J. Zapf

Radioimmunological determination of insulinlike growth factors I and II in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia.

Serum levels of immunoreactive insulinlike growth factors (IGF) I and II were determined by a modified IGF I and a new IGF II radioimmunoassay in normal children and adults, and in patients with acromegaly, isolated growth hormone deficiency, and extrapancreatic tumor hypoglycemia. Serum samples were gel filtered by a simple routine procedure at acidic pH to dissociate and separate IGF from the IGF carrier protein. Mean immunoreactive IGF I levels (+/- SD; corrected for crossreactivity of IGF II) were 193 +/- 58 ng/ml in normal adult subjects, 712 +/- 245 ng/ml in acromegalic patients and 24 +/- 14 ng/ml in patients with isolated growth hormone deficiency. The lack of growth hormone alone, irrespective of an otherwise normal hormonal status, appears to be responsible for the drastic decrease of IGF I levels. Oversecretion of growth hormone does not increase the levels of immunoreactive IGF II: mean levels (+/- SD; corrected for crossreactivity of IGF I) in normal and acromegalic subjects are virtually identical (647 +/- 126 and 641 +/- 189 ng/ml, respectively). Apparently, normal growth hormone levels stimulate IGF II production already maximally. However in growth hormone deficiency immunoreactive IGF II is significantly decreased (252 +/- 99 ng/ml). Thus, IGF II, like IGF I, is growth hormone dependent. But in contrast to IGF I, the growth hormone dependence of IGF II seems to become apparent only at subnormal growth hormone levels. In normal children IGF I is age dependent: it is low in newborn cord sera (51 +/- 20 ng/ml) and gradually rises into the adult range with increasing age. At the onset of and during puberty mean IGF I levels lie above prepubertal values. In contrast, IGF II levels in normal children are independent of age and pubertal stage beyond the first year of life, whereas newborns have significantly lower IGF II values. Hypoglycemia resulting from extrapancreatic tumors is not associated with increased immunoreactive IGF I or II levels. IGF I is decreased in most of the sera (mean level +/- SD:56 +/- 39 ng/ml) whereas IGF II lies in the normal range (556 +/- 195 ng/ml).

Acute metabolic effects and half-lives of intravenously administered insulinlike growth factors I and II in normal and hypophysectomized rats.

Insulinlike growth factors (IGF) act qualitatively like insulin on insulin target tissues in vitro. In the circulation in vivo they are bound to specific carrier proteins. In this form or when continuously infused into hypophysectomized (hypox) rats they do not exert acute insulinlike effects on glucose homeostasis. This study definitively shows that intravenous bolus injections of pure IGF I or II act acutely on glucose homeostasis: they lower the blood sugar, enhance the disappearance of U-[14C]glucose from serum and increase its incorporation into diaphragm glycogen in normal and hypox rats in the presence of antiinsulin serum. The same effects were obtained with recombinant human IGF I injected intravenously either with or without antiinsulin serum into normal rats. Free fatty acid levels decreased transiently only in normal animals. Lipid synthesis from glucose in adipose tissue was not stimulated in hypox and barely stimulated in normal rats. The half-life of injected IGF I or II in normal rats (approximately 4 h) is strikingly different from that in hypophysectomized rats (20-30 min) and appears to depend on the growth hormone-induced 150,000-200,000-mol wt IGF carrier protein that is lacking in hypophysectomized rats. 15 min after the bolus serum IGF I and II concentrations were similar to steady state levels during long-term infusion in hypox rats. Free IGF was barely detectable, however, in the infused animals, whereas 40-100% was found free 15 min after the bolus. These observations for the first time confirm the hypothesis that only free IGF, but not the IGF carrier protein complex, is bioavailable to insulin target tissues.

Insulin-like growth factors and insulin: comparative aspects

SummaryIGF I and IGF II are two insulin-like growth factors resembling insulin in many respects. They stem from a common precursor, act through receptors similar to the insulin receptor with which they cross-react. When administered in large amounts they produce hypoglycemia. Their major effects, however, are on replication and differentiation of cells of mesodermal origin. IGF I is the major growth promoting factor in vivo. The synthesis and secretion of IGF I by the liver depend on the growth hormone status, insulin and nutrition. In contrast to insulin, the IGFs circulate in blood bound to the carrier proteins. Their half-life in man is in the order of 16 h. IGF I deficiency results in dwarfism (pygmy, Laron dwarf, toy poodle) despite normal or elevated growth hormone secretion. The anabolic actions of insulin and of the IGFs appear to complement each other as shown in Figure 7.

Nonsuppressible insulin-like activity (NSILA) from human serum: Recent accomplishments and their physiologic implications☆

Abstract It is now known that nonsuppressible insulin-like activity extracted from human serum (NSILA-S) consists of at least two chemically and biologically closely related polypeptides with a molecular weight of 7500. Their primary and tertiary structures strikingly resemble that of human proinsulin and suggest a common phylogenetic ancestor. Beyond their acute insulin-like actions on insulin target tissues the two polypeptides exert pronounced effects on growth of cultured cells and on sulfation, RNA and protein synthesis of cartillage. For these reasons they have been termed insulin—like growth factors (IGF I and II). In adipose tissue, their biologic potency is ∼60 times lower than that of insulin and corresponds to their weak affinity for the insulin receptor rather than to their high affinity for a specific IGF-receptor whose functional role is still unclear. By contrast, their biologic potency in heart and skeletal muscle is close to that of insulin ( 1 2 – 1 5 ) although their affinity for the insulin receptor is ∼100 times lower than that of insulin. Therefore, the insulin-like actions of IGFs on muscle appear to be mediated by the IGF-receptor. In fibroblasts and chondrocytes, binding of IGFs to specific high affinity IGF-receptors correlates closely with their effects on growth indices and sulfation. The latter effects occur at ∼50 times lower concentrations than with insulin. In native blood, IGFs are tightly associated with a specific carrier protein and the free forms are barely detectable. Binding to the carrier blocks their action on insulin target tissues, which explains the absence of acute insulin-like effects of endogenous IGF in vivo. In contrast, tissues concerned with growth may possess mechanisms to “extract” IGFs from the carrier complex. IGFs are under the control of growth hormone: total IGF levels are increased in acromegalics and decreased in hypopituitary and Laron dwarfs. This characteristic and their distinct biologic properties coin IGFs as members of the somatomedin family. The close relationship is further underlined by the pronounced cross-reactivity with somatomedins in radioreceptor and radioimmunoassays. Low serum IGF values are also found in patients with liver cirrhosis. Among other evidence, this points to the liver as one site of production of IGFs. Total IGF levels were not found to be elevated in 15 patients with hypoglycemia caused by extrahepatic tumors. Antibodies that allow to discriminate between the two species of IGF have recently been raised in rabbits. From the results of the radioimmunoassays, it becomes apparent that normal human serum contains 4–6 times more IGF II than IGF I. In acromegalic patients, only IGF I is elevated, whereas IGF II lies within the normal range. In hypopituitary and in Laron dwarfs, but also in patients with liver cirrhosis, IGF I is more drastically reduced than IGF II. Thus, IGF I may be the more important growth factor, and the two seem to be regulated differently. Besides carrier-bound IGFs, another large molecular weight peptide with nonsuppressible insulin-like activity is present in human serum. It is not interconvertible to small molecular weight IGF and it is not active on sulfation of cartilage. Therefore, it represents an entity different from IGFs. Thus, bioassays carried out in whole serum detect different insulin-like activities: Since carrier-bound IGF is inactive on adipose tissue the fat pad assay measures large molecular weight NSILA rather than IGF, whereas the sulfation assay reflects the serum level of carrier-bound IGFs and other somatomedins.

The Insulin-Like Growth Factors (IGF) of Human Serum: Chemical and Biological Characterization and Aspects of Their Possible Physiological Role

Publisher Summary This chapter discusses the chemical and biological characterization, and properties of the insulin-like growth factors (IGF). The chapter also discusses their possible physiological role. Blood is a large reservoir of insulin-like activity. Insulin represents only a small portion of this activity. IGF I and II are polypeptides with molecular weights of 7649 and 7471. They contain 70 and 67 amino acid residues, respectively, each in a single polypeptide chain with three intra-chain disulfide bridges. Each of the two polypeptides contains two regions—domains—in which parts of the sequences can be aligned to identical parts in human pro-insulin: 38% of the sequence of residues 42 to 70 in IGF I and 44% of the sequence of residues 41 to 67 in IGF II (termed A-domains), are identical to the insulin A-chain. As in pro-insulin, the A- and B-domains are connected by a C-domain. However, in contrast to the C-peptide of pro-insulin, which consists of 35 amino acids, the C-regions of IGF I and II are only 12 and 8 residues long, respectively.

Binding of nonsuppressible insulinlike activity to human serum: Evidence for a carrier protein☆

Abstract When 125 I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of 125 I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound 125 I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of 125 I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely. These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other.

Insulin-like growth factor I stimulates lipid oxidation, reduces protein oxidation, and enhances insulin sensitivity in humans.

To elucidate the effects of insulin-like growth factor I (IGF-I) on fuel oxidation and insulin sensitivity, eight healthy subjects were treated with saline and recombinant human (IGF-I (10 micrograms/kg.h) during 5 d in a crossover, randomized fashion, while receiving an isocaloric diet (30 kcal/kg.d) throughout the study period. On the third and fourth treatment days, respectively, an L-arginine stimulation test and an intravenous glucose tolerance test were performed. A euglycemic, hyperinsulinemic clamp combined with indirect calorimetry and a glucose tracer infusion were performed on the fifth treatment day. IGF-I treatment led to reduced fasting and stimulated (glucose and/or L-arginine) insulin and growth hormone secretion. Basal and stimulated glucagon secretion remained unchanged. Intravenous glucose tolerance was unaltered despite reduced insulin secretion. Resting energy expenditure and lipid oxidation were both elevated, while protein oxidation was reduced, and glucose turnover rates were unaltered on the fifth treatment day with IGF-I as compared to the control period. Enhanced lipolysis was reflected by elevated circulating free fatty acids. Moreover, insulin-stimulated oxidative and nonoxidative glucose disposal (i.e., insulin sensitivity) were enhanced during IGF-I treatment. Thus, IGF-I treatment leads to marked changes in lipid and protein oxidation, whereas, at the dose used, carbohydrate metabolism remains unaltered in the face of reduced insulin levels and enhanced insulin sensitivity.

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

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.

Regulation of binding proteins for insulin-like growth factors (IGF) in humans. Increased expression of IGF binding protein 2 during IGF I treatment of healthy adults and in patients with extrapancreatic tumor hypoglycemia.

UNLABELLED Insulin-like growth factors (IGFs) in blood form two complexes with specific binding proteins (BPs): a large, growth hormone (GH)-dependent complex with restricted capillary permeability, and a smaller complex, inversely related to GH, with high turnover of its IGF pool and free capillary permeability. The distribution of BPs and of IGFs I and II between these complexes was studied in sera from healthy adults treated with IGF I or/and GH and from patients with extrapancreatic tumor hypoglycemia. Like GH, IGF I administration raises IGF I and two glycosylation variants of IGFBP-3 in the large complex, but unlike GH drastically reduces IGF II. During IGF I infusion, IGFBP-3 appears in the small complex whose IGFBP-2 and IGF I increase three- to fivefold and fivefold, respectively. GH treatment, associated with elevated insulin levels, suppresses IGFBP-2 and inhibits its increase owing to infused IGF I. The small complex of tumor sera contains increased amounts of IGFBP-2 and -3, and two- to threefold elevated IGF II. CONCLUSIONS low GH and/or insulin during IGF I infusion and in extrapancreatic tumor hypoglycemia enhance expression of IGFBP-2 and favor partition of IGFBP-3 into the small complex. Free capillary passage and high turnover of its increased IGF I or II pools may contribute to compensate for suppressed insulin secretion during IGF I infusion or to development of tumor hypoglycemia.

Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia?

The pathogenesis of extrapancreatic tumor hypoglycemia has been related to the secretion of big insulin-like growth factor (IGF) II by the tumor. In 25 of 28 patients with this type of hypoglycemia we found 1.5-8-fold elevated serum levels of immunoreactive big (15-25 kD), but decreased levels of normal IGF II. After removal of the tumor, big IGF II disappeared and normal IGF II increased. Tumors contained elevated levels of IGF II, 65-80% in the big form. The insulin-like bioactivity of big IGF II and its affinity towards IGF-binding proteins (IGFBP)-2 and -3 are similar to those of normal IGF II, but two- to threefold higher on a molar basis. Big IGF II is mainly bound to the 50-kD IGFBP complex. The latter contains approximately 10 times more of this peptide than in normal serum and displays three- to fourfold increased insulin-like bioactivity. The formation of the 150-kD IGFBP complex with 125I-recombinant human IGFBP-3 is impaired in tumor serum. This results in sequestration of IGFBP-3 and predominant association of big IGF II with IGFBP-2 and -3 in the 50-kD complex. Increased bioavailability of big IGF II in this complex due to unrestricted capillary passage and enhanced insulin bioactivity of this big IGF II pool provide a continuous increased insulin-like potential available to insulin and type 1 IGF receptors of insulin-sensitive tissues and thus may lead to sustained hypoglycemia.