It was one of the riddles of early insulin-like growth factor (IGF) research that the permanent insulin-like potential of circulating IGF by far exceeded that of fasting serum insulin levels without obvious effects on glucose homeostasis. The total serum level of IGF-I and -II in normal adults, but also in insulin-deficient diabetic patients, lies around 1 μg/ml, which corresponds to an insulin-like activity of 200–300 μU/ml of insulin equivalents (1). This insulin-like potential would be expected to cause permanent hypoglycemia and should prevent diabetes. The riddle was solved when highaffinity IGF-binding proteins (IGFBPs) which specifically bind IGFs and limit their bioavailability were discovered in serum. Circulating IGF is found in two major IGFBP complexes with molecular masses of 150 and 40–50 kDa. Approximately 80% of the total serum IGF is associated with the 150 kDa complex. It has a long serum half-life (∼12–16 h) due to restricted capillary permeability
U. Kaufmann, J. Zapf and E. R. Froesch
The effects of hypophysectomy and subsequent growth hormone (GH) treatment on the serum levels of NSILA and of its binding protein were studied in rats.
After hypophysectomy NSILA levels fall to 6 % of the normal. Under GH treatment they rise slowly to 65 % of normal after 12 d. In parallel with the changes of serum NSILA, [35S] sulphate incorporation into costal cartilage in vitro is markedly decreased in hypox animals and restored towards the normal by GH treatment. The relative binding activity of [125I] NSILA-S of serum "stripped" from endogenous NSILA is also reduced after hypophysectomy to approx. 30 % of normal, i. e. to a lesser extent than serum NSILA levels. This concentration of binding protein is sufficient to bind trace amounts of labelled NSILA-S. The half-life of an intravenously injected tracer of [125I]NSILA-S is not significantly decreased in hypox animals. Substitution with GH results in a rise of the relative binding activity to normal after 12 d.
Chromatography of serum, equilibrated with [125I]NSILA-S tracer, on Sephadex G-200 at neutral pH reveals different radiochromatographic patterns for serum from hypox and normal rats: In hypox rats the main peak of radioactivity appears at 60 % bed volume, in normal rats between 45 and 50 %. During GH treatment this main peak shifts from 60 back to 45–50 %.
The distribution of binding activity is similar for normal and hypox rat serum after chromatography on Sephadex G-200 at acidic pH. Furthermore, the binding characteristics of "stripped" hypox and normal serum are identical. This suggests that the same binding protein is present in the normal and hypox rat serum in different molecular forms. It is concluded that GH is a major factor regulating the level of NSILA and of its binding protein in the rat.
U. Kaufmann, J. Zapf and E. R. Froesch
The influence of Dowex-50 adsorption chromatography on the recovery of two different forms of serum NSILA, large and small mol. wt. NSILA, and on the recovery of the binding protein of the small mol. wt. form was studied and compared with another extraction procedure, gel filtration on Sephadex G-50 in 1 m acetic acid.
Partially purified NSILA-S is adsorbed to Dowex-50 at pH 6.8. It can be eluted with 20 mm NH4OH and appears unchanged with regard to its biological activity and molecular weight. Adsorption of 125I-labelled NSILA-S to Dowex-50 does not change its binding characteristics to serum.
When serum is chromatographed on Sephadex G-50 in 1 m acetic acid, NSILA is obtained in a large and in a small molecular weight form (NSILA-S). After recombination of the small molecular weight NSILA fraction with the "stripped" serum fraction, which contains large mol. wt. NSILA and a specific carrier protein for NSILA-S, re-chromatography of this mixture on Sephadex G-50 at neutral pH yields NSILA mostly in the void volume. It adsorbs to Dowex-50. After elution from Dowex, acidic gel filtration on Sephadex G-50 results in an elution pattern which is completely different from that of NSILA-S.
Adsorption of serum to Dowex-50 results in a dramatic decrease of the NSILA-S binding activity.
It is concluded that Dowex-50 adsorption chromatography of serum
- inactivates most of the serum NSILA-S binding protein
- leads to the loss of acid dissociable small mol. wt. NSILA (NSILA-S). Therefore, Dowex-50 adsorption chromatography is not suitable for the subsequent determination or further purification of NSILA-S from whole serum.
C Schmid, C Ghirlanda-Keller and J Zapf
OBJECTIVE: Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits cell growth. Previous reports have suggested the existence of plasma membrane IGFBP-3 receptors that could mediate direct, IGF-independent effects. Thus far, however, the only well-defined putative IGFBP-3 receptor is the type V transforming growth factor-beta (TGF-beta) receptor, a membrane glycoprotein that mediates TGF-beta-induced growth inhibition in selected cells. The aim of the study was to test whether IGFBP-3 and TGF-beta exert short-term effects in an osteosarcoma cell line that produces no IGF but contains type 1 IGF receptors. DESIGN: DNA synthesis and apoptosis in Saos-2/B-10 cells were measured in response to IGF-I, IGF-II, IGFBP-3 and TGF-beta2, and to type 1 IGF receptor ligands with poor affinity for IGFBP-3 ([QAYL]-IGF-I and insulin). RESULTS: IGF-I and IGF-II stimulated thymidine incorporation into DNA and suppressed apoptosis in a dose-dependent manner with maximal effects at 1 and 3 nM respectively. TGF-beta2 slightly increased thymidine incorporation into DNA but had no effect on apoptosis. IGFBP-3 had no effect by itself. Whereas it blocked the above effects of 1 nmol/l IGF-I, it did not block those of 1 nmol/l [QAYL]-IGF-I or 100 nmol/l insulin. CONCLUSIONS: IGFBP-3 does not affect DNA synthesis or apoptosis in an IGF-independent manner in IGF-responsive osteosarcoma cells. It therefore appears to act essentially by sequestration of IGF.
U. VETTER, W. HEIT, O. VELEZ, E. HEINZE and J. ZAPF
U. Widmer, Ch. Schmid, J. Zapf and E. R. Froesch
Abstract. Biological effects of insulin-like growth factors (IGF) I and II on primary cultures of chick embryo liver cells have been investigated and compared 1) with the biological effect of insulin and 2) with competitive binding of the three hormones to their respective binding sites.
IGF I and II stimulate the incorporation of d[U-14C]-glucose into liver cell glycogen in a time- and dose-dependent manner, but with a 5–10-fold lower potency than insulin: Both IGFs also lead to enhanced incorporation of 5-[3H]uridine and l[U-14C]valine into trichloroacetic acid (TCA) insoluble material and to activation of ornithine decarboxylase activity. Their potency in stimulating RNA synthesis and ornithine decarboxylase activity is comparable to that of insulin. Protein synthesis is maximally stimulated at 3 nm by all three hormones. In the competitive binding studies, IGF I and II are 10-fold less potent than insulin in competing for [125I]insulin binding, but 100-fold more potent than insulin in competing for [125I]IGF I or II binding.
These studies show that IGF I and II stimulate the same metabolic indices as insulin in the chick embryo liver. By comparing these biological effects with competitive binding data it appears that IGFs act on glucose metabolism in the chick embryo liver via the insulin receptor, whereas stimulation of growth indices by IGFs and insulin appears to be mediated by their own specific receptors.
U. Schlumpf, R. Heimann, J. Zapf and E. R. Froesch
Non-suppressible insulin-like activity (NSILA) is a term used for a variety of substances in serum, excluding insulin, which promote glucose uptake of adipose tissue and diaphragm in vitro. NSILA-S is a peptide with a molecular weight of 7000 which is soluble in acid ethanol and which has been purified on a large scale from human serum.
This study describes a simple chromatographic one step procedure by which NSILA-S can be extracted and quantitatively measured in individual sera. Using Sephadex G-75 equilibrated with 1 m acetic acid, NSILA-S was detected only in one peak containing small molecular peptides. NSILA-S obtained with this one step chromatographic procedure exerted all the effects of purified NSILA-S including sulphation activity on the rat cartilage. All chromatographic fractions with NSILA-S also had sulphation activity.
Both, NSILA-S and sulphation activity were increased in acromegalics and decreased in pituitary dwarfs. It is suggested that one molecule in serum is responsible for both activities which are, at least in part, under the control of growth hormone.
I. K. Ashton, J. Zapf, I. Einschenk and I. Z. MacKenzie
Abstract. IGF-1 and IGF-2 were measured by specific radioimmunoassay after acid-ethanol extraction of plasma obtained by foetoscopy from 20 normal foetuses aged 15–23 weeks. IGF-1 and IGF-2 levels were 36 ± 11 and 162 ± 55 ng/ml, respectively. In comparison, levels in cord blood were 84 ± 58 and 264 ± 176 ng/ml, respectively, and in adult plasma were 410 ± 106 and 818 ± 272 ng/ml. Both IGF-1 and IGF-2 were in the normal foetal range in a further three foetuses with anencephaly and two foetuses with spina bifida. No sex difference was observed. IGF-1 was positively correlated with foetal body weight (P <0.001), placenta weight (P <0.02) and with body length measured crown-rump (P<0.01) or crown-heel (P < 0.02). No correlation between IGF-2 and body weight, length, placenta weight or gestational age was found.
Both IGF-1 and IGF-2 are present in the human foetal circulation earlier in gestation than has previously been demonstrated, the levels being low throughout this period of gestation in comparison with adult plasma.
J. E. Eigenmann, M. Becker, B. Kammermann, J. Zapf, W. Leemann and E. R. Froesch
Non-suppressible insulin-like activity (NSILA) was determined in 5 dogs before and after hypophysectomy. All NSILA determinations were carried out on serum samples after acidic Sephadex G-50 chromatography by two different assay systems, i. e. a bioassay and a protein binding assay. The levels of NSILA decreased significantly after hypophysectomy and returned to near normal levels after 2 weeks. T3−, T4− and cortisol levels were drastically reduced during the entire period of the experiment. Several GH determinations after hypophysectomy revealed very low levels. Insulin-induced hypoglycaemia failed to provoke a rise of GH levels as late as 4 months after hypophysectomy.
These findings indicate that:
1) The pituitary gland cannot be the site of synthesis of NSILA.
2) NSILA concentrations in the dog are maintained at a near normal level in the presence of very low growth hormone and thyroid hormone concentrations, so that these latter hormones do not appear to be the only regulatory factors concerned in NSILA synthesis.
J. E. Eigenmann, M. Francke-Mäder, J. Zapf and E. R. Froesch
Non-suppressible insulin-like activity (NSILA-S) was determined in dogs after acute changes in the blood sugar and after injection of growth hormone. Serum was chromatographed over Sephadex G-50 columns equilibrated in 1 m acetic acid and NSILA-S was determined in the fractions between 55 and 85 % column volume using a protein binding assay and the conventional fat pad assay. The results obtained with these two methods correlated rather well (r = 0.74). Hyperglycaemia induced by an intravenous glucose load, by intravenous administration of mannoheptulose or both was not followed by an increase in NSILA-S levels. The injection of insulin and human growth hormone did not lead to alterations of the NSILA-S levels. It is concluded that total NSILA-S levels in the dog do not change acutely following manipulations of the blood sugar and that, in all likelihood, NSILA-S plays no role in the regulation of blood glucose.