Three different lines of research and three entirely different observations have led to the discovery of the insulin-like growth factors (IGFs). In 1957, Salmon & Daughaday carried out an important experiment (44). They observed that serum stimulated the incorporation of 35S into incubated cartilage. Serum of hypophysectomized rats was devoid of this sulfation activity. It could not be reconstituted by addition of growth hormone to the incubation medium but reappeared after administration of growth hormone to hypophysectomized rats (44). These observations led Daughaday to postulate that growth hormone itself does not stimulate growth processes in vitro and in vivo, but rather induces the formation of factors that mediate the message of growth hormone. These factors-first called sulfation factors, later soma­ tomedins-Iead to sulfation of cartilage in vitro, which reflects growth in vivo. As we shall see, this hypothesis has been proven to be valid. Most cultured cells need serum to grow. Several growth factors in serum are responsible for growth-promoting effects, an aspect that will be discussed below in more detail. In 1972, Pierson & Temin extracted factors from serum, which they called multiplication-stimulating activity (MSA) (37). These fac­ tors had molecular weights below 10,000. When added to culture medium, they stimulated cells to replicate. Later it was found that cultured liver cells secrete MSA into the culture medium (10). This was the first demonstration that MSA is produced in the liver and that MSA may lead to autocrine stimulation of the same cells by which it is secreted. The third road towards the discovery of insulin-like growth factors departed from an entirely different observation: Serum exerts insulin-like effects on

Actions of Insulin-Like Growth Factors

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb15595.x

Insulin-like growth factors I and II

No agreement exists concerning the necessity for extracting serum somatomedin (Sm) before radioreceptor assay (RRA) and RIA. We have developed a simplified system of Sm extraction which has permitted a quantitative comparison of native and extracted Sm. To 0.8 ml of a mixture of 87.5% ethanol and 12.5% 2 N HC1, 0.2 ml serum is added. After centrifugation, 0.5 ml supernatant is neutralized with 0.2 ml 0.855 M Tris base (XT). Recovery of added 125I-labeled insulin-like growth factor I ([125I]IGF-I) was nearly quantitative. Neutral gel filtration studies established that virtually all of the binding complex was removed by acid-ethanol precipitation. The addition of 100 µl Tris-neutralized acid-ethanol buffer (AE B) to the [125I]IGF-I human placental membrane RRA produced an insignificant shift in the displacement curve with partially purified IGF reference preparation; the shift of the [125I]Sm C RIA displacement curve was greater but did not prevent accurate measurement when AE B was added to blank and stan...

Inhibition of Access of Bound Somatomedin to Membrane Receptor and Immunobinding Sites: A Comparison of Radioreceptor and Radioimmunoassay of Somatomedin in Native and Acid-Ethanol-Extracted Serum

Binding proteins for the insulin-like growth factors: structure, regulation and function

IGF-I and -II share specific serum carrier proteins which elute on neutral Sephadex G-200 gel permeation chromatography at apparent molecular masses of 50 and 200 kD. The half-lives of free and carrier protein-bound 125I-IGF-I and -II were determined after bolus injections of the tracers into two normal adults. Labelled IGF-I and -II migrated first with the 50-kD and later with the 200-kD complex. In these complexes their apparent half-lives were 20-30 min and 12-15 h, respectively. The apparent half-life a free 125I-IGF-I and -II was 10-12 min. In a second set of experiments, recombinant human insulin-like growth factor I was infused during 6 days in two healthy adults at a dose of 20 micrograms.kg-1.h-1 (corresponding to around 30 mg/day). Serum obtained before and during the infusion was subjected to neutral Sephadex G-200 gel permeation chromatography and fractions were pooled according to the apparent molecular masses at which the carrier protein complexes elute. IGF-I and -II in these pools were determined by RIA. Before the IGF-I infusion, 92 and 272 micrograms/l of IGF-I and -II were found in the 200-kD complex, 45 and 91 micrograms/l in the 50-kD complex, and 15 and 5 micrograms/l were present in the free form. Corresponding figures during the IGF-I infusion were 389 and 18 micrograms/l for the 200-Kd complex, 201 and 54 micrograms/l for the 50-kD complex, and 80 and less than 1 microgram/l for free IGF-I and -II. Using the half-lives of the tracer studies and the levels of the different molecular weight forms of IGF in serum, the production rates for IGF-I and -II were calculated to be 10 mg and 13 mg per day.

Insulin-like growth factors I and II in healthy man. Estimations of half-lives and production rates.

Nonsuppressible insulin-like activity extracted and purified from human serum (NSILA-S) mimics all insulin-like effects in vitro and, after injection, in vivo in the presence of excess insulin antibodies. However, there is no evidence that it exerts acute insulin-like effects in its native form in the circulation, where it is almost completely bound to a specific large molecular weight carrier protein. In this paper we show that partially purified NSILA-S-carrier protein, devoid of endogenous insulin-like activity, inhibits the stimulatory effect of NSILA-S, but not of insulin, on 3-0-methylglucose transport and on lipogenesis from [U-(14)C]glucose in isolated rat fat cells. Concomitantly, it prevents binding of (125)I-labeled NSILA-S to the insulin receptor and to the NSILA-S-binding site. The following explanation is, therefore, offered for the absence of acute insulin-like effects of native NSILA-S in vivo: In native serum NSILA-S occurs almost exclusively as NSILA-S-carrier complex. According to recent findings the passage of this complex through blood capillaries is restricted. The present results indicate that, in addition, it is metabolically inactive, or, at least, possesses reduced metabolic activity. The well-known phenomenon that whole serum, nevertheless, exerts pronounced nonsuppressible insulin-like effects on adipose tissue in vitro seems, therefore, to be mainly caused by the presence of a large molecular weight insulin-like protein not identical to the NSILA-S-carrier complex.

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J. Grunwald

Papers

Inhibition of the Action of Nonsuppressible Insulin-Like Activity on Isolated Rat Fat Cells by Binding to its Carrier Protein