Studies of liver lipids in normal, alloxan-diabetic and pregnancy-toxaemic sheep.

Triacylglycerols were the major lipid class in the fatty livers from alloxan-diabetic sheep and those suffering from pregnancy toxaemia, with the concentrations increased by 15- and 25-fold, respectively, compared with the normal state. Analysis of the fatty acid composition of total liver triacylglycerols in these animals showed a significant decrease in the proportion of saturated fatty acids, 16:0 and 18:0, and increase in the proportion of polyunsaturated fatty acids (18:2 omega 6, 18:3 omega 3 and 20:4 omega 6), particularly in those with pregnancy toxaemia. In contrast, total liver phospholipids showed a significant increase in the proportion of 18:0 in ewes with pregnancy toxaemia and a significant decrease in a range of polyunsaturated fatty acids in both the diabetic and toxaemic animals. Also, although the concentration of both phosphatidylcholine and phosphatidylethanolamine increased in the diabetic livers the ratio of phosphatidylcholine/phosphatidylethanolamine fell significantly, from 2.22 in the control animals to 1.59. The data suggest that, following the large influx of plasma fatty acids into the ovine liver in diabetes and pregnancy toxaemia, there is a diversion of polyunsaturated fatty acids from phospholipids to triacylglycerols. In diabetic sheep these changes may in turn affect phosphatidylcholine synthesis via the methylation pathway in liver. These changes in lipid composition may, in part, explain the degenerative changes in membrane and subcellular organelle structure and the failure of liver function observed both in advanced diabetes and in severe pregnancy toxaemia.

Des(1-3)IGF-I: a truncated form of insulin-like growth factor-I.

Des(1-3)IGF-I, a truncated variant of human IGF-I with the tripeptide Gly-Pro-Glu absent from the N-terminus, has been isolated from bovine colostrum, human brain and porcine uterus. This protein probably results from post-translational cleavage of IGF-I. Des(1-3)IGF-I generally is about 10-fold more potent than IGF-I at stimulating hypertrophy and proliferation of cultured cells, a consequence of much reduced binding to IGF-binding proteins, in turn caused by the absence of the glutamate at position 3. The increased potency is retained in part when the variant is administered in vivo, with selective anabolic effects particularly evident in gut tissues. Clinical opportunities for des(1-3)IGF-I have not yet been evaluated, but could apply in catabolic states as well as for the treatment of inflammatory bowel diseases.

Preferential intestinal delivery of long[Arg3] insulin-like growth factor (LR3IGF-I) over IGF-I in preweaning and adult rats.

During early postnatal development, the intestine is highly responsive to LR(3)IGF-I administration but refractory to IGF-I, in contrast to the mature intestine. Given that LR(3)IGF-I is an IGF-I analog that binds poorly to IGF binding proteins, the response of the intestine is likely to reflect regulation of IGF-I bioactivity by IGF binding proteins. This study measures the delivery of exogenous IGF-I peptides to the intestine in preweaning (d-19) and adult rats to determine whether a correlation exists with the potency advantage of LR(3)IGF-I in the intestine during postnatal development. IGF-I or LR(3)IGF-I (2.6 microg/kg) was spiked with corresponding (125)I-labeled peptide (10 x 10(6) cpm) and administered iv as a bolus (n = 5-6/group) with blood and tissue samples collected 5 and 10 min post injection. In both age groups, the levels of (125)I-IGF-I retained in the blood at both 5 and 10 min were higher than the levels of (125)I-LR(3)IGF-I, consistent with the slower clearance rate for the native peptide. In the gastrointestinal tract, the levels of (125)I-LR(3)IGF-I per gram of tissue were 37-50% higher than (125)I-IGF-I. Surprisingly, there was little difference in the relative delivery of LR(3)IGF-I to IGF-I to the intestine, across developmental age. Although bolus iv-injected LR(3)IGF-I was cleared more rapidly from the circulation than IGF-I and was subsequently delivered to the intestine in higher amounts than the native peptide, the ratio of LR(3)IGF-I to IGF-I in gut tissues was approximately 2:1 in both age groups. Hence, selective delivery to the gut is unlikely to explain the markedly higher potency of (125)I-LR(3)IGF-I in stimulating growth of the preweaning vs. adult intestine.

Protein synthesis and breakdown rates associated with the insulin resistance of fibroblasts from patients with leprechaunism.

Postreceptor defects in insulin action have been reported in fibroblasts isolated from two patients with Leprechaunism, Leprechaun/Ark-1 and Leprechaun NC-1. We have extended the published reports on glucose, aminoisobutyric acid, and thymidine uptake in these cells to measurements of protein synthesis and protein breakdown. We found a remarkably consistent pattern of responsiveness between the two Leprechaun fibroblast lines. First, protein synthesis proceeded at a low basal rate that was only slightly stimulated by insulin. Second, basal rates of protein breakdown were significantly higher than in normal skin fibroblasts, with approximately equal inhibitory effects produced by 100 nM insulin. Third, the responses of protein synthesis and protein breakdown to insulin required higher concentrations of the hormone to elicit half-maximal effects. Fourth, both Leprechaun cell lines were slow growing in complete medium, a situation that results from low rates of protein synthesis and high rates of protein breakdown. Fifth, the abnormal rates of protein metabolism in the presence of serum were caused not by the inability of serum to produce anabolic responses but because the unstimulated rates reflect a more catabolic basal state. Taken together with previous published results, our measurements suggest a generalized metabolic defect in Leprechaun fibroblasts that can only partly be explained by the reduced sensitivity of the cells to insulin.

A role for epidermal growth factor in the morphological and functional maturation of the adrenal gland in the fetal rhesus monkey in vivo.

We determined the effects of epidermal growth factor (EGF) and beta-methasone on the growth and development of the adrenal gland of the fetal rhesus monkey in vivo between 121-128 days of gestation. The adrenal to body weight ratio was significantly greater (P < 0.05) in EGF-treated fetuses (0.988 +/- 0.046 x 10(-3) g/g) and significantly reduced (P < 0.05) in beta-methasone-treated fetuses (0.401 +/- 0.056 x 10(-3) g/g) compared with that in control fetuses (0.689 +/- 0.050 x 10(-3) g/g). The increase in adrenal weight with EGF administration was due to hypertrophy of definitive zone cells of the adrenal cortex, whereas the reduction in adrenal weight after beta-methasone treatment was due to a decrease in the size of definitive and fetal zone cells of the adrenal cortex. By Western analysis, EGF treatment induced a significant (P < 0.05) 2.8-fold increase in the amount of protein for 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) in the fetal adrenal. EGF also stimulated the induction of immunocytochemical staining for 3 beta HSD in transitional zone cells of the adrenal cortex. In contrast, beta-methasone resulted in 2.6-, 4.5-, and 6.6-fold significant decreases (P < 0.05) in the amount of protein for cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/17,20-lyase, and 3 beta HSD in the fetal adrenal. After beta-methasone treatment. 3 beta HSD staining was detected in some of the definitive zone cells, with no 3 beta HSD staining in the transitional zone. In conclusion, growth and functional differentiation of fetal primate adrenal gland can be accelerated prematurely by EGF and inhibited by glucocorticoid negative feedback.

Changes in protein synthesis and breakdown rates and responsiveness to growth factors with ageing in human lung fibroblasts.

The effects of insulin, epidermal growth factor, insulin-like growth factor-2 and fetal bovine serum on protein synthesis and protein breakdown have been measured in mid-passage and senescent cultures of human diploid lung fibroblasts. Each of the individual growth factors was a potent inhibitor of protein breakdown with no difference in either the maximum response or sensitivity evident between senescent cells and mid-passage cultures. Binding of 125I-labelled epidermal growth factor per mg of cell protein similarly showed no difference between senescent and confluent mid-passage fibroblasts, although sparse mid-passage cells bound more of the ligand. These results indicate normal binding and normal responsiveness to growth factors in senescent cultures. However, rates of protein synthesis are higher in confluent mid-passage cells and especially so in sparse mid-passage cells than in senescent cells of intermediate density. Furthermore, senescent cells differ from either growth state of mid-passage cells because protein synthesis in aged cells is much less responsive to any of the growth factors or to fetal bovine serum. It is suggested from these results that the reduced ability of serum or growth factors to initiate DNA synthesis or growth in ageing cells may be a consequence of an unresponsive protein synthesis pathway rather than a generalised defect in growth factor action.

Production of a human epidermal growth factor fusion protein and its degradation in rat gastrointestinal flushings.

This study describes the biosynthesis of a human epidermal growth factor fusion protein, Long EGF, that has a 53 amino acid extension peptide derived from the 46 N-terminal amino acids of porcine GH. The approach allowed the production of Long EGF at high efficiency due to the expression of the fusion protein in high yield as inclusion bodies in Escherichia coli. Long EGF had a slightly lower potency compared with native EGF in a range of assays, including binding to anti-EGF antibodies or the EGF receptor, stimulation of Balb/3T3 fibroblast and rat intestinal epithelial cell growth, as well as counteracting the inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta 1. Degradation of Long EGF and native EGF was compared in gastrointestinal flushings as an indication of whether the EGF domain of the fusion protein would be protected from proteolytic cleavage and be useful as a trophic agent in the gut. Incubation with flushings from the stomach or jejunum of rats caused rapid cleavage of the extension peptide, releasing native EGF. A C-terminal truncation of Arg53 in the stomach and a removal of the C-terminal pentapeptide (49 Trp-Trp-Glu-Leu-Arg53) in the small bowel was demonstrated by N-terminal sequencing and mass spectrometry. The degradation patterns were reflected by changes in migration of products on SDS-PAGE and in subsequent binding activities to the EGF receptor and anti-EGF antibodies. The data show that a human EGF fusion protein can be produced efficiently in a bacterial expression system and that it retains biological activity in vitro. Although the extension peptide was rapidly cleaved from Long EGF in both stomach and small bowel producing similar biological activity to native EGF, it could not prevent subsequent degradation of the EGF domain. Other strategies are being investigated to develop an effective oral form of EGF that resists digestion by proteases in the gastrointestinal tract.

Specificity of the localization of transforming growth factor-alpha immunoreactivity in colon mucosa.

Transforming growth factor-alpha (TGF-alpha) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-alpha immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-alpha knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-alpha) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-alpha) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-alpha knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-alpha molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-alpha should be used for TGF-alpha immunohistochemistry in the colon.

Degradation of IGF-I in the adult rat gastrointestinal tract is limited by a specific antiserum or the dietary protein casein.

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 (t1/2) of 2 min by all three methods. IGF-I was slightly more stable in the stomach (t1/2 = 8, 5 and 2.5 min by the above three methods), and considerably more stable in the colon (t1/2 = 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.

Enhanced potency of truncated insulin-like growth factor-I (des(1-3)IGF-I) relative to IGF-I in lit/lit mice.

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 micrograms IGF-I, 30 micrograms des(1-3)IGF-I or 3 micrograms 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 micrograms 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.

Differences in the association of insulin-like growth factor-I (IGF-I) and IGF-I variants with rat, sheep, pig, human and chicken plasma-binding proteins.

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 LR3IGF-I binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer of insulin-like growth factor (IGF)-I from blood to intestine: comparison with IGFs that bind poorly to IGF-binding proteins.

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, radiolabelled 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 radiolabelled 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 radiolabelled 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.

Effects of insulin-like growth factor-I peptides in rats with acute renal failure.

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.

Superior potency of infused IGF-I analogues which bind poorly to IGF-binding proteins is maintained when administered by injection.

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 micrograms/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 micrograms/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 LR3IGF-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, LR3IGF-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 tau-methylhistidine excretion by dexamethasone-treated rats was reduced to a threefold greater extent by injected LR3IGF-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.

Insulin-like growth factor-binding proteins in tissue fluids from the lamb.

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, 32, 28 and 23.5 kDa in plasma and all lymphs as well as an IGF-II-specific band of 22 kDa in prescapsular 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 tissue-specific release of binding proteins into extracellular fluid.

Rapid changes in plasma concentrations of insulin-like growth factor-I (IGF-I), IGF-II and IGF-binding proteins during anaesthesia in young sheep.

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.

Insulin-like growth factor-I and its N-terminal modified analogues induce marked gut growth in dexamethasone-treated rats.

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 micrograms/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.

Effects of full-length and truncated insulin-like growth factor-I on nitrogen balance and muscle protein metabolism in nitrogen-restricted rats.

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 less than 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 less than 0.01). The rate of muscle protein breakdown calculated from the urinary excretion of 3-methyl-histidine 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 less than 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.

Properties of synthetic-gene recombinant human epidermal growth factor: comparison with the natural growth factor from human urine and milk.

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 radio-receptor 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.

A milk growth factor extract reduces chemotherapeutic drug toxicity in epithelial cells in vitro.

Transforming growth factor-beta (TGF-beta) and insulin-like growth factor (IGF-I) can attenuate drug-induced cell death in epithelial cells. Since milk whey contains a mixture of these and other growth factors, we evaluated mitogenic bovine whey extract (MBWE) for protective activity against chemotherapy drug damage in cultured epithelial cells (mink lung, MvlLu). Etoposide and vinblastine reduced cell survival by up to 90%. This was attenuated by the addition of MBWE before and during drug exposure, but not following drug removal. MBWE was compared with individual growth factors known to be present in the mixture. IGF-I and platelet-derived growth factor were ineffective, whereas TGF-beta2 induced growth inhibition and cell survival, with a maximum response at 3 ng/ml. TGF-beta2 bioactivity was also demonstrated by showing that acidification of MBWE (A-MBWE), to activate TGF-beta2, enhanced its growth inhibitory and chemoprotective activities 60- and 12-fold, respectively. However, MBWE contained additional protective factors. When TGF-beta2 and the MBWE preparations were compared, on the basis of growth inhibition equivalents, MBWE protected cells against drug toxicity at concentrations an order of magnitude lower than with TGF-beta2 or A-MBWE. Immunoneutralization of the TGF-beta present in MBWE and A-MBWE eliminated all growth inhibitory activity but not all cell survival activity. We conclude that the MBWE preparations are cytoprotective against two chemotherapy drugs when added before and during drug exposure. TGF-beta contributes to this activity, but the extracts contain other factors that promote the survival of epithelial cells after chemotherapy drug exposure.