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

The relative potency of IGF-I and the analogue LR³IGF-I to either promote growth or reverse catabolism in rats when administered by injection rather than by continuous infusion has been examined. LR³IGF-I has very low affinity for the IGF-binding proteins in the rat and hence is cleared from the circulation more quickly than is IGF-I. Experiments were performed in normal growing rats (150 g body weight) and in rats made catabolic by dexamethasone infusion (20 μg/day). IGFs or vehicle were delivered subcutaneously for 7 days either by continuous infusion via osmotic pumps or by injection once or twice daily at 320 and 400 μg/day in normal and catabolic rats respectively.

As expected, continuous infusion of IGFs showed greater efficacy than either of the injection modes especially in its anti-catabolic actions. When infused continuously LR³ IGF-I was generally 1·5- to 2-fold more potent than IGF-I for changes in body weight gain, visceral organ weights and feed use efficiency. Notably, LR³ IGF-I remained more potent than IGF-I in several of these effects even when the peptides were given by once-daily injection. In addition, N^τ-methylhistidine excretion by dexamethasone-treated rats was reduced to a threefold greater extent by injected LR³ IGF-I than by injected IGF-I. Notwithstanding these effects, LR³IGF-I was barely equipotent with IGF-I for reversal of carcass muscle loss in dexamethasone-treated rats.

Despite its more rapid clearance from the circulation, injected LR³IGF-I retains superior potency to injected IGF-I for several actions, albeit the potency is much reduced compared with continuous infusion. Thus our data indicate that use of IGF analogues which have low affinity for binding proteins may have advantages in potency and/or tissue specificity where IGFs are necessarily administered by injection.

Journal of Endocrinology (1996) 150, 77–84

ABSTRACT

The effects of insulin-like growth factor-I (IGF-I) on the gut of 150 g dexamethasone-treated rats were compared with those of two analogues with reduced affinity for IGF-binding proteins, des(1–3)IGF-I and LR³IGF-I, an N-terminal-extended variant. Administration of IGF-I for 7 days to rats made catabolic by co-treatment with dexamethasone induced a dose-dependent increase in total gut weight, with the highest dose of IGF-I (695 μg/day) increasing gut weight by up to 60%, and gut weight as a fraction of body weight by up to 32%. Effects were apparent in all regions of the gut examined, including the stomach, small intestine and colon. Histological and biochemical analyses of the intestine showed that cross-sectional mass, rather than gut length, was increased, and proportional increases in wet weight, protein and DNA content per unit length were measured in both the mucosa and muscularis layers. The rate of duodenal protein synthesis measured on day 7 of treatment was not increased by IGF-I treatment. The IGF-I analogues had qualitatively similar effects to IGF-I, but were consistently severalfold more potent, providing evidence that IGF-binding proteins reduce the biological activity of exogenous IGF-I in the gut. The results indicate that the gut is one of the most sensitive IGF-I target tissues, and that potency in vivo correlates with a reduced interaction with IGF-binding proteins.

Journal of Endocrinology (1992) 133, 421–431

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

We have tested whether an animal with substantial amounts of both IGF-I and IGF-II in circulation, such as the guinea pig, would respond to chronic IGF infusion in the same manner as the adult rat, which has negligible amounts of IGF-II in blood. Female guinea pigs of 350 g body weight were continuously infused for 7 days with recombinant guinea pig IGF-I or -II (120 or 360 μg/day) or long R³ IGF-I (LR³IGF-I) (120 μg/day), an analogue which has much reduced affinities for IGF binding proteins. IGF-I or IGF-II infusion led to substantial increases in plasma IGF-I or IGF-II respectively in comparison with vehicle-infused animals. Nevertheless, body weight gain, feed intake, feed conversion efficiency and carcass composition were not significantly affected by any treatment (significance was deemed to be P<0·05). Amongst the tissues examined only the fractional weight (g/kg body weight) of the adrenals was increased, and that only by the higher dose (360 μg/day) of IGF-I. However, the fractional weight of adrenals, gut, kidneys and spleen were significantly increased by LR³IGF-I, but again overall growth was not stimulated. A possible explanation for the lack of IGF-I effects is that total circulating IGF concentrations were not increased by these treatments. IGF-II significantly raised total IGF concentrations at the higher dose only. Plasma IGF-I was reduced by IGF-II infusion, as was plasma IGF-II by IGF-I infusion. LR³IGF-I treatment lowered both plasma IGF-I and IGF-II concentrations, a response probably related to a reduction in total plasma IGF binding protein (IGFBP), especially IGFBP-3, concentrations. We conclude that although the guinea pig is responsive to IGF treatment, the effects differ markedly from those elicited in rats.

Journal of Endocrinology (1995) 146, 247–253

ABSTRACT

The ability of insulin-like growth factor-I (IGF-I) to protect against losses of body protein during periods of dietary nitrogen restriction has been evaluated in young rats. Recombinant human IGF-I was administered by osmotic pumps at dose rates of 0, 1·2 or 2·9 mg/kg per day over a 7-day period beginning with the transfer of animals from an 18% to a 4% protein diet. A fourth group received the potent truncated IGF-I analogue, des(1–3)IGF-I, at a dose of 1·2 mg/kg per day over a comparable 7-day period. Plasma IGF-I levels were reduced by 60% following nitrogen restriction, a reduction that was partly prevented by IGF-I administration, especially at the higher dose, but not measurably by des(1–3)IGF-I. The major IGF-binding protein circulating in blood, IGFBP-3, demonstrated a similar pattern of change.

A significant (P<0·05) protection of body weight was achieved in the low dose IGF-I and des(1–3)IGF-I groups, but only after differences in food intake had been eliminated by analysis of covariance. Nitrogen balances were not significantly different unless analysis of covariance was used to adjust for the nitrogen intakes, whereupon all treatment groups showed improved balance, especially the animals treated with the low IGF-I dose and des(1–3)IGF-I (both P<0·01). The rate of muscle protein breakdown calculated from the urinary excretion of 3-methylhistidine was not significantly altered by the treatments, but fell progressively throughout the 7 days. The fractional rate of muscle protein synthesis measured on the final day was increased by 31, 26 and 21% respectively by the low and high doses of IGF-I and by des(1–3)IGF-I. Organ weights (g/kg body weight) showed no effects of IGF-I treatment except for 16% increases in the weight of kidneys in the high dose IGF-I and the des(1–3)IGF-I groups. Carcass analyses demonstrated higher water and lower fat contents (all P< 0·01) in the same groups. These results suggest that exogenous IGF-I and especially des(1–3)IGF-I can partly protect body protein reserves during nitrogen restriction.

Journal of Endocrinology (1991) 128, 97–105

Abstract

We have examined the influence of nutrition on plasma IGF-I, IGF-II and IGF-binding protein (IGFBP) levels and on hepatic IGF-I gene expression in young meat-type chickens. Plasma IGF concentrations were measured by using RIA with recombinant chicken IGFs as standards.

In chickens fed the control diet containing 200 g/kg dietary protein ad libitum for 7 days, plasma IGF-I concentrations increased significantly from those found in the initial control group. Food restriction for either 4 or 7 days decreased plasma IGF-I by 30% from the initial control. When chickens were refed ad libitum for 3 days after 4 days of restricted feeding, plasma IGF-I levels recovered to those of the control birds fed ad libitum. In chickens eating a low protein diet (100 g/kg protein), the plasma IGF-I tended to be lowered but the decrease was not significant. Although the intensity of IGF-I and β-actin mRNA bands protected in the RNase protection assay was changed by nutrition, no statistical effect of nutrition on the ratio of IGF-I to β-actin was observed. The nutritional treatments had no effect on plasma IGF-II concentrations.

Western ligand blot and chromatographic analyses were used to investigate the influence of nutrition on IGFBP profiles. Both IGF-I and IGF-II ligands in the Western ligand blot revealed the most intense binding at 30 kDa for plasma obtained from chickens with restricted food intake. The 30 kDa band also appeared at a lower intensity in the group fed a low protein diet but not in any other groups. These observations were confirmed by neutral gel chromatography. The chicken IGF-II ligand revealed an intensely labelled band corresponding to 75 kDa and this was not affected by nutrition.

IGF-I and IGFBP concentrations in the plasma of young broiler chickens were influenced by nutritional state but IGF-II concentrations were not. The lack of a response in circulating IGF-II levels may have been due to the presence of high concentrations of a 75 kDa specific binding protein which did not respond to nutrition in this experiment.

Journal of Endocrinology (1996) 149, 181–190

ABSTRACT

Administration of IGF-I over a 14-day period to growing female rats via s.c. implanted osmotic pumps led to an increased body weight gain, an improved N retention and a greater food conversion efficiency. The effects were dose-dependent, with the highest daily dose tested, 278 μg/day, producing 18–26% increases in these measurements. LR³IGF-I, a variant of human IGF-I that contains an amino terminal extension peptide as well as glutamate-3 replaced by arginine and exhibits very weak binding to IGF-binding proteins, was substantially more potent than the natural growth factor, in that 44 μg/day of this peptide produced similar effects to the high IGF-I dose. Organ weight and carcass composition measurements showed that the two IGF peptides generally maintained body proportions at those existing when the experiment began. Muscle protein synthesis and myofibrillar protein breakdown were both slightly increased by IGF treatment, so that the observed improvement in N retention could not be explained through protein accretion rates calculated from these measures. Infusion of human GH at a dose of 213 μg/day did not stimulate body growth. This investigation establishes that IGF peptides stimulate the growth of normal growing animals, with IGF-I variants that bind less well to IGF-binding proteins being more active than IGF-I.

Journal of Endocrinology (1993) 137, 413–421