The effect of a chronic maternal cortisol infusion on the late-gestation fetal sheep.

Exposure of the fetus to excess maternal glucocorticoids has been postulated to alter fetal growth and development, and thus provide a possible mechanism for the link between impaired fetal growth and altered postnatal physiology. However, the effects of exposure to excess maternal glucocorticoids on fetal physiology and metabolism in utero have not been described. We therefore studied the effects of chronic maternal cortisol infusion on fetal growth, blood pressure, metabolism and endocrine status in chronically catheterised fetal sheep. We infused hydrocortisone (80 mg/day, n=6) or saline (n=8) for 10 days into the pregnant ewes beginning at 119 days of gestation. Maternal cortisol infusion reduced fetal growth rate by 30% (girth increment 2.9+/-0.3 vs 1.8+/-0.4 mm/day, P=0.03). Maternal cortisol infusion increased fetal heart weight by 15% relative to body weight and increased ventricular wall thickness by 30% in the left and 50% in the right ventricle. The weight of the spleen was reduced by 30% and placental weight reduced by 25%. Fetal blood pressure increased by approximately 10 mmHg (20%) during maternal cortisol infusion. Maternal cortisol infusion did not alter amino-nitrogen concentrations. However, maternal lactate concentrations increased by 80% and fetal lactate concentrations increased by 74% with maternal cortisol infusion, and both maternal and fetal urea concentrations increased by 40%. Circulating maternal IGF-binding protein (IGFBP)-3 levels had increased by 20% by the end of the maternal cortisol infusion. Fetal IGF-I concentrations decreased during cortisol infusion and fetal IGFBP-1 concentrations were negatively correlated with fetal weight (r=-0.76, P=0.02). We conclude that even a modest elevation of maternal cortisol levels affects fetal growth, cardiovascular function, metabolism and endocrine status which may have long-term consequences.

Apoptosis: live or die--hard work either way!

This review presents a brief overview of the cell's apoptotic machinery, including specific and indirect death signals. Specific death signals are transferred via death ligands, death receptors, and their intracellular signalling pathways. Indirect death signals cumulate a wide range of stimuli that potentially harm survival of cells. These include intercalating drugs, irradiation or altered intracellular signalling. Herein, a focal point is the mitochondrial control of specific death enzymes--so called caspases--by members of the pro-apoptotic Bax and BH3 subfamily or the anti-apoptotic Bcl-2 subfamily. While the initiation of cell death happens through a variety of signalling systems, the activation of caspases plays a pivotal role in the progression towards the final morphologic findings in cells undergoing apoptosis. Caspases appear to directly cleave and inactivate substrates that are clinical for the maintenance of cell structure and function but also regulate the activity of other enzymes that induce the apoptotic phenotype within the cell. The insulin-like growth factors (IGFs) are potent proliferation factors and potently inhibit apoptosis acting via the ubiquitously expressed IGF-I receptor. Within IGF-I receptor signalling, key to the inhibition of apoptosis are the RAS/RAF/mitogen-activated protein (MAP)-kinase pathway and the PI 3'-kinase pathway. To give an example of high clinical relevance of apoptosis within endocrine disorders, apoptotic death of pancreatic beta cells in type 1 diabetes disease and the involvement of IGF-II in beta cell survival and beta cell function is discussed in detail. Finally, further understanding of signalling systems that are involved in proliferation or in apoptosis might provide novel tools to treat or even heal disorders like type I diabetes.

Growth hormone treatment of breeding bulls used for artificial insemination improves fertilization rates.

To evaluate new therapeutical concepts for male subfertility, we tested the effects of exogenous recombinant bovine growth hormone (rbGH) on various endocrine and metabolic parameters both in blood and in seminal plasma of bulls. Sperm quality was assessed morphometrically and by monitoring the number of successful artificial inseminations (AIs) defined as non-return rates (NRR). Aliquots of 450 semen samples were used from each bull and each experimental period (4 wk before, 14 weeks during and 6 wk after treatment). Six out of ten sires (average age 8.4 years) were treated every two weeks with 640-mg depot formulated rbGH (Eli Lilly). Four bulls received vehicle only. Blood plasma bGH, IGF-I, insulin and glucose concentrations were increased with rbGH treatment. In seminal plasma there was no effect of rbGH treatment on fructose and citrate or on testosterone concentrations. With one exception, rbGH-treated bulls had greater IGFBP-3 concentrations in seminal plasma. Motility of spermatozoa after freezing and thawing was increased compared with pretreatment rates. Most interestingly, the number of successful AIs was increased by an average of 6.0% NRR when ejaculates from rbGH-treated bulls were used.

Insulin-like growth factor-I inhibits the progression of human U-2 OS osteosarcoma cells towards programmed cell death through interaction with the IGF-I receptor.

Insulin-like growth factor-I exerts potent mitogenic effects through the type I IGF receptor, a member of the insulin receptor family, and exhibits at the same time some insulin-like metabolic activities. We have questioned whether IGF-I presents moreover a modulatory effect upon programmed cell death (PCD)(apoptosis) in serum-deprived human osteosarcoma U-2 OS cells, a cell line synthesizing IGF-II and exhibiting an increased DNA synthesis following treatment with IGF-I. U-2 OS cells were cultured in a medium containing 0.8% FCS and growth arrest was induced by transfer to serum-free growth conditions. PCD was measured using a commercially available DNA degradation ELISA while viable cell numbers were counted microscopically after trypan exclusion to estimate net proliferative activity. Following serum withdrawal for 24 hrs., the level of PCD in U-2 OS cells was increased six-fold while cell number was reduced by approximately 35% compared to cells grown in the presence of 15% serum. Incubation with recombinant human IGF-I for 24 hrs. caused a dose-dependent inhibition of the level of programmed cell death. Co-incubation with an IGF-I receptor monoclonal antibody (alphaIR3) dose-dependently blocked the effects of 10 ng/ml IGF-I on PCD, with an ED50 of 1-10 ng/ml of alphaIR3 immunoglobulin. Conversely IGF-1 provoked a significant cell number increase, an effect blocked by addition of alphaIR3. The addition of an inhibitor of caspase 1 (ICE) had little effect on PCD but resulted in a net increase in the number of viable cells. In summary, IGF-I treatment of U-2 OS cells at the same time inhibits the induced programmed cell death and increases the cell number, effects which are blocked by addition of IGF-I receptor antibodies. These data support the hypothesis that IGF-I affects cells in a dual way, both by enhancing proliferative responses and by suppressing programmed cell death. The differential response between PCD and cell number to ICE inhibitors suggests the existence of independent control systems for these processes although the role of IGF-I in this study has yet to be determined.

Expression of apoptosis and cell cycle related genes in proliferating and colcemid arrested cells of divergent lineage.

Progression through the cell cycle and redirection of cells towards programmed cell death (apoptosis) are tightly inter-related processes. However the requirement for tissue and cell type specificity suggests that a wide variety of mechanisms are used to achieve the same purpose. To examine this issue, we investigated cell cycle (c-myc, p53, p21/WAF) and apoptosis related (bcl-2, bcl-X(L), bax-alpha) gene expression in two cell lines of very different origin under proliferating and apoptosis-inducing conditions. Transformed human osteosarcoma cells (MG63) and non-transformed human kidney embryonal fibroblasts (293-0) were kept in culture in medium containing 10% FCS and growth arrest was induced by the addition of 50 ng/ml colcemid. Colcemid treatment caused growth arrest and elevated expression of cyclin B1 protein in both cell lines. Apoptosis was significantly elevated in both cell lines after colcemid exposure for at least one cell cycle. However the pattern of expression of cell cycle and apoptosis related genes, determined by RT-PCR, was quite different between the two cell lines during exponential growth and cell cycle arrest. Colcemid treatment did not markedly influence c-myc, p53 and p21/WAF expression in MG63 cells but did suppress c-myc and increase p21/WAF in 293-0 cells. Furthermore colcemid treated MG63 cells exhibited elevated bcl-2 and bax-alpha expression while similar treatment of 293-0 cells resulted in decreased bcl-X(L) and slightly increased bax-alpha expression. While growth arrest and apoptosis were induced in both MG63 and 293 cells following colcemid treatment, the differences in gene expression suggest that the mechanism by which these cells determine cell fate is quite different and may determine the sensitivity of different cell populations to anti-neoplastic drug therapy. The distinct patterns of gene expression should be carefully defined before mechanisms of apoptotic cell death are studied.

Fetal programming of insulin-like growth factor (IGF)-I and IGF-binding protein-3: evidence for an altered response to undernutrition in late gestation following exposure to periconceptual undernutrition in the sheep.

It has been demonstrated in several animal models that undernutrition in utero has significant long lasting effects on subsequent fetal and postnatal development. To address the hypothesis that the insulin-like growth factors (IGFs) may mediate such effects, our study examined whether a period of periconceptual maternal undernutrition could have a lasting influence on the IGF axis in the fetal sheep. Ewes were either allowed to feed ad libitum or kept undernourished from day 60 prior to mating until day 30 after conception, and then both groups were allowed to feed ad libitum. These groups were further divided at day 105 of gestation, either being fed ad libitum or undernourished until day 115 of gestation. Fetal and maternal blood samples were obtained at both day 105 and 115 of gestation. We describe the development of a specific homologous RIA to measure ovine IGF-binding protein-3 (IGFBP-3) in fetal and maternal sheep plasma. Fetal plasma IGFBP-3 and IGF-I concentrations were significantly (P<0.05) reduced at day 115 of gestation after maternal undernutrition. The fetal plasma IGFBP-2 levels were unchanged. The degree of reduction in fetal plasma IGFBP-3 and IGF-I between day 105 and 115 of gestation as a response to acute maternal undernutrition was significantly greater (P<0.05) in fetuses of mothers receiving low periconceptual nutrition. The response of maternal plasma IGFBP-3 and IGF-I to undernutrition did not depend on the level of periconceptual nutrition. Western blot data indicate that changes in either maternal or fetal plasma IGFBP-3 concentrations were not the result of increased proteolytic activity. These results suggest that exposure to maternal periconceptual undernutrition reprograms IGFBP-3 and IGF-I regulation in the developing sheep fetus, altering its response to undernutrition in late gestation.

IGF-I and IGF-binding protein-3 in plasma of GH-deficient rats.

The majority of IGF-I circulates in a large (150 kDa) ternary complex with IGF-binding protein-3 (IGFBP-3) and a non-IGF-binding acid-labile subunit. The secretion of ternary complex into the circulation from liver has been considered to be GH-dependent; however, recent data indicate that GH does not directly regulate hepatic IGFBP-3 synthesis. To examine the role of insulin in regulating plasma IGFBP-3 levels, postpubertal male GH-deficient (dw/dw) rats were treated every 8 h with injections (s.c.) of 0.9% saline, 20 micrograms insulin/day, 200 micrograms hIGF-I/day, or 20 micrograms insulin/day plus 200 micrograms hIGF-I/day, for 10 days with the animals being killed 2-3 h after the final injection. Hypoglycaemia was not observed in any of the treatment groups. hIGF-I treatment increased longitudinal growth and weight gain (P < 0.05), while insulin treatment had no effect. Plasma IGF-I levels were increased in groups treated with hIGF-I (P < 0.05), while insulin treatment resulted in a reduction (P < 0.05): saline = 267.1 +/- 15.6 (ng/ml +/- S.E.M.), insulin = 219.3 +/- 17.5, hIGF-I = 391.7 +/- 17.6, insulin plus hIGF-I = 357.5 +/- 31.8. Hepatic IGF-I mRNA expression was increased in insulin-treated dw/dw rats in comparison with hIGF-I-treated animals (P < 0.05) but not in comparison with saline control or the combined treatment groups. Plasma levels of intact IGFBP-3, measured by ligand blot analysis, were increased in all treatment groups compared with saline (P < 0.05): saline = 100.0 +/- 9.4% (% of saline +/- S.E.M.), insulin = 149.9 +/- 17.5%, hIGF-I = 191.4 +/- 17.3%, insulin plus hIGF-I = 205.4 +/- 15.3%. The levels of the 28/32 kDa IGFBPs and IGFBP-4 in plasma were increased by hIGF-I treatment (P < 0.05) but not by insulin treatment. Hepatic specific 125I-bovine GH binding was not significantly different in any of the treatment groups. This study provides the first evidence in nondiabetic animals that insulin regulates hepatic IGF-I mRNA expression, plasma IGF-I and plasma IGFBP-3 levels in the GH-deficient state without changes in hepatic GH receptors. The divergent response of plasma IGF-I and IGFBP-3 levels to insulin treatment in the present study may indicate an effect of insulin on the clearance of IGF-I from the circulation.

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.

The effects of ovine placental lactogen infusion on metabolites, insulin-like growth factors and binding proteins in the fetal sheep.

It has been suggested, but not shown, that in the fetus placental lactogen (PL) may affect the regulation of the IGFs and fetal metabolism. To examine the effects of PL on the circulating concentrations of the IGFs, IGF-binding proteins (IGFBPs), glucose, free fatty acids (FFAs) and amino nitrogen (AN), we infused late gestation sheep fetuses with recombinant ovine PL (roPL). Five chronically-catheterised sheep fetuses were infused intravenously with three 24 h infusions of saline, roPL (100 micrograms bolus then 500 micrograms over 24 h) and then saline again. Fetal roPL infusion increased plasma oPL from 0.4 +/- 0.1 to 3.3 +/- 0.5 nM (mean +/- S.E.M.; P < 0.05; factorial analysis of variance and Scheffé's test). Fetal plasma IGF-I, IGF-II, insulin, FFAs and blood glucose were unaffected by the roPL infusion. Fetal plasma IGFBP-3, as measured by Western ligand blotting, decreased by 30% during fetal roPL infusion while other fetal plasma IGFBPs were unaffected. Fetal roPL infusion decreased fetal blood AN from 7.3 +/- 0.5 to 6.6 +/- 0.2 mM (P < 0.05). Maternal plasma IGF-I, IGF-II, IGFBPs, insulin, FFAs, blood glucose and AN were unaffected by the fetal roPL infusion. Saline infusion had no effect on any parameter. The data suggest that PL is not a significant determinant of plasma IGFs in the late gestation sheep fetus although there may be an indirect effect via alterations in levels of IGFBP-3. The effect of fetal roPL infusion on fetal blood AN concentrations may suggest some role for PL in the regulation of fetal amino acid metabolism.

A homologous radioimmunoassay for ovine insulin-like growth factor-binding protein-2: ontogenesis and the response to growth hormone, placental lactogen and insulin-like growth factor-I treatment in sheep.

Although insulin-like growth factor-binding protein-2 (IGFBP-2) is an abundant IGFBP in fetal and postnatal plasma, its regulation is not yet clearly understood. To address this question in sheep, we purified ovine IGFBP-2 and developed a homologous radioimmunoassay. We have studied its ontogenesis and measured serum concentrations of ovine IGFBP-2 after bovine growth hormone (bGH), ovine placental lactogen (oPL) and IGF-I treatment. Concentrations of IGFBP-2 were high at 125 days of gestation (550 +/- 15 micrograms/l) but fell after birth (P < 0.05) and plateaued after 1 year of age (340 +/- 20 micrograms/l). In lactating ewes, bGH treatment for 7 days significantly reduced (21%; P < 0.05) IGFBP-2 relative to the saline-treated group. Similarly, in neonatal lambs, bGH treatment from day 3 to day 23 of life reduced (P < 0.05) IGFBP-2 by 23% relative to the saline-treated group. oPL had no effect on serum levels of IGFBP-2 in the ewe or the neonatal lamb. In well-fed yearling lambs, treatment with IGF-I reduced IGFBP-2 values by 27% (P < 0.05) relative to control animals. In yearling lambs, reduced nutrition increased plasma IGFBP-2 (41%; P < 0.05). However this increase was abolished by IGF-I treatment. The changes in plasma levels of IGFBP-2 were positively related to changes in IGF-II while there was a negative relationship between circulating IGF-I and IGFBP-2 such that both IGF-I and IGF-II may play a role in the regulation of IGFBP-2 in serum.

Periconceptual undernutrition resets plasma IGFBP levels and alters the response of IGFBP-1, IGFBP-3 and IGF-1 to subsequent maternal undernutrition in fetal sheep.

Maternal undernutrition inhibits fetal growth and alters circulating levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs). This study investigates whether the fetal IGF axis could be reprogrammed by maternal undernutrition and hence be a potential contributing factor to changes in fetal and postnatal metabolism. Ewes were either fed a lib. or undernourished from day-60 to day 30 of gestation, and then both groups were fed ad lib. These groups were further divided at day 105, either being fed ad lib or undernourished until day 115. Fetal blood samples were obtained at day 105 and day 115. IGFBP-1 and IGFBP-3 levels were lower at day 105 in the periconceptually undernourished fetuses. Levels of IGFBP-1 were increased and IGFBP-3, IGFBP-4, IGF-1, glucose and insulin were reduced at day 115 after undernutrition. The degree of change in IGFBP-1, IGFBP-3 and IGF-I between day 105 and day 115 was greater in fetuses receiving low periconceptual nutrition. These results indicate that periconceptual undernutrition is able to reprogramme the fetal IGF axis such that the responses of IGF-I and the IGFBPs to undernutrition in late gestation are markedly altered.

Circulating insulin-like growth factor II/mannose-6-phosphate receptor and insulin-like growth factor binding proteins in fetal sheep plasma are regulated by glucose and insulin.

We have reported previously that levels of insulin-like growth factor I (IGF-I) and IGF-II in fetal sheep plasma decrease with maternal starvation and increase following an infusion of glucose to the starved fetus, while a fetal infusion of insulin elevates UGF-I alone. We now report the changes in the circulating IGF-II/M6P receptor and plasma IGF binding proteins (IGFBPs), as measured by western blotting and ligand blotting, respectively, in fetus and mother during this study. In fetal plasma, the circulating IGF-II/mannose-6-phosphate (M6P) receptor, IGFBP-3 and IGFBP-4 were reduced during starvation. While circulating IGF-II/M6P receptor and IGFBP-4 levels were increased following the fetal insulin or glucose infusion, IGFBP-3 was unchanged and increased only after 48 h of maternal refeeding. Both IGFBP-1 and IGFBP-2 increased with starvation but while IGFBP-1 levels returned to control values following both insulin and glucose infusion, levels of IGFBP-2 were not reduced significantly by either infusion or by refeeding. In maternal plasma, levels of IGFBP-3 and IGFBP-4 decreased while IGFBP-1 and IGFBP-2 increased after 48 h of starvation. Levels of each IGFBP were unaltered following the fetal infusions but returned to values obtained during the control period after refeeding. These data show that each of the IGF carrier proteins is sensitive of changes in nutrition, either acutely, such as IGFBP-1, or chronically, as for IGFBP-3. This suggests that the circulating IGF-II/M6P receptor and the IGFBP's may modulate IGF activity in the fetus during different nutritional states.

Insulin-like growth factors and their binding proteins in plasma and milk after growth hormone-stimulated galactopoiesis in normally lactating women.

We performed a double-blind randomized placebo-controlled trial of recombinant human growth hormone (hGH) in normally lactating women (N = 8 per group) to investigate the endocrine mode of action of the galactopoietic effect of this hormone. Insulin-like growth factors I (IGF-I) and II (IGF-II) and their binding proteins (IGFBP-1, IGFBP-2 and IGFBP-3) were measured by radioimmunoassay in plasma and milk samples collected throughout the study. All assays were validated for human plasma and milk. Human GH treatment (0.1 IU.kg-1 body wt.day-1 for 7 days) increased plasma concentrations of IGF-I from 22.1 +/- 1.3 nmol/l (mean +/- SEM) to 59.7 +/- 2.5 nmol/l (p < 0.01). At the end of the study the increase in plasma IGF-I correlated significantly with the increase in milk volume (r = 0.67, p < 0.005, N = 16). The IGF-I levels were considerably lower in milk, with 0.14 +/- 0.03 nmol/l before and 0.31 +/- 0.04 nmol/l after hGH treatment. The increase in milk IGF-I levels (134.0 +/- 14.5%) with hGH treatment was significant (p < 0.01) and plasma and milk IGF-I concentrations correlated significantly when considering all samples of the study (r = 0.45, p < 0.001, N = 56). The concentrations of IGF-II were not changed significantly with hGH treatment in plasma (52.5 +/- 2.5 nmol/l before and 42.6 +/- 3.9 nmol/l after treatment) or milk (2.1 +/- 0.29 nmol/l before and 2.3 +/- 0.49 nmol/l after hGH treatment).(ABSTRACT TRUNCATED AT 250 WORDS)

Passive immunization against circulating insulin-like growth factor-I (IGF-I) increases protein catabolism in lambs: evidence for a physiological role for circulating IGF-I.

Primed constant infusions of [14C]urea were used to determine the acute effect of passive immunization against circulating free and protein-bound insulin-like growth factor-I (IGF-I) on the rate of net protein catabolism (NPC) in castrated male lambs fasted for 48 h. Following an intravenous bolus of 50 ml IGF-I antiserum, the rate of NPC increased to a peak 30 min after injection of 1.69 +/- 0.16 g/kg per day from a baseline value of 1.45 +/- 0.22 g/kg per day (P < 0.05, n = 4). In three animals given 50 ml equivalents of the purified immunoglobulin fraction, NPC increased from 1.31 +/- 0.20 to 1.59 +/- 0.16 g/kg per day (P < 0.05). A similar trend was observed in animals given 25 ml antiserum (n = 4). The rate of NPC did not increase following a bolus of non-immune serum in control animals and the rate of NPC in the treated lambs returned to control levels within 60 min of antibody injection. Plasma insulin and glucose concentrations in both the treated and control groups were unchanged throughout the study. These data suggest that circulating IGF-I has a physiological role in regulating whole body protein turnover during starvation and possibly other catabolic states. The effect of immunoneutralization of circulating IGF-I is transient and this suggests that while IGF-I has an endocrine role in the regulation of protein turnover, other regulatory mechanisms are involved.

Growth hormone stimulates galactopoiesis in healthy lactating women.

Sixteen normally lactating women underwent a double-blind randomized placebo-controlled trial of recombinant human growth hormone (hGH) to assess the effect of hGH on milk production in early lactation. Milk volumes were measured by test weighing procedures of the infants and removal of residual milk on a control day and after 7 days of treatment with recombinant hGH (0.1 IU.kg-1 body weight.d-1) or placebo treatment. Although all women were lactating normally before the study commenced, milk volume in 8 hGH treated mothers was increased (p < 0.02) by 18.5 +/- 1.4% (mean +/- SEM) compared to 11.6 +/- 2.0% in the placebo-treated group (N = 8). No adverse effects were seen with hGH treatment and no major changes noted in milk constituents. The hGH concentrations in milk were low and did not change with therapy. Plasma concentrations of IGF-1 increased significantly within 24 h of hGH treatment and increased further towards the end of the trial to values of 2.6-fold above the pretreatment values. The concentration of IGF-1 in milk was approximately 100-fold lower than those observed in plasma and could only be reliably measured after size exclusion chromatography to remove the interfering influence of IGF binding proteins in the radioimmunoassay. All women treated with hGH showed a small increase in milk IGF-1 concentrations but the values remained within the range of values observed in women receiving the placebo treatment (1.2-4.4 micrograms/l). Growth hormone treatment increased milk volume in normal lactating women during early lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Body growth, carcass composition, and endocrine changes in lambs chronically treated with recombinantly derived insulin-like growth factor-I.

Castrate yearling male sheep were treated for 8 weeks with either 50 micrograms/kg body wt/8 hourly sc insulin-like growth factor-I (IGF-I) (n = 10) or with saline (n = 9). IGF-I treatment increased plasma IGF-I from 235 +/- 17 to 347 +/- 16 ng/ml (P less than 0.001). There was a gradual divergence in body wt (P less than 0.10) between treatment groups. Food intake did not change significantly. The weight of the spleen corrected for body wt increased by 40% (P less than 0.001) and there was a marginal increase in adjusted kidney wt (P less than 0.1). There was no effect of IGF-I on carcass weight or dimensions, or on long bone length, although the weight per unit length of the tibia (P less than 0.05) and femur (P less than 0.10) were increased. There was no effect on wool growth. Plasma IGF binding proteins (IGFBPs) were quantified by ligand blot analysis. In the IGF-I treated group, IGFBP-1 showed a transient increase (P less than 0.05) at day 3 but was similar in both groups at day 55 of treatment. IGFBP-2 was suppressed (P less than 0.05) by day 55 and IGFBP-3 and 4 did not change. Plasma glucose was elevated (P less than 0.05) and plasma insulin was suppressed (P less than 0.01) from 280 +/- 32 pg/ml to 124 +/- 30.4 pg/ml, plasma urea (P less than 0.01) and creatinine (P less than 0.05) were reduced in the IGF-I treated group. The somatogenic effect of IGF-I in this study was minimal suggesting that in the well fed animal with an intact somatotropic axis IGF-I treatment at doses which double plasma IGF-I does not enhance somatic growth performance. However, the marked splenomegaly shows the sensitivity of splenic growth to systemic IGF-I. The suppression of insulin with chronic IGF-I treatment was accompanied by hyperglycaemia--this may explain in part the lack of a significant anabolic response and may limit the utility of IGF-I therapy unless higher doses with insulin-like effects are used.

Ontogenic differences in the nutritional regulation of circulating IGF binding proteins in sheep plasma.

Well-fed castrated male sheep (N = 3) and 125 days gestation pregnant ewes (N = 6) with chronically catheterized fetuses were fasted for 72 h. Insulin-like growth factor-binding protein (IGFBP) levels in fed and starved fetal, maternal and castrated male sheep plasma were measured using ligand blot analysis. IGFBPs in adult and fetal sheep differed in distribution both before and after 72 h starvation. IGFBP-3 was the major postnatal binding protein, while in the fetus IGFBP-2, IGFBP-3 and the circulating IGF type 2 receptor fragment each contributed 25-30% of total IGF binding capacity. After starvation, total IGF binding capacity and IGFBP-3 fell in plasma of maternal and castrated male sheep (p less than 0.05). Total IGF binding capacity rose with starvation in fetal plasma (p less than 0.05) as a result of an increase in IGFBP-1 (p less than 0.01) and IGFBP-2 (p less than 0.05). The different nutritional control of the IGFBPs in the fetus and the adult may reflect ontogenic differences in the regulation and function of circulating IGFs and their binding proteins.

The circulating molecular weight forms of infused recombinant insulin-like growth factor-I and effects on glucose and fat metabolism in lambs.

We have investigated the relationship between the plasma distribution of infused recombinant insulin-like growth factor-I across the insulin-like growth factor binding proteins and the resultant effects on glucose and fat metabolism. The studies were performed in 24-h fasted ram lambs which received primed constant infusions of 3H labelled glucose tracer. When isotopic equilibrium had been reached, the animals received 90-min infusions of human insulin-like growth factor-I at various doses (2.5, 20, 40 and 120 micrograms.kg-1.h-1, n = 3 for each dose). Total plasma insulin-like growth factor-I was significantly elevated by infusion at a rate of 40 micrograms.kg-1.h-1 (from 185 +/- 14 micrograms/l to 442 +/- 41 micrograms/l, p less than 0.05) and 120 micrograms.kg-1.h-1 (from 181 +/- 2 micrograms/l to 953 +/- 39 micrograms/l, p less than 0.005). The plasma concentrations of insulin-like growth factor-I not associated with binding proteins remained undetectable (less than 15 micrograms/l) at the end of the 2.5 and 20 micrograms.kg-1.h-1 doses, but were significantly elevated at the end of the 40 and 120 micrograms.kg-1.h-1 infusions (to 71 +/- 14 micrograms/l, p less than 0.05 and 176 +/- 55 micrograms/l, p less than 0.01 respectively). The infused insulin-like growth factor-I associated primarily with 35-60 kilodalton binding proteins. Glucose kinetics were significantly altered only by the highest dose infusion, during which there was a fall in plasma glucose concentration from 3.5 +/- 0.2 mmol/l to 1.9 +/- 0.2 mmol/l (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoassay for insulin-like growth factor-I: solutions to some potential problems and pitfalls.

This report describes essential requirements for the validation of a radioimmunoassay (RIA) for insulin-like growth factor-I (IGF-I) and presents solutions to some problems and pitfalls commonly observed. The preparation of IGF-I to be used as radioligand or standard has to be selected carefully since some IGF-I preparations are contaminated with variants which demonstrate different potencies for different antisera used in the RIA. Accurate assessment of IGF-I levels in blood plasma requires an efficient extraction method for the IGF-binding proteins (IGFBPs). Extraction methods to remove the influence of IGFBPs in the RIA were compared using blood plasma of considerable differences in IGF-I/IGFBP ratios. Acidification of plasma before column chromatography on Sephadex G-75 (G75) is generally considered to be the most reliable extraction method, but it is very time-consuming. The acid-ethanol extraction (AE) of plasma is not valid in many situations. Non-parallel displacement to the IGF-I standard was observed with AE-extracted plasma samples in the RIA. In addition, a comparison of IGF-I values obtained in the RIA after AE or G75 extraction of fetal ovine plasma has shown no significant correlation. We report an extraction technique based on a modified AE extraction followed by cryo-precipitation (AEC). AEC extraction on blood plasma reduced residual IGFBPs to a level that did not interfere in the assay. Furthermore, AEC-extracted plasma samples showed parallel displacement in the RIA to highly purified preparations of authentic IGF-I. We observed high correlations, with a slope close to unity, of IGF-I values obtained in the RIA using the AEC or G75 extraction for plasma from different species including adult and fetal sheep, rat, mouse and man. The AEC extraction provides a rapid and simple alternative to G75 extraction for blood plasma from a variety of species provided that high-affinity antisera are used for the RIA.