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C J Xian, C A Shoubridge, and L C Read

Abstract

To investigate the potential of IGF-I peptides as therapeutics in the gut, the survival profiles of a bolus of 125I-labelled IGF-I (8·6 ng) in vivo in various ligated gut segments of fasted adult rats have been examined. The intactness of IGF-I tracer in the flushed luminal contents was estimated by trichloroacetic acid precipitation, antibody and receptor binding assays. It was found that IGF-I was degraded very rapidly in duodenum and ileum segments with a half-life (t 1/2) of 2 min by all three methods. IGF-I was slightly more stable in the stomach (t 1/2=8, 5 and 2·5 min by the above three methods), and considerably more stable in the colon (t 1/2=38, 33 and 16 min as judged by the three methods). Rates of degradation in gut flushings in vitro were similar to the in vivo rates except for the colon, where IGF-I was proteolysed more rapidly in vivo.

As a means of developing gut-stable and active forms of IGF-I, several approaches were examined for their effectiveness in prolonging IGF-I survival in the upper gut. It was found that the extension peptide on the analogue, LR3IGF-I did not protect IGF-I, nor did association with IGF-binding protein-3. However, an IGF-I antiserum was effective in prolonging IGF-I half-life in duodenum fluid by 28-fold. Charge interaction between IGF-I and heparin could also protect IGF-I in the stomach but not in duodenum flushings. Furthermore, casein (a non-specific dietary protein) and to a lesser extent, BSA and lactoferrin, were effective in preserving IGF-I structural integrity and receptor binding activity in both stomach and duodenum fluids. It can be concluded that IGF-I cannot be expected to retain bioactivity if delivered orally because of rapid proteolysis in the upper gut, but the use of IGF antibodies and casein could represent useful approaches for IGF-I protection in oral formulae.

Journal of Endocrinology (1995) 146, 215–225

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A A Martin, C M Gillespie, L Moore, F J Ballard, and L C Read

Abstract

The effect of insulin-like growth factor-I (IGF-I) administration on body weight gain and the rate of recovery of renal function was investigated in rats following an acute episode of renal ischaemia. Since the des(1–3)IGF-I and LR3IGF-I variant forms of IGF-I have been shown to be more potent than IGF-I, their effects were also examined. Acute renal failure was produced in male Sprague–Dawley rats by clamping both renal arteries for 45 min. Treatment was commenced at the time of renal artery occlusion with vehicle (0·1 mol acetic acid/l; control group), IGF-I (2·0 mg/kg per day), des(1–3)IGF-I (2·0 mg/kg per day) or LR3IGF-I (1·5 mg/kg per day) by s.c. osmotic pump, and continued for 7 days, with rats being held in metabolism cages. Glomerular filtration rate (GFR) was estimated by the use of 51Cr-EDTA continuously infused i.p. via osmotic pump. Following the episode of renal ischaemia, body weight gain and nitrogen retention were significantly improved in all three peptide-treated groups, and serum urea concentrations were reduced in the groups treated with IGF-I and des(1–3)IGF-I. However, there was no evidence of the variants having any increased potency over the growth effects of IGF-I itself. GFR was significantly reduced, urine output was increased and urinary concentrating ability was reduced in all groups compared with normal rats, with no significant effect of the IGF peptides being apparent.

A closer examination of the acute effects of LR3IGF-I on renal function was undertaken by measuring GFR for 3 days before and 3 days after renal ischaemia in two groups of rats, treated for the latter 3 days with either vehicle (controls) or LR3IGF-I (1·5 mg/kg per day). LR3IGF-I treatment following renal ischaemia resulted in a significantly greater fall in GFR than in controls, urinary osmolality was also significantly reduced, and fractional excretion of sodium was increased. In addition, there was histological evidence of a greater degree of tubular epithelial calcification in the kidneys of the rats treated with LR3IGF-I.

This study showed that administration of IGF peptides at doses sufficient to cause significant improvement in anabolic status did not improve renal function in rats following an acute episode of renal ischaemia. Indeed the LR3IGF-I variant of IGF-I had a deleterious effect on renal function in the early stage of the recovery period.

Journal of Endocrinology (1994) 140, 23–32

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C. Gillespie, L. C. Read, C. J. Bagley, and F. J. Ballard

ABSTRACT

The relative potencies of insulin-like growth factor (IGF-I) and the N-terminal truncated derivative, des(1–3)IGF-I, have been compared in lit/lit mice. Injection of 30 μg IGF-I, 30 μg des(1–3)IGF-I or 3 μg des(1–3)IGF-I daily for 3 weeks increased total length and nose-rump length of the animals substantially more than in controls or animals treated with 3 μg IGF-I daily.

Body weight changes were not statistically significant. The lower dose of des(1–3)IGF-I, but not that of IGF-I, led to increases in kidney and heart weights relative to controls, while the higher dose of either IGF-I or des(1–3)IGF-I also increased the weights of liver, lungs and stomach. These results indicate that the higher potency of des(1–3)IGF-I demonstrated in cultured cells also applies in vivo to at least one strain of GH-deficient animals.

Journal of Endocrinology (1990) 127, 401–405

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L. C. Read, L. Summer, S. M. Gale, C. George-Nascimento, F. J. Ballard, and J. C. Wallace

ABSTRACT

The receptor-binding, immunological and biological properties of synthetic-gene human epidermal growth factor (EGF), produced by recombinant techniques in yeast have been compared with those of mouse submaxillary gland EGF and partially purified EGF from human urine and milk. The three forms of human EGF produced parallel concentration curves in radioreceptor assays using AG2804 Simian virus 40-transformed human lung fibroblasts and iodinated mouse or recombinant human EGF. Equivalent receptor-binding activities of urine and milk EGF were equipotent with recombinant human EGF in an homologous radioimmunoassay using recombinant human EGF antiserum with 125I-labelled recombinant human EGF, while none of the preparations was effective in competing for binding of 125I-labelled mouse EGF to mouse EGF antiserum. Urine, milk and recombinant human EGF stimulated protein synthesis and inhibited protein degradation in cultured AG2804 fibroblasts with identical potency. On a weight basis, mouse EGF was equipotent with recombinant human EGF in competitive binding to cell receptors and in effects on protein synthesis or protein degradation in AG2804 fibroblasts. It is concluded that recombinant human EGF is indistinguishable from the natural growth factor from urine or milk and shares similar biological properties with mouse EGF.

J. Endocr. (1986) 109, 245–250

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A P D Lord, A A Martin, F J Ballard, and L C Read

Abstract

The net transfer of 125I-labelled insulin-like growth factor (IGF)-I from the blood to the distal small intestine was measured in anaesthetized lambs using a non-recirculating vascular-perfused intestine. To determine whether IGF-binding proteins (IGFBPs) reduce net IGF transfer, radio-labelled IGF-I was compared with two analogues, des(1–3)IGF-I and LR3IGF-I, which show reduced affinity for IGFBPs. Radiolabelled IGF-I, des(1–3)IGF-I or LR3IGF-I (1 ng/ml plasma) was infused for 45 min into the arterial supply of a 10 cm intestinal segment, either in the absence of added unlabelled peptide (high specific activity) or in the presence of a 100-fold excess of unlabelled homologous peptide (low specific activity) to achieve different proportions of free and complexed peptide. Very little degradation of radiolabelled peptides was detected in plasma, with 3–10% degradation in the intestinal tissue. Less than 5% of radiolabelled IGF-I remained as free peptide in the efferent venous plasma of the perfused segment at both specific activities. Bound radio-labelled IGF-I was found by size-exclusion chromatography mainly in the 30–50 kDa region, with a smaller proportion in the 150 kDa peak. The net intestinal transfer of IGF-I, calculated as the sum of the proportions of infused tracer recovered from intestinal tissue, luminal contents and lymph, was 3·46 ± 0·22% (s.e.m.) and 3·49 ± 0·93% when infused at high and low specific activities respectively. The analogues differed from IGF-I with up to ninefold higher concentrations of free radio-labelled peptide in venous plasma of the perfused intestinal segment, and corresponding decreases in binding to the 30–50 kDa binding proteins. Notwithstanding these marked differences in the plasma levels of free peptide, net intestinal transfer was very similar for the three peptides, as was the extent of degradation in the intestinal tissue. The lack of correlation between binding to 30–50 kDa binding proteins and net intestinal transfer suggests that association with 30–50 kDa plasma binding proteins is not a rate-limiting determinant of net IGF transfer to intestinal tissue.

Journal of Endocrinology (1994) 141, 505–515

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F M Tomas, A B Lemmey, L C Read, and F J Ballard

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, Nτ-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

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A. P. D. Lord, A. A. Martin, P. E. Walton, F. J. Ballard, and L. C. Read

ABSTRACT

Heparinized samples of plasma, cerebrospinal fluid (CSF) and lymph from intestinal, prescapular and popliteal lymph nodes were collected from young lambs in order to characterize and compare the insulin-like growth factor-binding proteins (IGFBPs) in extracellular fluids with those from the circulation. Prior to collection and analysis, the superiority of heparin for plasma preparation was established over EDTA and citrate or the use of serum, according to the extent of IGF-I and IGF-II binding achieved in the high molecular mass IGFBP region in vitro. The IGFBPs were characterized by ligand blotting and competitive binding techniques using radiolabelled IGF-I, IGF-II and the truncated IGF analogue, des(1–3)IGF-I, as well as by ligand blotting of fractions after Superose 6 chromatography of incubations of fluids with labelled factors. This combined analysis demonstrated an IGF-II-specific binding protein at approximately 250 kDa that was present in plasma and each lymph type and presumably represented the soluble type-2 IGF receptor; a complex of 130 kDa containing 52 kDa and 46 kDa binding proteins that was labelled by all three IGF peptides was particularly evident in plasma and intestinal lymph and was probably a complex between IGFBP-3 and the acid-labile subunit; and a group of binding proteins that chromatographed as IGF complexes at approximately 50 kDa. This last group contained IGFBP bands of 52, 46, 35, 28 and 23·5 kDa in plasma and all lymphs as well as an IGF-II-specific band of 22 kDa in prescapular and popliteal lymphs. CSF differed qualitatively from plasma and lymph in that the 52/46 kDa IGFBP bands were undetectable in this fluid; the 35 kDa band was the predominant binding protein, and neither this nor the 28, 23·5 and 22 kDa proteins bound des(1–3)IGF-I to any significant extent. The 52,46 and 28 kDa bands in plasma and lymph did bind this ligand. Immunoblots using antisera against bovine IGFBP-2 showed binding at 35 kDa in all fluids as well as several bands at lower molecular masses. Taken together these results show not only marked differences in the binding protein profiles of sheep plasma, lymph and CSF, but both qualitative and quantitative differences between intestinal, prescapular and popliteal lymphs. We speculate that the differences between lymphs may result from tissuespecific release of binding proteins into extracellular fluid.

Journal of Endocrinology (1991) 129, 59–68

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A P D Lord, L C Read, P C Owens, A A Martin, P E Walton, and F J Ballard

Abstract

Halothane anaesthesia in young sheep results in greatly increased plasma binding capacity for radiolabelled insulin-like growth factor (IGF), as demonstrated using size-exclusion chromatography. Most of the increased binding was at an estimated molecular mass range of 30–50 kDa, with a smaller increase evident at 130–150 kDa. These changes were not evident in control animals which had food withheld for the same period. The progressive increase in plasma radioligand binding during anaesthesia was the net result of a rise in circulating levels of a 29–31 kDa IGF-binding protein (IGFBP), as shown by ligand blotting, and declining plasma concentrations of IGF-I and IGF-II. Recovery from anaesthesia was accompanied by the restoration of plasma IGFs and the IGFBP towards pre-anaesthesia concentrations. The induced IGFBP was provisionally identified as IGFBP-1 because it bound anti-IGFBP-1 antiserum but not antibodies against IGFBP-2, IGFBP-3 or IGFBP-4. The elevation of plasma IGFBP-1 immunoreactivity was associated with reduced concentrations of glucose and insulin, the regulators of IGFBP-1 in humans and rats. These results suggest that IGF experiments that require anaesthesia but assume that the anaesthetised state is representative of conscious sheep should be reassessed. A similar situation may occur with other mammalian species.

Journal of Endocrinology (1994) 141, 427–437

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A P D Lord, S E P Bastian, L C Read, P E Walton, and F J Ballard

Abstract

Associations between labelled insulin-like growth factors (IGFs) and IGF-binding proteins in plasma have been compared in the rat, sheep, human, pig and chicken. The IGFs tested were recombinant human IGF-I, the truncated variant, des(1–3)IGF-I, and LR3IGF-I, an extended form that had been engineered so as to minimize interactions with IGF-binding proteins. Marked species differences were demonstrated, notably that the IGF-I variants which exhibited extremely weak binding in rat plasma bound significantly in plasma from the other species. This result was shown both by size-exclusion chromatography of labelled IGFs added to plasma, in which the extent of variant IGF-I binding decreased in the order sheep>human>pig=chicken>rat, and by competition for labelled IGF-I binding in vitro, in which the order was pig=chicken>sheep>human>rat. Notwithstanding these differences, the two IGF-I variants showed only slight between-species binding differences when tested with purified rat, sheep and human IGF-binding protein-3. Ligand blotting experiments with plasma from the five species similarly showed a consistent pattern in that IGF-I binding was much greater than des(1–3)IGF-I binding, which in turn was greater than LR3 IGF-I binding. These experiments suggest first that IGF-binding properties measured after the removal of endogenous IGFs do not always reflect the situation with untreated plasma or in vivo, and secondly, the increased potencies of des(1–3)IGF-I and LR3 IGF-I in rat growth studies that have been ascribed to higher concentrations of these peptides in the free form cannot necessarily be extended to other species.

Journal of Endocrinology (1994) 140, 475–482

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F. M. Tomas, S. E. Knowles, P. C. Owens, L. C. Read, C. S. Chandler, S. E. Gargosky, and F. J. Ballard

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