is regulated in a different manner than plasma IGF-I. The fasting-induced fall (P < 0.05) in plasma concentrations of IGF-binding protein (IGFBP)-3 was reduced with rbGH treatment, and plasma concentrations IGFBP-2 were altered in an inverse manner. This study suggests that fasting-induced GH resistance can be alleviated by rbGH treatment independent of whether treatment is commenced before or after the onset of catabolic stress. Our observation of prolonged anticatabolic action of prophylactic rbGH treatment supports the proposal that prophylactic use of GH may reduce the degree of catabolism associated with subsequent interventions and, thus, improve clinical outcome.

Effect of restriction of placental growth on expression of IGFs in fetal sheep: relationship to fetal growth, circulating IGFs and binding proteins.

To determine whether tissue production of the IGFs is altered when fetal growth is retarded, IGF-I and -II mRNAs were measured in tissues of fetal sheep subjected to placental restriction and the relationships between IGF gene expression, circulating IGF protein and fetal growth were examined. The majority of potential placental attachment sites were surgically removed from the uterus of 12 non-pregnant ewes to restrict placental size in a subsequent pregnancy. Blood and tissues were collected at 121 days of gestation (term = 150) in 12 fetuses with restricted placental size and eight normal fetuses. IGF-I and IGF-II mRNA was detected by solution hybridization/ribonuclease protection assay in placenta and all fetal tissues studied. IGF-I mRNA was most abundant in skeletal muscle and liver and IGF-II mRNA was highest in kidney and lung. Restriction of placental size reduced fetal weight by 17% and reduced the pO2 (18%) and glucose concentration (23%) of fetal blood. Placental restriction also reduced IGF-I mRNA in fetal muscle (P < 0.002), lung (P < 0.05) and kidney (P < 0.01) but had no significant effect on IGF-II mRNA in any tissue. IGF-I mRNA in fetal liver, kidney and skeletal muscle correlated positively with the concentration of IGF-I protein in fetal blood (P < 0.01). There was no relationship between the concentration of IGF-II protein in fetal blood and IGF-II mRNA in any fetal tissue examined. The concentration of IGF-binding protein-3 (IGFBP-3) in fetal arterial blood plasma measured by RIA correlated positively with fetal weight and with plasma IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs) and tissue mRNA levels of IGFBP-2 and IGFBP-4 in the ovine fetus.

The IGF-binding proteins (IGFBPs) are a family of at least six structurally related proteins, which bind the IGFs and modulate their actions, including the regulation of pre- and postnatal growth. In this study we have examined the relationship between circulating and tissue mRNA levels of IGFBPs and related this to circulating IGFs in the fetal sheep over the gestational period when rapid growth and development occurs. Circulating IGFBP-2, as measured by Western ligand blot (WLB), increases between early and mid gestation, remains high, then declines throughout late gestation (P = 0.0002). Circulating IGFBP-3 increases throughout gestation, as measured by WLB or RIA (P = 0.04 and P = 0.0001 respectively), as does circulating IGFBP-4 (P = 0.004). These ontogenic changes in circulating IGFBPs-2 and -4 are paralleled by changes in liver mRNA for these proteins and, for IGFBP-2, by those in kidney IGFBP-2 mRNA also. This suggests that liver and kidney may be the primary contributors to circulating IGFBP-2 and the liver to circulating IGFBP-4, IGFBP-2 mRNA is present in the heart and lung in early gestation but barely detectable in these tissues after approximately 60 days gestation. IGFBP-4 mRNA is also present in the heart in early but not late gestation, but is abundant in the lung throughout gestation. These results demonstrate tissue specific and developmental regulation of IGFBPs-2 and -4 at the mRNA level. To assess any role the circulating IGFs may play in mediating these changes in IGFBPs, or vice versa, both plasma IGF-I and IGF-II were measured by RIA. Circulating IGF-I increases as gestation progresses (P = 0.0001), while circulating IGF-II increases between early and mid gestation, remains high (P = 0.01), then declines. Circulating IGF-I is positively correlated with fetal weight (r = 0.66, P = 0.03), circulating IGFBP-3 (r = 0.54, P = 0.01) and IGFBP-4 (r = 0.52, P = 0.01). Circulating IGF-II positively correlates with circulating IGFBP-2 (r = 0.48, P = 0.02) throughout gestation and at 1 day postnatally. These relationships are consistent with circulating IGF-I influencing IGFBPs-3 and -4, and similarly, IGF-II determining IGFBP-2, or vice versa. Alternatively, these correlations may reflect coordinate regulation of IGF and IGFBP by a common factor.

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.

Isolation and characterization of ovine IGFBP-4: protein purification and cDNA sequence.

Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7.5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino-terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3' non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 2.6 kb major transcript and two minor transcripts of approximately 2.1 and 1.8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver > kidney > lung >> heart and also in several fetal tissues, thus suggesting tissue-specific and developmental regulation. The availability of purified oIGFBP-4 and oIGFBP-3 as well as DNA probes for oIGFBP-4 will enable further study of the properties and functions of these proteins, as well as the establishment of specific assays for these IGFBPs.