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 2 5–30% of total IGF binding capacity. After starvation, total IGF binding capacity and IGFBP-3 fell in plasma of maternal and castrated male sheep (p<0.05). Total IGF binding capacity rose with starvation in fetal plasma (p <0.05) as a result of an increase in IGFBP-1 (p< 0.01) and IGFBP-2 (p<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.
Brian W Gallaher, Bernhard H Breier, Mark H Oliver, Jane E Harding and Peter D Gluckman
Helga Sauerwein, Bernhard H. Breier, John J. Bass and Peter D. Gluckman
We evaluated the effect of chronic bovine growth hormone treatment on the hepatic somatotropic receptor. Growing lambs were treated with bGH at 0, 0.05, 0.15, 0.25 or 0.55 mg · kg−1 · day−1 daily (N=5/group) for 56 days. The binding of ovine GH to hepatic membranes washed with 4 mol/l MgCl2 and prepared in the presence of aprotinin was examined. The specific binding of oGH was increased (p<0.01) from 7.1±1.2% in saline-treated controls to 17.4±1.5% in the 0.55 mg · kg−1 · day−1 group. Scatchard analysis showed curvilinear plots that best fitted a two-site model in 22/25 livers. The two sites had estimated dissociation constants (Kd) of 3 to 13 nmol/l for the low-affinity site and a Kd ranging from 0.17 to 0.31 nmol/l for the high-affinity site. Treatment with bGH had no consistent effect on the affinity of either binding site. However, bGH therapy was associated with a dose-dependent increase (p<0.01) in the number of high-affinity somatotropic receptors. There was no effect of bGH therapy on the concentration of low-affinity binding sites. The concentration of high-affinity receptors correlated with weight gain (r=0.54, p<0.01), fat content (r=−0.54, p<0.01), protein content (r=0.40, p<0.05), and plasma IGF-I (r=0.57, p<0.005). The concentrations of low-affinity binding sites showed no such correlations. These observations demonstrate that an important effect of chronic GH therapy in animals with an intact somatotropic axis is to increase significantly the concentration of high-affinity somatotropic receptors. It is suggested that enhancement of the number of high-affinity somatotropic receptors is central to the determination of the efficacy of GH therapy.
Geoffrey R Ambler, Bernhard H Breier, Andrzej Surus, Hugh T Blair, Stuart N McCutcheon, Aviva Silbergeld and Peter D Gluckman
We evaluated the interrelationship between, and regulation of, the hepatic growth hormone receptor and serum GH binding protein (GH BP) in pigs treated with recombinant porcine growth hormone (rpGH). Infant and pubertal male pigs (N = 5 per group) received either rpGH 0.15 mg/kg daily or diluent intramuscularly for 12 days. Somatic growth, serum IGF-I and GH BP and [125I]bovine GH (bGH) binding to MgCl2-treated hepatic membrane homogenates were examined. Marked age-related increases were seen in serum GH BP (p<0.001) and [125I]bGH binding to hepatic membranes (p<0.001). GH BP was increased in rpGH treated animals (p = 0.03), from 13.8±1.2 (mean±1 x sem) (controls) to 17.8±2.0% in infants, and from 35.2±2.6 (controls) to 41.8±3.4% in pubertal animals. [125I]bGH binding to hepatic membranes was also increased by rpGH treatment (p<0.05), from 7.0±1.6 (controls) to 15.4±3.6% in infants and from 53.7±7.1 (controls) to 65.1±11.8% in pubertal animals. No significant interaction between age and treatment was seen. Overall, serum GH BP correlated significantly with [125I]bGH membrane capacity (r=0.82, p<0.001), with a correlation of r= 0.83 in the infant animals but no significant correlation in the pubertal animals considered alone (r=0.13). Serum IGF-I correlated significantly with serum GH BP (r=0.93, p<0.001) and bGH membrane binding capacity (r = 0.91, p< 0.001). These observations suggest that serum GH BP levels reflect major changes of hepatic GH receptor status. In addition, the present study demonstrates that the hepatic GH receptor can be induced by GH in the infant pig, despite a developmentally low GH receptor population at this age, suggesting potential efficacy of GH at earlier ages than generally considered.
Bernhard H Breier, Stella R Milsom, Werner F Blum, Jürg Schwander, Brian W Gallaher and Peter D Gluckman
We performed a double-blind randomized placebo-controlled trial of recombinant human growth hormone (hGH) in normally lactating women (N = 8 per group) to investgate 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). The IGFBP-1 levels were not changed with hGH treatment in plasma (approximately 1.3 nmol/l) or milk (approximately 0.2 nmol/l). Although IGFBP-2 concentrations in plasma were reduced significantly (p<0.05) after hGH treatment (11.1±1.5 before and 8.4±0.9 nmol/l after hGH treatment), milk IGFBP-2 levels did not respond to hGH treatment. Milk levels were markedly higher (sevenfold) in comparison to plasma levels. Plasma IGFBP-3 showed a delayed and smaller rise with hGH treatment in comparison to the rise observed in IGF-I. However, at the end of the study the response (38.6±4.9%) to hGH was significant (p<0.01) and a significant correlation was observed also between the increase in IGFBP-3 and the increase in milk volume (r=0.55, p =0.03, N=16). Plasma IGF-I and IGFBP-3 concentrations correlated significantly when considering all samples of the study (r=0.61, p<0.001, N=63). Milk IGFBP-3 levels were approximately 100-fold lower in comparison to plasma levels and did not correlate with any other measurements. Our data show that hGH-stimulated galactopoiesis in normally lactating women is mediated by significant elevations of plasma and milk IGF-I and plasma IGFBP-3. While IGF-I may be a principal mediator of the galactopoietic effect of hGH, we cannot simply attribute the action of hGH solely to a systemic rise in IGF-I. The increase in plasma IGFBP-3 with hGH treatment suggests that IGFBP-3 could facilitate the delivery of IGF-I to the mammary gland. The high concentrations of IGFBP-2 in milk suggest that mammary epithelial IGFBP-2 may direct regional tissue distribution of IGF-I to the site of milk synthesis.
Stella R Milsom, Bernhard H Breier, Brian W Gallaher, Vanessa A Cox, Alistair J Gunn and Peter D Gluckman
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 μg/l). Growth hormone treatment increased milk volume in normal lactating women during early lactation. This galactopoietic effect of hGH treatment was accompanied by an increase in plasma IGF-1 with no major change in milk constituents and no side effects to the mothers and the babies. Since our results show that a moderate dose of hGH can increase milk volume in healthy, normally lactating women during peak lactation, we suggest that hGH therapy should be considered in future trials for treatment of lactational insufficiency or lactational failure.
Rafat A. Siddiqui, Stuart N. McCutcheon, Duncan D. S. Mackenzie, Hugh T. Blair, J. Eldon Ormsby, Peter D. Gluckman and Bernhard H. Breier
A study was undertaken to investigate the role of testosterone in regulating growth and circulating levels of insulin-like growth factor-I in male mice from lines divergently selected on the basis of plasma IGF-I. Controls of each lines were sham-operated at 10 days of age and treated with peanut oil from day 14 to day 70. A second group, which was castrated at 10 days and treated with testosterone enanthate (0.5 μg · (g body weight) −1 · day−1) from day 14 to 70, did not differ from controls in body weight but had higher plasma IGF-I concentrations. Delaying testosterone therapy until day 42 in a third group retarded growth, with body weights being significantly lower than those of other two groups from days 35 to 56. However, plasma IGF-I levels in this group were not different from those of controls. Effects of line and treatment were additive. It is concluded that the greater pubertal growth of high-line compared to low-line males is not due to greater stimulation of circulating IGF-I by testosterone. Furthermore, testosterone does not appear to influence pubertal growth by acting on circulating levels of IGF-I.