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F Rivero, L Goya, and A M Pascual-Leone

Abstract

Insulin-like growth factors (IGFs) are considered to be endocrine regulators during development, and different species have been used for the study of these factors during the perinatal period. The neonatal rat is a very useful model widely utilized to study endocrine alterations throughout the developmental period; few references in the literature present the neonatal rat as a model for the study of IGFs however.

This study was undertaken to compare two extraction methods, acid–ethanol cryoprecipitation (AEC) and formic acid–acetone (FA), for the removal of IGF-binding proteins (IGFBPs) from neonatal and adult rat serum in fed (control) and undernourished populations prior to measurement of IGF-I by radioimmunoassay (RIA). The IGF-I values obtained by RIA following AEC or FA extraction were compared with those obtained following gel filtration (GF), which is considered to be the reference method. Western ligand blotting was used to determine IGFBPs in unextracted serum and after AEC or FA extraction of serum from rats of 10 and 20 days of age and adult rats in both populations. Although serum IGF-I levels after AEC or FA in adult control rats were comparable with those obtained following GF, a significant correlation was found only after AEC extraction both in fed and undernourished adult rats. During the neonatal period, at 10 and 20 days, serum IGF-I levels after AEC or FA extraction were very different from those obtained after GF, especially in undernourished populations, and the correlation was very poor at 10 and 20 days of age in both populations studied.

The IGFBP radioligand blots showed a different rate of change in control and undernourished rats during the period studied. Changes in IGFBP-3 and the band at 30 kDa are reported in both control and undernourished animals. The different proportions of the three major IGFBPs remaining after AEC or FA extraction were also shown. We conclude that AEC extraction, originally validated for use in human serum, is also satisfactory for use in adult but not in neonatal rat serum. However, FA extraction was not a useful method for the rat serum. In neonatal rat serum, the only reliable extraction method was GF.

Journal of Endocrinology (1994) 140, 257–263

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F Rivero, L Goya, C Aláez, and A M Pascual-Leone

Abstract

The purpose of the present study was to investigate the influence of nutrients and insulin on IGFs and their binding proteins (IGFBPs) during the fetal and neonatal periods of three rat populations: (a) rats undernourished by a 35% reduction in the diet from day 16 of gestation, (b) streptozotocin-induced diabetic rats from the same day, or 4 days after birth, and (c) control rats. Fetuses from the diabetic population showed a decrease in insulinemia at 19 and 21 days, along with an increase in glycemia at all stages. Neither glycemia nor insulinemia changed in the fetuses of undernourished mothers, but body weight was decreased at birth. Serum IGF-II decreased at 18 and 19 days of gestation in fetuses from undernourished mothers, and increased at 18, 19 and 21 days in fetuses from diabetic mothers. Serum IGFBPs of low molecular weight (IGFBP-1 and IGFBP-2) increased in the three fetal populations studied, although no changes in serum IGFBPs were found from the effect of undernutrition or diabetes, but fetal liver IGFBP-1 mRNA expression was found to be decreased in undernourished and diabetic animals as compared with controls. In neonatal rats, body weight, insulinemia and serum GH decreased in both undernourished and diabetic rats vs controls, while glycemia decreased in the undernourished and increased in the diabetic group. Serum IGF-II decreased only in diabetic rats and serum IGF-I decreased in both groups. The neonatal serum 30 kDa complex (IGFBP-1 and -2) also increased in undernutrition and diabetes parallel to the expression of mRNA. But, taken together, the changes in IGFBP peptide levels and liver mRNA expression strongly suggest that the 30 kDa complex seems to be composed mostly of IGFBP-1 in the diabetic group and of both IGFBP-1 and -2 in the undernourished animals. The studies of liver mRNA expression of IGFs and IGFBPs confirm the different metabolic control mechanism for the availability of IGFs by the IGFBPs, depending on the animal's maturity. The different adaptation shown by the diabetic neonatal population was confirmed by correlation studies between body weight, glycemia, insulinemia, IGF-I and IGFBPs. The different mechanism of adaptation in diabetic vs undernourished rats seems to be probably due to the decisive role played by hyperglycemia in the diabetic population, and also shows the crucial influence of nutritional status on IGFs and IGFBPs.

Journal of Endocrinology (1995) 145, 427–440

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S Ramos, L Goya, MA Martin, F Escriva, and AM Pascual-Leone

The aim of this work was to study the influence of the endocrine balance between thyroid hormones, insulin and growth hormone (GH) on the regulation of insulin-like growth factor binding proteins (IGFBPs), complementing a study previously reported for insulin-like growth factors (IGFs) in similar populations. Serum concentrations of IGFBPs-1 to -3 were assayed by Western ligand blot and their mRNA expression in the liver assayed by RNase protection assay in the hypothyroid populations: thyroidectomized and mercapto-1-methylimidazole (MMI)-treated neonates, and thyroidectomized adult rats at different periods after thyroidectomy. Serum concentrations of insulin, GH and IGF-I were increased in thyroidectomized neonates and decreased in the other populations. IGFBPs-1 and -2 increased 79% and 50% respectively in thyroidectomized neonatal rats compared with control at 15 days after thyroidectomy, whereas only IGFBP-2 increased (87%) in MMI-treated neonates, which had low serum insulin and GH compared with control on the same days. In thyroidectomized adult rats, IGFBPs-1 and -2 decreased 60% compared with controls on all days studied. Furthermore, when streptozotocin was administered to thyroidectomized neonates and insulin was given to thyroidectomized adult rats to restore insulin to control values in both groups, a differential regulation was found for IGFBPs-1 and -2. The transcriptionally induced decrease in IGFBP-3 (20-25% compared with control in neonates and 50% in adult rats), however, seemed to be regulated by GH and IGF-I. The similarity of changes in IGFBPs found in hypothyroid, undernourished and streptozotocin-induced diabetic neonatal rats suggests that the regulatory effect of insulin or GH on the IGFBPs requires the reduced biologically active thyroid hormone that is found in these three populations.

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S Ramos, L Goya, C Alvarez, MA Martin, and AM Pascual-Leone

The effects of different doses of thyroxine (T(4)) delivered by injection or s.c. pellet implantation on alterations of the IGF/IGF binding protein (IGFBP) system were studied in neonatal and adult thyroidectomized (Tx) rats. Body weight, blood glucose, plasma insulin, TSH and GH and pituitary GH content, as well as serum IGF-I, IGF-II, IGFBP-1, -2 and -3 and their liver mRNA expression were assayed. Pellet implantation with the smaller dose of T(4) (1.5 microg/100 g body weight (b.w.) per day) in Tx neonatal rats decreased serum IGF-I, -II and the 30 kDa complex of IGFBPs (IGFBP-1 and -2), and increased serum IGFBP-3. Only the larger dose of T(4) (3 microg/100 g b.w. per day) recovered liver mRNA expression of IGF-I and ensured euthyroid status as shown by the normalized levels of plasma TSH. The rapid increase of body weight and serum GH after T(4) administration indicated a high sensitivity to T(4) during the neonatal period. Serum and liver mRNA expression of IGFs and plasma insulin and GH recovered in adult Tx rats after pellet implantation of 1.75 microg/100 g b.w. per day throughout 10 days. The continuous replacement of T(4) by pellet seems to be the most suitable method for thyroid rehabilitation. A very good correlation was found between insulin and IGF-II in Tx neonates treated with T(4) but not between insulin and IGF-I in Tx adults. IGFBP-2 seems to be up-regulated by T(4) deprivation in neonatal and adult rats. Finally, a good correlation as well as a partial correlation were found between IGFs and thyroid hormones in both neonatal and adult Tx populations, suggesting a direct effect in vivo of T(4) on the hepatic secretion of IGFs, as previously suggested in vitro.