ABSTRACT

Rat pituitary FSH was fractionated by chromatofocusing between pH 6 and 3. Ten components were resolved having apparent isoelectric points between 3·1 and 5·1. A comparative study of pituitary FSH and FSH secreted in vitro by quartered pituitary glands in the presence and in the absence of gonadotrophin-releasing hormone (GnRH) revealed similar patterns of charged species of intracellular and released FSH. Although GnRH increased FSH secretion about fourfold, no influence on the pattern of charged species was observed. Utilizing exclusion chromatography and chromatofocusing, pituitary FSH was compared to serum FSH which had been extracted by immunoaffinity chromatography. The results demonstrate for serum FSH a larger molecular size and a relative shift to more acidic components. Metabolic clearance rates of eight FSH components separated by chromato-focusing were measured in adult male rats. Half-lives varied between 13 min and several hours. A correlation existed between decrease of isoelectric points and decrease of metabolic clearance rates. These findings suggest that (1) all hypophysial FSH components are secreted into the circulation at similar rates and (2) the more acidic FSH components which appear to contain increased sialic acid, have a longer circulatory half-life and are more abundant in serum. It is concluded that sialylation may be involved in modulating serum FSH levels.

J. Endocr. (1985) 105, 29–37

ABSTRACT

Insulin-like growth factor-II (IGF-II) may be one of the most important local growth factors in human fetal adrenals (HFAs), where its mRNA levels are upregulated by ACTH. We have investigated whether protein kinase C (PKC)-dependent mechanisms and various polypeptide growth factors participate in the regulation of IGF-II gene expression in cultured HFA cells, and whether HFA cells secrete IGF-II peptide into the culture medium. ACTH enhanced IGF-II mRNA accumulation dose- and time-dependently, maximally four- to sixfold, and this increase was inhibited dose-dependently (0·01-100 μg/l) by 12-O-tetradecanoyl phorbol-13-acetate (TPA), a PKC activator. TPA decreased basal IGF-II mRNA levels by approximately 55%. Staurosporine, a PKC inhibitor, abolished the inhibitory effects of TPA and induced accumulation of IGF-II mRNA. Dibutyryl cyclic AMP, cholera toxin and forskolin increased IGF-II mRNA accumulation as much as ACTH, and TPA inhibited these stimulations in a similar way. ACTH increased the IGF-II peptide concentration in most experiments, but this increase was modest in comparison with IGF-II mRNA changes. TPA, although it decreased IGF-II mRNA levels, tended to increase IGF-II peptide in the medium. Additions of GH, IGF-I and IGF-II to the cell culture medium also increased IGF-II mRNA accumulation. Transforming growth factor-β1 inhibited IGF-II mRNA accumulation to the same extent as TPA. Epidermal growth factor and basic fibroblast growth factor did not change IGF-II mRNA levels. Our results confirm previous reports that ACTH is an important regulator of IGF-II in human fetal adrenals, and show that IGF-II gene expression is under multifactorial control, which includes the PKC system and polypeptide growth factors.

Journal of Endocrinology (1993) 137, 533–542

ABSTRACT

This study concerned the resolution of rat pituitary FSH utilizing chromatofocusing. Among the 11 components resolved and positively identified, ten had apparent isoelectric points (pI) between 3·1 and 5·1. Approximately 1% of pituitary FSH eluted at pH 9·4. Treatment with varying amounts of neuraminidase followed by refocusing generated FSH components of higher pI values. Treatment with other glycosidases did not alter the elution characteristics in chromato-focusing, while exclusion chromatography established an inverse relationship between apparent molecular weight and pi. Dose–response curves of various FSH components and of the reference preparation in the current radioimmunoassay system were parallel to each other. A study of their in-vitro bioactivity, utilizing granulosa cells which produce a plasminogen activator due to FSH in a dose-dependent manner, provided the following evidence: increased acidity of the components led to (1) an increase of maximum response and (2) an increase of the dose necessary for half-maximum response. Considering the observed alterations in the hetereogeneity of FSH with changing physiological states of the animal, it is concluded that qualitative changes of the FSH molecule are perhaps involved in a modulatory role in the biopotencies of the hormone.

J. Endocr. (1985) 105, 17–27

Abstract

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 μg/l) but fell after birth P<0·05) and plateaued after 1 year of age (340 ± 20 μg/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.

Journal of Endocrinology (1995) 144, 75–82

Abstract

Human phaeochromocytomas abundantly express insulin-like growth factor-II (IGF-II), but its regulation and biological role in these neoplasms is not known at present. To clarify the regulation of IGF-II gene expression in phaeochromocytomas, we studied the effects of glucocorticoids, nerve growth factor (NGF), and protein kinase A and C regulators in primary cultures of human phaeochromocytoma cells. Cytoplasmic RNA was extracted and analysed by Northern and dot blotting with a ³²P-labelled cDNA probe for IGF-II. Dexamethasone treatment (500 ng/ml) for 3 and 7 days resulted in a 260% and 515% increase in the accumulation of IGF-II mRNA respectively. The stimulatory effect of dexamethasone was time-and dose-dependent. The increases in the 6·0 and 2·2 kb species of IGF-II mRNAs were the most apparent. Cortisol (1 μg/ml) increased the amount of IGF-II mRNA by threefold compared with the control. NGF (200 ng/ml), dibutyryl cyclic AMP (1 mm) and 12-O-tetradecanoyl phorbol-13-acetate (a protein kinase C activator; 100 ng/ml) had no significant effect on IGF-II mRNA levels. These data suggest that IGF-II gene expression in human phaeochromocytomas may be regulated by microenvironmental glucocorticoids.

Journal of Endocrinology (1994) 142, 29–35

Abstract

Insulin-like growth factor-II (IGF-II) and IGF-binding protein-2 (IGFBP-2) were measured in amniotic fluid, extraembryonic fluid and maternal serum from 20 women with apparently normal first trimester pregnancies prior to termination. A further 111 specimens of amniotic fluid were collected from women at 10–20 weeks of pregnancy. Levels of IGFBP-2 were similar in coelomic fluid and maternal serum. Levels in amniotic fluid were lower than those in serum and coelomic fluid (Mann–Whitney test; P=0·0002 and P<0·0001 respectively). The levels of IGF-II were much higher in maternal serum than in coelomic fluid, and higher in the latter than in amniotic fluid (Mann–Whitney test; P<0·0001 for both situations). The levels of IGFBP-2 were relatively low at 10–11 weeks (medians 19·8 and 61·1 μg/l) but thereafter increased to 20 weeks (median 1400 μg/l). The levels of IGF-II showed a similar pattern. The findings suggest that the role of IGF-II and IGFBP-2 in the regulation of growth or differentiation of the fetus or of its surrounding membranes may change with advancing pregnancy.

Journal of Endocrinology (1994) 142, 379–383

Abstract

Inhibition of growth in man and laboratory animals by glucocorticoid treatment is well recognized, yet we have previously shown that glucocorticoids may paradoxically enhance GH secretion and increase serum insulin-like growth factor (IGF) levels. IGFs circulate bound to high-affinity binding proteins (IGFBPs) which modulate their actions, and circulating GH may be associated with two binding proteins (GHBPs) of which the high-affinity GHBP has been characterized and is structurally identical to the extracellular domain of the GH receptor.

We have investigated the time-course of changes in GH, IGFs and their binding proteins induced by glucocorticoid treatment in normal male volunteers (n=12, age range 22–31 years) sampled at 0800 h daily before and during treatment with dexamethasone (2 mg twice daily) for 5 days. In addition, subjects were sampled at 30-min intervals over 7-h periods (0730–1430 h) during the day prior to dexamethasone (day 0), on day 1 following the first dose of dexamethasone and on day 5 following the last dose of dexamethasone. Mean serum IGF-I rose over the initial 72 h and remained elevated at 96 h (297 ± 11·5 compared with basal levels of 215·5 ± 9·3 μg/l, P<0·001) whereas IGF-II levels did not change (472·6 ± 20·5 vs 450·3 ± 21·7 μg/l, P=0·97). There was a concomitant rise in serum IGFBP-3 from basal levels of 3·69 ± 0·23 mg/l to a peak at 5 days of 4·16 ± 0·21 (P=0·003 vs day 1). Mean fasting IGFBP-1 levels fell significantly within 24 h, remaining low throughout the 5-day period whilst fasting insulin and C-peptide levels increased. IGFBP-2 rose within 24 h from basal levels of 315·5 ± 27·9 to 407·8 ± 27·3 μg/l at 24 h (P=0·01), then fell steadily to reach a nadir at 5 days of 240·4 ± 18·9 μg/l (P=0·02 vs basal levels). Peak GH secretion on day 1 (14·5 ± 3·7 μg/l) occurred between 4 and 5 h after dexamethasone administration. On day 5, the time of peak GH secretion was similar but the peak was attenuated (7·9 ± 1·2 μg/l, P<0·01 vs day 1). No morning rise in GH secretion had been observed prior to dexamethasone (day 0). GHBP fell steadily during the 5-day treatment period from basal levels of 29·3 ± 1·9% to day 5 levels of 24·4 1·6%, P=0·001.

The fall in GHBP as a result of dexamethasone treatment is in accordance with the reported inverse relationship between 24-h GH secretion and GHBP in health and disease states in man. This study has demonstrated clear temporal relationships between alterations in circulating levels of GH, IGF-I and IGFBP-1, -2 and -3 during dexamethasone treatment which support possible mechanisms whereby glucocorticoids induce a catabolic state through their endocrine and local tissue effects.

Journal of Endocrinology (1994) 142, 547–554

ABSTRACT

Glucocorticoids inhibit somatic growth in man and laboratory animals, and have long been regarded as suppressors of both stimulated GH secretion and insulin-like growth factor (IGF) activity. Recent evidence suggests, however, that glucocorticoids can be potent GH secretagogues in their own right with concomitant increases in circulating IGF-I levels. IGFs circulate tightly bound to a group of high-affinity binding proteins (IGFBPs) which modulate their actions. In order to investigate the effects of glucocorticoids on serum levels of IGFs and IGFBPs, normal male volunteers were sampled over 24-h periods before and directly after treatment with dexamethasone (2 mg twice daily) for 96 h. Following dexamethasone administration, all volunteers showed a marked increase in mean± s.e.m. IGF-I levels over the 24-h sampling period (292·2±31·8 before dexamethasone, 425·9 ±37 μg/l after dexamethasone, P<0·005); there was no change in mean IGF-II levels. Integrated mean insulin levels were raised by dexamethasone treatment (50 ±4·6 before dexamethasone, 117±13·4 mU/l after dexamethasone, P= 0·002) and IGFBP-1 was significantly suppressed (42·9±8·2) before dexamethasone, 28·0±7·9 μg/l after dexamethasone, P<0·001). IGFBP-2 levels were similarly suppressed after dexamethasone (319·5±24·5 before dexamethasone, 214·8±8·5 μg/l after dexamethasone, P=0·002), and there was a significant increase in IGFBP-3 levels from 3·24 ±0·25 to 3·67±0·32 mg/l (P=0·0153). Mean IGF bioactivity over the sampling period after dexamethasone was reduced by approximately 60% (0·93 ±0·39 before dexamethasone, 0·39 ±0·05 U/ml after dexamethasone, P <0·0001).

There were strong negative correlations between both insulin and IGF-I levels and those of IGFBP-2, suggesting the presence of a novel regulation mechanism for this binding protein. The established inverse relationship between insulin and IGFBP-1 was maintained after dexamethasone. These results suggest that glucocorticoids alter the bioactivity of IGFs, possibly by induction of inhibitors, but that neither IGFBP-1 nor IGFBP-2 can be implicated as glucocorticoiddependent inhibitors of IGF bioactivity in man. The study also demonstrates for the first time a strong negative correlation between IGF-I, insulin and IGFBP-2.

Journal of Endocrinology (1993) 136, 525–533