Insulin-like growth factor characteristics of an acidic non-suppressible insulin-like activity.

The biological activities of an acidic form of non-suppressible insulin-like activity (ILA pI 4.8) have been studied. ILA pI 4.8 was isolated from Cohn fraction IV-1 of human serum by pH 5.5 ion-exchange chromatography on SP-Sephadex. Carrier-bound ILA was eluted at pH 9.7 and then sequentially gel chromatographed in 1% formic acid on Sephadex G-75 and Bio-Gel P-30. The low-Mr (7000) active material was subjected to flat bed isoelectric focusing. Overall recovery was 87 munit of insulin equivalents/100 g of Cohn fraction IV-1, with a specific activity in the range 4-10 munit/mg of protein, representing a purity of 1-6%. This material has been tested in a variety of insulin-like growth factor (IGF)/somatomedin assay systems. It stimulated, in a dose-related manner, [14C]glucose conversion into lipid by isolated rat adipocytes, 35SO4(2-) incorporation into weanling rat costal cartilage and [3H]thymidine incorporation into DNA of cultured human fibroblasts. Like IGF-I and -II, ILA pI 4.8 was able to inhibit degradation of 125I-insulin by crude homogenates of rat liver. In addition, the biological activity of ILA pI 4.8 was completely suppressible by a recently described inhibitor of IGF-I and IGF-II. ILA pI 4.8 was able to compete, in a parallel manner, with 125I-IGF-I and 125I-IGF-II and, at higher doses, with 125I-insulin in a placental radioreceptor assay. No cross-reactivity was seen in a radioimmunoassay for IGF-I and -II C-peptides, but at higher concentrations parallel displacement was observed in a somatomedin C/IGF-I radioimmunoassay using two different antisera. These data indicate that ILA pI 4.8 does possess many of the biological activities previously reported for the IGFs. Since ILA pI 4.8 does occur naturally in serum, it would appear reasonable to tentatively include it as one of the IGF/somatomedin family.     

Recent advances in the biochemistry and physiology of the insulin-like growth factor/somatomedin family

The insulin-like growth factors (IGF) or somatomedins (Sm) are a family of low molecular weight circulating growth factors which have a major, but not absolute, dependence on GH, and have been shown to stimulate body growth and skeletal metabolism in vivo. They are thus considered to mediate the effects of GH on skeletal growth. In humans, the family consists of two well-characterized forms--IGF-I or SmC (a basic peptide) and IGF-II (a "neutral" peptide)--as well as perhaps two less well characterized forms--SmA (a neutral peptide) and an acidic insulin-like activity (ILA pI 4.8). Similar IGF/Sm species have been found and well-characterized in rat serum. Some higher mol wt forms also exist in tissues and body fluids and may represent possible precursor forms. On the basis of in vitro, clinical and in vivo evidence it has been postulated that IGF-I is the primary growth factor in the adult, whilst IGF-II is probably a major foetal growth factor. In vitro the IGF/Sms have a variety of effects including (1) acute insulin-like metabolic actions, which are observed primarily in insulin target tissues and are initiated largely at insulin receptors, and (2) longer term effects, associated with cell growth and skeletal tissue metabolism, and which occur in traditionally non-insulin target tissues, primarily via IGF/Sm receptors. These observations, together with the circumstantial clinical evidence favouring a close association between IGF levels and growth status, clearly indicate a role for IGF/Sms in growth regulation. This concept is now fully supported by the demonstration that IGF-I infused into hypophysectomized (GH-deficient) rats results in increased growth and skeletal metabolism. The physiological regulation of the expression of net IGF activity in vivo is complex and is controlled by the following three determinants: the levels of IGFs, the levels of the specific carrier-proteins and the levels of IGF inhibitors. Both IGFs and their carrier-proteins are influenced by the GH status of the animal as well as by other hormones, nutritional status and chronic illness. Little is known yet about the control of the various IGF inhibitors that have been described. Of importance, however, is the general concept that normal growth is dependent on an adequate balance between all three determinants and that some regard must be had for the contribution of each in determining the overall potential for growth under given circumstances.     

Production of insulin-like growth factor II (IGF-II) and different forms of IGF-binding proteins by HT-29 human colon carcinoma cell line.

The serum-free medium conditioned by the human colon cancer cell line HT-29 contains insulin-like growth factors (IGF) that are entirely complexed to binding proteins (IGF-BP). Gel filtration in acid conditions of the cell-conditioned medium permits separation of IGF-BP from two molecular forms of IGF of 15,000 and 7,500 Mr. As determined by ligand blotting, IGF-BP are heterogeneous and constituted of three molecular forms of 31,000, 28,000, and 26,000 Mr. Using IGF-I and IGF-II radioreceptor assays, IGF-I radioimmunoassay (RIA), and competitive protein-binding assay specific for IGF-II, it is shown that the IGF-type eluting in 15 K and 7.5 K position from gel filtration is restricted to IGF-II. Its concentration is approximately 6 ng/10(6) HT-29 cells with 60% present as a high-molecular-weight form of IGF-II. This large 15 K IGF molecule is devoided of any IGF-binding activity and might represent incomplete processing of pro-IGF-II peptide. By contrast, the level of IGF-I detected by RIA is barely measurable and considered negligible (0.57 pg/10(6) HT-29 cells). Although these IGF-II-like peptides exhibit a growth-promoting activity on FR3T3 fibroblasts, they cannot stimulate, as recombinant IGF-I or IGF-II, 3H-thymidine incorporation into DNA of HT-29 cells, whatever the experimental conditions used. Finally, we have shown that IGF binding is restricted predominantly to the basolateral domain of the cell membrane by using HT-29-D4 clonal cells, derived from the parental HT-29 cell line, maintained in a differentiated state by culture in a medium in which glucose is replaced by galactose.     

Simultaneous isolation of insulin-like growth factors I and II from adult sheep serum

Ovine insulin-like growth factors I and II (oIGF-I and oIGF-II) have been purified from adult sheep serum. oIGF-II-like receptor-binding activity and IGF-I-like immunoactivity were enriched on SP-Sephadex C-25, then purified using HPLC in the presence of a variety of counter ions. IGF-I- and IGF-II-like activities were separated using HPLC in the presence of 0.2% tetrabutylammonium phosphate at pH 7.0. The final recovery of oIGF-I was 82.6 micrograms from 3.2 litres of adult sheep serum (a yield of 17.6%), and the recovery of oIGF-II was 388 micrograms (a yield of 13.3%). Both IGF preparations were considered to be homogeneous as judged by single sharp peaks during analytical HPLC, and unique N-terminal amino acid sequences. Purified ovine IGFs had molecular weights similar to that of other IGFs (approximately 7000), and the first 30 N-terminal amino acids of both peptides were identical to their human counterparts. The isoelectric points of oIGF-I (pI approximately 8.2) and oIGF-II (pI approximately 6.8) were similar to those of human (h) IGFs (hIGF-I pI approximately 8.2; hIGF-II pI approximately 6.5), and the overall amino acid content of the ovine IGFs was also similar to that of IGFs from other species. oIGF-II preparations from fetal sheep and from adult sheep appeared to be identical. The isolation procedure represents one of general utility that can be easily modified to facilitate the isolation of recombinant IGFs from culture fluid.     

Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.     

Insulin-like growth factor binding protein (IGFBP)-3-bound IGF-I and IGFBP-3-bound IGF-II in growth hormone deficiency

In blood, circulating IGFs are bound to six high-affinity IGFBPs, which modulate IGF delivery to target cells. Serum IGFs and IGFBP-3, the main carrier of IGFs, are upregulated by GH. The functional role of serum IGFBP-3-bound IGFs is not well understood, but they constitute the main reservoir of IGFs in the circulation. We have used an equation derived from the law of mass action to estimate serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II, as well as serum free IGF-I and free IGF-II, in 129 control children and adolescents (48 girls and 81 boys) and in 13 patients with GHD. Levels of serum total IGF-I, total IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 were determined experimentally, while those of IGFBP-4, IGFBP-5 and IGFPB-6, as well as the 12 affinity constants of association of the two IGFs with the six IGFBPs, were taken from published values. A correction for in vivo proteolysis of serum IGFBP-3 was also considered. In controls, serum total IGF-I, total IGF-II, IGFBP-3, IGFBP-3-bound IGF-I, IGFBP-3-bound IGF-II and free IGF-I increased linearly with age, from less than 1 to 15 years, in the two sexes. The concentrations of serum free IGF-I and free IGF-II were approximately two orders of magnitude below published values, as well as below the affinity constant of association of IGF-I with the type-1 IGF receptor. Therefore, it is unlikely that these levels can interact with the receptor. In the 13 patients with GHD, mean (+/- SD) SDS of serum IGFBP-3-bound IGF-I was -2.89 +/- 0.97. It was significantly lower than serum total IGF-I, free IGF-I or IGFBP-3 SDSs (-2.35 +/- 0.83, -1.12 +/- 0.78 and -2.55 +/- 1.07, respectively, p = 0.0001). The mean SDS of serum total IGF-II, IGFBP-3-bound IGF-II and free IGF-II were -1.25 +/- 0.68, -2.03 +/- 0.87 and 0.59 +/- 1.10, respectively, in GHD. In control subjects, 89.8 +/- 4.47% of serum total IGF-I and 77.3 +/- 9.4% of serum total IGF-II were bound to serum IGFBP-3. In patients with GHD, the mean serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II were 8.63 +/- 8. 53 and 19.1 +/- 14.7% below the respective means of control subjects (p < 0.02). In conclusion, in GHD there was a relative change in the distribution of serum IGFs among IGFBPs, due to the combined effects of the decrease in both total IGF-I and IGFBP-3. As a result, serum IGFBP-3-bound IGF-I and IGFBP-3 bound IGF-II, the main reservoirs of serum IGFs, were severely affected. This suggests that the decrease in serum IGFPB-3-bound IGF-I and IGFBP-3-bound IGF-II might have a negative effect for growth promotion and other biological effects of IGF-I and IGF-II. Finally, the estimation of serum IGFBP-3-bound IGF-I, or the percentage of total IGF-I and IGF-II bound to IGFBP-3, might be useful markers in the diagnosis of GHD.     

Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis.

Insulin-like growth factor (IGF) binding to the type 1 IGF receptor (IGF1R) elicits mitogenic effects, promotion of differentiation and protection from apoptosis. This study has systematically measured IGF1R binding affinities of IGF-I, IGF-II and 14 IGF analogues to a recombinant high-affinity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those designed to investigate binding of IGF-binding protein, which exhibited IGF1R-binding affinities similar to those of IGF-I or IGF-II; (b) those generated to probe IGF1R interactions with greatly reduced IGF1R-binding affinities. The relative binding affinities of IGF-I analogues and IGF-I for the IGF1R determined by BIAcore analysis agreed closely with existing data from receptor-binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring affinities of IGFs for IGF1R. In parallel studies, IGF1R-binding affinities were related to ability to protect against serum withdrawal-induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromocytoma cell line, PC12. In this model system, IGF-I and IGF-II at low nanomolar concentrations are able to prevent apoptosis completely. We conclude that ability to protect against apoptosis is directly related to ability to bind the IGF1R.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.     

Insulin-like growth factors (IGF-I and IGF-II) inhibit C2 skeletal myoblast differentiation and enhance TNF alpha-induced apoptosis

IGF-I and IGF-II are thought to be unique in their ability to promote muscle cell differentiation. Murine C2 myoblasts differentiate when placed into low serum media (LSM), accompanied by increased IGF-II and IGF binding protein-5 (IGFBP-5) production. Addition of 20 ng/ml TNF alpha on transfer into LSM blocked differentiation, IGF-II and IGFBP-5 secretion and induced apoptosis. We, therefore, wished to assess whether IGFs could protect against the effects of TNF alpha. Neither inhibition of differentiation or induction of apoptosis was rescued by co-incubation with IGF-I or IGF-II. A lower dose of TNF alpha (1 ng/ml) while not inducing apoptosis still inhibited myoblast differentiation by 56% +/- 12, (P < 0.001), indicating that induction of apoptosis is not the sole mechanism by which TNF alpha inhibits myoblast differentiation. Addition of IGF-I or IGF-II alone reduced differentiation by 49% +/- 15 and 33% +/- 20, respectively, (P < 0.001), although neither induced apoptosis. For muscle cells to differentiate, they must arrest in G0. We established that addition of IGF-I, IGF-II or TNF alpha to the myoblasts promoted proliferation. The myoblasts could not exit the cell cycle as efficiently as controls and differentiation was thus reduced. Unexpectedly, co-incubation of IGF-I or IGF-II with 1 ng/ml TNF alpha enhanced the inhibition of differentiation and induced apoptosis. In the absence of apoptosis we show an association between IGF-induced inhibition of differentiation and increased IGFBP-5 secretion. These results indicate that the effects of the IGFs on muscle may depend on the cytokine environment. In the absence of TNF alpha, the IGFs delay differentiation and promote myoblast proliferation whereas in the presence of TNF alpha the IGFs induce apoptosis.     

Insulin-like growth factor (IGF)-binding protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can stimulate apoptosis and inhibit cell proliferation directly and independently of binding IGFs or indirectly by forming complexes with IGF-I and IGF-II that prevent them from activating the IGF-I receptor to stimulate cell survival and proliferation. To date, IGF-independent actions only have been demonstrated in a limited number of cells that do not synthesize or respond to IGFs. To assess the general importance of IGF-independent mechanisms, we have generated human IGFBP-3 mutants that cannot bind IGF-I or IGF-II by substituting alanine for six residues in the proposed IGF binding site, Ile(56)/Tyr(57)/Arg(75)/Leu(77)/Leu(80)/Leu(81), and expressing the 6m-hIGFBP-3 mutant construct in Chinese hamster ovary cells. Binding of both IGF-I and IGF-II to 6m-hIGFBP-3 was reduced >80-fold. The nonbinding 6m-hIGFBP-3 mutant still was able to inhibit DNA synthesis in a mink lung epithelial cell line in which inhibition by wild-type hIGFBP-3 previously had been shown to be exclusively IGF-independent. 6m-hIGFBP-3 only can act by IGF-independent mechanisms since it is unable to form complexes with the IGFs that inhibit their action. We next compared the ability of wild-type and 6m-hIGFBP-3 to stimulate apoptosis in serum-deprived PC-3 human prostate cancer cells. PC-3 cells are known to synthesize and respond to IGF-II, so that IGFBP-3 could potentially act by either IGF-dependent or IGF-independent mechanisms. In fact, 6m-hIGFBP-3 stimulated PC-3 cell death and stimulated apoptosis-induced DNA fragmentation to the same extent and with the same concentration dependence as wild-type hIGFBP-3. These results indicate that IGF-independent mechanisms are major contributors to IGFBP-3-induced apoptosis in PC-3 cells and may play a wider role in the antiproliferative and antitumorigenic actions of IGFBP-3.     

Insulin-like growth factors induce apoptosis as well as proliferation in LIM 1215 colon cancer cells

The insulin-like growth factor (IGF) system plays an important role in cell proliferation and survival. However, more recently, a small number of studies have shown that IGFs induce apoptosis in some cells. Our initial studies showed this occurred in LIM 1215 colon cancer cells but not RD rhabdomyosarcoma cells. IGFs induced both proliferation and apoptosis in LIM 1215 cells, and the induction of apoptosis was dose-dependent. [R54, R55]IGF-II, which binds to the IGF-I receptor with normal affinity but does not bind to the IGF-II receptor, induced apoptosis to the same extent as IGF-II, whereas [L27]IGF-II, which binds to the IGF-I receptor with 1000-fold reduced affinity, had no effect on apoptosis. These results suggest that the IGF-I receptor is involved in induction of apoptosis. Western blot analyses demonstrated that Akt and Erk1/2 were constitutively activated in RD cells. In contrast, phosphorylation of Akt and Erk1/2 were transient and basal expression of Akt protein was lower in LIM 1215 cells. Analysis of apoptosis-related proteins showed that IGFs decreased pro-caspase-3 levels and increased expression of pro-apoptotic Bad in LIM 1215 cells. IGFs co-activate proliferative and apoptotic pathways in LIM 1215 cells, which may contribute to increased cell turnover. Since high turnover correlates with poor prognosis in colorectal cancer, this study provides further evidence for the role of the IGF system in its progression.     

The role of the IGF axis in hepatocarcinogenesis.

Primary hepatocellular carcinoma (HCC) is one of the most common forms of malignant cancer with the fourth highest mortality rate worldwide. Major risk factors for the development of HCC include chronic infections with the hepatitis B or C virus, alcohol consumption, exposure to dietary aflatoxin B1, hereditary liver disease or liver cirrhosis of any etiology. Recent studies have discovered changes in the insulin-like growth factor (IGF) axis that affect the molecular pathogenesis of HCC, including the autocrine production of IGFs, IGF binding proteins (IGFBPs), IGFBP proteases, and IGF receptor expression. Characteristic alterations detected in HCC and hepatoma cell lines comprise the overexpression of IGF-II and the IGF-I receptor emerging as critical events in malignant transformation and growth of tumors. Simultaneous reduction of IGFBP expression and the increase in proteolytic cleavage of IGFBPs result in an excess of bioactive IGFs. Finally, defective functions of the IGF-II/mannose 6-phosphate receptor involved in degradation of IGF II, the activation of the growth inhibitor TGF-beta1, and the lysosomal targeting of cathepsin proteases capable to degrade extracellular matrix proteins may contribute to the development of HCC.     

Receptor Binding, Endocytosis, and Mitogenesis of Insulin-Like Growth Factors I and II in Fetal Rat Brain Neurons

Cell surface binding, internalization, and biological effects of insulin-like growth factors (IGFs) I and II have been studied in primary neuronal cultures from developing rat brain (embryonic day 15). Two types of IGF binding sites are present on the cell surface. The IGF-I receptor alpha-subunit (Mr 125,000) binds IGF-I with a KD of 1 nM and IGF-II with 10 times lower affinity. The mannose-6-phosphate (Man-6-P)/IGF-II receptor (Mr 250,000) binds IGF-II with a KD of 0.5 nM and IGF-I with 100 times lower affinity. Surface-bound IGF-I and IGF-II are internalized by their respective receptors binding and internalization of IGF-II but not those of IGF-I. Neuronal synthesis of RNA and DNA is increased twofold by IGF-I with 10 times higher potency than IGF-II. Antibody 3637, which blocks receptor binding of IGF-II, has no effect on the DNA response to IGF-I or IGF-II. Double immunocytochemical staining with antibodies to bromodeoxyuridine and neurofilament shows that greater than 80% of the bromodeoxyuridine-positive cells become neurofilament positive. It is concluded that IGF-I and IGF-II bind to two receptors on the surface of neuronal precursor cells that mediate endocytosis and degradation of IGF-I and IGF-II. Proliferation of neuronal precursor cells is stimulated by IGF-I and IGF-II via activation of the IGF-I receptor.     

Regulation of breast cancer growth by insulin-like growth factors

The IGFs may be important autocrine, paracrine or endocrine growth factors for human breast cancer. IGF-I and II stimulate growth of cultured human breast cancer cells. IGF-I is slightly more potent, paralleling its higher affinity for the IGF-I receptor. Antibody blockade of the IGF-I receptor inhibits growth stimulation induced by both IGFs, suggesting that this receptor mediates the growth effects of both peptides. However, IGF-I receptor blockade does not inhibit estrogen (E₂)-induced growth suggesting that secreted IGFs are not the major mediators of E₂ action. Several breast cancer cell lines express IGF-II mRNA by both Northern analysis and RNase protection assay. IGF-II activity is found in conditioned medium by radioimmuno and radioreceptor assay, after removal of somatomedin binding proteins (BP) which are secreted in abundance. IGF-I is undetectable. BPs of 25 and 40 K predominate in ER-negative cell lines while BPs of 36 K predominate in ER-positive cells. Blockade of the IGF-I receptor inhibits anchorage-independent and monolayer growth in serum of a panel of breast cancer cell lines. Growth of one line (MDA-231) was also inhibited in vivo by receptor antibody treatment of nude mice. The antibody had no effect on growth of MCF-7 tumors. These data suggest the IGFs are important regulators of breast cancer cell proliferation and that antagonism of this pathway may offer a new treatment strategy.     

Circulating levels of insulin-like growth factors increase and molecular forms of their serum binding proteins change with human pregnancy.

Insulin-like growth factors (IGF) and binding proteins were measured in serum from pregnant and nonpregnant women. IGF-I determined by immunoassay after acid-ethanol extraction was increased by pregnancy (p less than 0.005) and was highest in the third trimester (p less than 0.01). Size exclusion chromatography of serum in acid before assay (i) gave a very similar IGF-I pattern, (ii) showed that IGF-II was much higher than IGF-I and (iii) revealed less serum IGF-binding protein activity in pregnancy and lactation. All IGF-binding proteins except binding protein-1 were markedly reduced by pregnancy. This indicates a major change in the main carrier protein for IGFs in the circulation and suggests that tissue targetting of IGFs may be altered during pregnancy.     

The insulin-like growth factors and their binding proteins

1. This review provides a brief overview of the structure of the insulin-like growth factors (IGFs or somatomedins), their mRNA and genes; the regulation and sites of production of these peptides; their binding and actions in target tissues; and the structure and biological role of their binding proteins. 2. Molecular cloning techniques have allowed the prediction of precursor forms of IGF-I and IGF-II, have provided tools to study the regulation of the synthesis and translation of IGF mRNAs, and have recently yielded the primary sequence of the IGF-I receptor, supplementing other rapidly-accumulating structural data. 3. Several of the IGF binding proteins have also been purified, and initial structural studies performed. 4. The increased knowledge of the structures of the IGFs, their receptors and binding proteins should now permit rapid progress in understanding the physiology and functions of these proteins.     

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

Insulin-like growth factor (IGF)/somatomedin receptor subtypes: structure, function, and relationships to insulin receptors and IGF carrier proteins

The insulin-like growth factors IGF-I and IGF-II are mitogenic polypeptides with a high degree of chemical homology. Two distinct subtypes of receptors for the IGFs have been identified on the basis of structure and binding specificity. Type I IGF receptors bind IGF-I with equal or greater affinity than IGF-II, and also bind insulin with a low but definite affinity. They are structurally homologous to insulin receptors, containing disulfide-linked a-subunits that bind the peptides and beta-subunits that have intrinsic tyrosine-specific kinase activity. Type II IGF receptors typically bind IGF-II with greater affinity than IGF-I, and do not interact with insulin. They consist of a single polypeptide and lack tyrosine kinase activity. Because of the extensive cross-reactivity of IGF-I and IGF-II with both type I and type II receptors, we believe that potentially either receptor may mediate the biological responses of either peptide. Type I IGF receptors have been shown to mediate the mitogenic effects of the IGFs in some cell types. Whether type II IGF receptors mediate the same or different functions remains to be elucidated.