Growth regulation最新文献

The aim of this study was to investigate the influence of hemodialysis on insulin-like growth factor-I (IGF-I) and the IGF binding proteins (IGFBPs) in patients with end-stage renal disease (ESRD). IGF-I and IGF-II circulate bound to IGFBPs which are known to influence the IGF-I bioavailability. Ten ESRD patients were studied before and after hemodialysis on low flux filters. IGF-I, insulin and IGFBP-I were measured by specific RIAs, and IGFBP-2 and IGFBP-3 were quantified by densitometry after Western ligand blotting. Diurnal curves of IGFBP-1 were performed in two additional patients. Before dialysis, the mean (+/- SEM) IGF-I level was 202.2 +/- 12.1 micrograms/l corresponding to a SD-score of 1.8 +/- 0.3. Basal IGFBP-1 was increased 2-fold compared to normal levels (82.4 +/- 24.1 micrograms/l) and increased further during hemodialysis to 118.1 +/- 28.5 micrograms/l (P < 0.007). The mean increase during dialysis in IGFBP-1 was 74 +/- 24%. Predialysis IGFBP-2 was increased to 184.8 +/- 32.5% of the reference serum and was not significantly changed by dialysis. The predialysis IGFBP-3, 38.5 kDa band was within normal levels 90.1 +/- 18.8% of the reference serum while the IGFBP-3, 41.5 kDa band was decreased to 62.4 +/- 11.3% of the reference serum. Both IGFBP-3 bands were not significantly changed after dialysis. The mean basal insulin level was high, 38.2 +/- 3.0 mU/L, in spite of normal glucose levels suggesting insulin resistance. The mean values of IGF-I, insulin and glucose were unchanged after dialysis. The ratio between IGF-I and IGFBP-1 decreased significantly after dialysis to 53% of the ratio before dialysis (P < 0.005). The ratio between IGF-I and IGFBP-2 or IGFBP-3 did not change after dialysis. The circadian variation of IGFBP-1 during dialysis days was impaired with a delayed decrease of IGFBP-1 compared to the non-dialysis day. In ESRD patients predialysis mean values of insulin, IGF-I SD-score, IGFBP-1 and IGFBP-2 were increased, while the mean densitrometric values of the IGFBP-3 bands on Western ligand blot were either normal or reduced. IGFBP-1 was raised significantly with a mean of 74% after dialysis, the predialysis level was more than 2-fold elevated with impaired circadian variation of IGFBP-1 on dialysis days. High levels of IGFBPs may bind free IGF-I and decrease IGF-I bioavailability thus contributing to the catabolism associated with dialysis.

The growth-promoting and metabolic effects of recombinant ovine placental lactogen (oPL) were compared with those of recombinant bovine growth hormone (bGH) in young lambs. Lambs were treated by twice daily subcutaneous injection with oPL (n = 16) or bGH (n = 16) at a dose of 0.1 mg/kg live weight/day or with saline (n = 16) for 21 days commencing on day 3 of life. Jugular blood samples were taken on days 0, 10 and 20 of treatment. Half the lambs in each group were slaughtered at 24 days, and the other half at 9 months of age. Both bGH and oPL treatments induced small but significant (P < 0.05) increases in circulating concentrations of insulin-like growth factor-I (IGF-I) on day 10 of treatment, but not on day 20. Neither treatment altered plasma concentrations of glucose, non-esterified fatty acids, urea or creatinine compared to those in saline-treated lambs. Relative to those of bGH-treated (0.24 +/- 0.01 kg/day) or saline-treated (0.25 +/- 0.01 kg/day) lambs, live weight gains of oPL-treated lambs (0.28 +/- 0.01 kg/day) were significantly (P < 0.05) increased during treatment and differences in live weight were still apparent at 9 months of age. Similarly, treatment with oPL, but not bGH, significantly (P < 0.01) increased daily energy intake. It is concluded that placental lactogen and growth hormone do not have identical biological actions. While oPL is growth-promoting in young lambs, this effect may be mediated by stimulating voluntary feed intake rather than by elevating circulating concentrations of IGF-I.

Measurements of serum levels of insulin-like growth factor (IGF)-I, IGF-II and IGF binding protein (IGFBP)-1 have been carried out in conjunction with Western ligand blot analysis of serum IGFBPs in 39 constitutionally short children and adolescents and compared with those of 27 age-matched normal subjects (and also with 23 hypopituitary patients). Estimated amounts of the two forms of IGFBP-3 (42 and 39 kDa) and of IGFBP-2 (34 kDa) were obtained by laser densitometry scanning. Mean serum levels of IGF-I were decreased by 46% +/- 5% in short, compared to normal, prepubertal children (P < 0.01) and reduced slightly, but not significantly, in short pubertal children. IGFBP-1 levels decreased with age in short children, as they did in normals, but average values were significantly higher in short children (P < 0.001). There was also a tendency for higher IGFBP-2 levels in short prepubertal and pubertal children. IGFBP-3 bands were of equal intensity in short and normal subjects. Physiologically, IGFBP-3 undergoes limited proteolysis which results in facilitated dissociation of the IGFs, particularly IGF-I, and an increase in their turnover. Western immunoblotting detects proteolytic fragments of IGFBP-3 (the major one being of 30 kDa) that are not detected by ligand blotting. The ratio of proteolysed to total IGFBP-3 in short prepubertal children (36.8% +/- 2.6%) was significantly lower (P < 0.01) than in normal prepubertal subjects (60.6% +/- 8.9%). This lesser proteolysis of IGFBP-3 would explain the excessive levels of IGFBP-3 (detected by ligand blotting) relative to IGF levels in short children. These results suggest that growth retardation in short children involves IGF-I deficiency resulting from both decreased IGF-I synthesis and lesser bioavailability of the circulating IGF-I bound to IGFBP-3. High IGFBP-1 levels may also contribute towards limiting the availability of IGF-I to its target cells.

Production of insulin-like growth factor binding protein (IGFBP)-2 and accumulation of IGFBP-2 mRNA was determined in six leukaemic T-, B- or promyelocytic cell lines. Cell growth was compared in serum free medium M-3 and in medium M-9 containing 5% FCS. In both media, high amounts of IGFBP-2 as measured by radioimmunoassay were detectable in culture supernatant of T-cell lines and promyelocytic HL-60 cells, whereas only small amounts of IGFBP-2 were secreted by the B-cell lines. Production of IGFBP-2 in M-9 was approximately 20-fold higher (up to 195 ng ml-1) than in M-3, partially reflecting higher proliferation. However, quantitative reverse transcriptase polymerase chain reaction analysis revealed that, independent of the culture medium 10(6) T-cells contained between 30 and 48 units IGFBP-2 mRNA relative to the glycerol aldehyde phosphate dehydrogenase control gene, but B-cells contained less than 1 unit. Since IGF-II is known to be a major regulator of IGFBP-2, its influence on IGFBP-2 expression has to be investigated.

The role of placental lactogen (PL) in the regulation of maternal metabolism and fetal growth is not understood. Both PL and growth hormone (GH) have been suggested as possible regulators of mammogenesis. Our aim was to compare the effects of recombinant ovine placental lactogen (oPL) and bovine growth hormone (bGH) on maternal mammary gland development and fetal growth. Pregnant ewes were treated from day 101 to 107 of gestation with twice daily subcutaneous injections of recombinant oPL (n = 7), bGH (n = 8) (0.15 mg/kg live weight/day) or saline (n = 8). On day 108 of gestation, fetal and maternal tissues were collected. The relative abundance of growth hormone receptor (GHR), insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) mRNA was assessed in mammary gland, maternal liver and heart, and in fetal and placental tissues. There was no detectable change in mammary tissue GHR, IGF-1 or IGFBP-3 gene expression with either bGH or oPL treatment. Maternal administration of bGH, but not oPL, during pregnancy caused an increase in maternal hepatic IGF-1 gene expression (P < 0.005). Treatment with oPL, but not bGH, resulted in a significant increase (P < 0.025) in the relative abundance of fetal hepatic IGFBP-3 mRNA. Maternal hepatic GHR gene expression was not affected by treatment. This study suggests that while bGH treatment of pregnant ewes induces characteristic somatogenic responses, oPL treatment does not have comparable effects. However, oPL may indirectly influence the fetal somatotropic axis by altering fetal hepatic IGFBP-3 production.

Genetic differences in growth potential could result from changes in the levels of growth stimulatory factors or in the response of target tissues. The latter possibility was tested in adult myoblasts prepared from chickens selected for high (HG) or low growth rate (LG). Stimulation of [3H]-thymidine incorporation into DNA by serum was of higher amplitude in HG than LG muscle cells irrespective of whether the cell preparations were enriched in myoblasts or fibroblasts. HG myoblasts were also more responsive to insulin-like growth factor-I (IGF-I) in terms of [3H]-thymidine incorporation. IGF analogues with a reduced affinity for IGF binding proteins gave similar results suggesting that activity of binding proteins could not explain the difference between cells from the HG and LG lines. This difference was restricted to the proliferative stage because in myotubes, basal or IGF-I stimulated glucose and amino acid transports, tyrosine incorporation and protein degradation were not different.

Both articular cartilage and the central nervous system are target organs for insulin-like growth factors (IGFs). We have previously described the hormonal regulation of IGF binding proteins (IGFBPs) in the conditioned media (CM) of rat articular chondrocytes and in a rat neuroblastoma cell line (B104). In the studies presented here, we have investigated the role of IGFBP-5 proteases in these complex systems. Proteolysis of [125I] IGFBP-5 was maximal after 2-3 h incubation with CM of either cell type and did not further increase, even with an incubation of 12 h. Assessment of the effect of pH on protease activity showed that proteolysis was active between pH 6 and pH 9, but not at more acidic pH. Among the various protease inhibitors, serine protease inhibitors [benzamidine (100 mM), aprotinin (1 mg/ml), PMSF (10 mM)] and metalloprotease inhibitors [EDTA (1 mM), 1,10-phenanthroline (10 mM)] were the most effective in inhibiting the proteolysis of IGFBP-5, whereas aspartic and cysteine protease inhibitors were ineffective. These results indicate that the IGFBP-5 protease in the conditioned medium of rat articular chondrocytes and B104 cells belongs to a family of serine-metallo proteases. Interestingly, divalent cations, such as Zn+2 (1 mM) and Ca+2 (10 mM) also inhibited the IGFBP-5 proteolysis. This effect was not observed with monovalent ions, such as Na+ and K+. We also examined the effect of IGFs on IGFBP-5 protease activity, and found that IGF-I and -II inhibited the proteolysis in cell-free conditioned medium, while des(1-3) IGF-I was less effective. The IGFs may act to protect [125I] IGFBP-5 from the proteases in the CM, although the precise mechanism remains unknown. Thus, IGFBP-5 protease activity produced by both rat articular cartilage and B104 cells is a serine-metallo protease, that is inhibited by divalent cations and in the presence of IGF.

Insulin-like growth factor-binding proteins (IGFBPs) modulate IGF action at cellular level, through either inhibition or potentiation, and they also have intrinsic activity that is independent of their binding to IGFs. In prostate carcinoma (PC-3) cells, which are capable of growth for several days in serum-free medium, non-glycosylated recombinant human IGFBP-3 (rhIGFBP-3) had a biphasic mitogenic effect, stimulation being dose-dependent up to 20 ng/ml, followed by progressive depression down to zero stimulation at 150-200 ng/ml. This mitogenic effect was not intrinsic activity, but involved IGF-II secreted by the cells, since stimulation was abolished in the presence of anti-type 1 IGF receptor antibody (alpha IR-3). Western ligand- and immunoblot analysis of the culture media revealed several IGFBP species, in particular IGFBP-3 which exhibited an electrophoretic profile characteristic of limited proteolysis. The amounts of the proteolytic fragments increased in parallel with the concentrations of added rhIGFBP-3, but a large amount of intact protein remained at the highest concentrations added. When a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulphonyl fluoride (Pefabloc SC), was added at concentrations demonstrated to be non-toxic to the cells, IGFBP-3 proteolysis was diminished and rhIGFBP-3-induced stimulation of proliferation was suppressed. Conversely, in the presence of plasminogen transformed to plasmin by urokinase secreted by the cells, proliferation stimulated by rhIGFBP-3 and its proteolysis were enhanced. Our results suggest that the biphasic mitogenic effect of rhIGFBP-3 on PC-3 cells reflects changes in the availability to the cells of the IGF-II they secrete. This availability depends on the extent of IGFBP-3 proteolysis (which promotes release of bound IGF-II) and on the proportion of intact forms (which sequestrate secreted IGF-II).

The control of embryonic growth in vertebrates appears to rely on the orchestrated action of several families of growth factors and hormones. The contribution of insulin-like growth factor (IGF-I) to prenatal growth regulation is better established in mammals than in other vertebrate species. The status of (pro)insulin gene product(s) in the pancreas and non-pancreatic tissues may be another important contribution to embryonic growth signals. We have characterized tissue sources of IGF-I gene and (pro)insulin gene mRNAs in normal chicken embryogenesis and their changes in a model of avian growth retardation. We studied, by a highly sensitive reverse-transcription coupled to polymerase chain reaction (RT-PCR), the expression of IGF-I and (pro)insulin genes in brain, pancreas, liver and eye in embryos from late organogenesis (E8) to late development (E17); hatching is at E20-21, a period of fast embryonic growth. In brain, pancreas and eye, growth-retarded embryos had lower IGF-I mRNA expression. In contrast, in the liver, little IGF-I mRNA was found during normal embryogenesis, but some early induction occurred in E17 growth-retarded embryos. (pro)insulin gene expression was much lower in absolute levels in non-pancreatic tissues than in pancreas. However, it was developmentally regulated in brain, liver and eye. The growth-retarded, IGF-I-deficient embryos had an increased expression of (pro)insulin mRNA in the brain. While IGF-I treatment of growth-retarded embryos increased their serum IGF-I values, only partial recovery of embryonic weight was obtained. Since abnormalities in other hormones may contribute to the failure of systemic IGF-I to reverse the retarded phenotype, thyroid hormones (T3 and T4) levels were determined in liver, brain and eye. They were markedly altered only in the liver of growth-retarded embryos, where an increase in thyroid hormone content was observed. We conclude that, in chicken embryos and possibly other vertebrates, normal growth may implicate multiple hormones, including the concerted action, endocrine/paracrine, of IGF-I and (pro)insulin gene products.

This study was designed to investigate the feedback loop between insulin-like growth factor-I (IGF-I) and IGF-II and the hypothalamic hormones growth hormone-releasing hormone (GHRH) and somatostatin (SS) using an in vitro rat hypothalamic model. IGF-I and, to lesser extent, IGF-II, both activate type 1 IGF receptors, while type 2 receptors are activated by IGF-II alone. IGF-I, IGF-II, their various specific analogues (Des[1-3]IGF-I, [Arg54/Arg55]IGF-II and [Leu27]IGF-II), insulin and the type 2 receptor antagonist beta-galactosidase were used on their own or in combination to study their effect on GHRH and SS release. Our results suggest that the simultaneous activation of type 1 and type 2 IGF receptors is needed for the negative feedback effect of IGFs on GHRH release in this in vitro system, in agreement with earlier findings in vivo. Somatostatin was not altered by any combination of peptides.