Insulin regulation of insulin-like growth factor action in rat hepatoma cells

We have reported previously that insulin causes a complete but reversible desensitization to insulin action in rat hepatoma HTC cells in tissue culture, and that this insulin resistance is mediated by postbinding mechanisms rather than receptor down-regulation (Heaton, J. H., and Gelehrter, T. D. (1981) J. Biol. Chem. 256, 12257-12262). We report here that insulin causes a similar desensitization to the induction of tyrosine aminotransferase by the insulin-like growth factors IGF-I and IGF-II isolated from human plasma, and by multiplication-stimulating activity, the rat homologue of IGF-II. The results of both competition-binding studies and affinity cross-linking experiments indicate that insulin-like growth factors (IGFs) bind primarily to IGF receptors rather than to insulin receptors. The low concentrations at which these factors induce transaminase is consistent with their acting primarily via IGF receptors. This is confirmed by experiments utilizing anti-insulin receptor antibody which both inhibits 125I-insulin binding and shifts the concentration dependence of insulin induction of tyrosine aminotransferase to the right. This same immunoglobulin does not inhibit 125I-multiplication-stimulating activity binding and only minimally inhibits 125I-IGF-I binding. Anti-insulin receptor antibody also does not significantly shift the concentration dependence for the IGFs, suggesting that IGFs induce transaminase by acting via IGF receptors. Although insulin down regulates insulin receptors, it does not decrease IGF-I or IGF-II binding. We conclude that insulin causes desensitization of HTC cells to IGFs by affecting a postbinding step in IGF action, which may be common to the actions of both insulin and insulin-like growth factors.     

Characterization of the insulin and insulin-like growth factor receptors and responsitivity of a fibroblast/adipocyte cell line before and after differentiation

TA1 cells, like 3T3-L1 cells, undergo a differentiation process in vitro from a fibroblast to an adipocyte phenotype. The TA1 pre-adipocytes were found to have low numbers of insulin receptors but high numbers of receptors for insulin-like growth factors (IGF) I and II. Also, the pre-adipocytes were more responsive to IGF than insulin as measured by either stimulation of glucose or amino acid uptake. After differentiation, the adipocytes had higher numbers of insulin receptors and a better responsitivity to insulin than to IGF-I. These results indicate that insulin-like growth factors are the primary regulators of the pre-adipocytes whereas insulin regulates the adipocytes.     

Insulin-like growth factor I in cultured rat astrocytes: expression of the gene, and receptor tyrosine kinase.

Gene expression, receptor binding and growth-promoting activity of insulin-like growth factor I (IGF I) was studied in cultured astrocytes from developing rat brain. Northern blot analysis of poly(A)+ RNAs from astrocytes revealed an IGF I mRNA of 1.9 kb. Competitive binding and receptor labelling techniques revealed two types of IGF receptor in astroglial cells. Type I IGF receptors consist of alpha-subunits (Mr 130,000) which bind IGF I with significantly higher affinity than IGF II, and beta-subunits (Mr 94,000) which show IGF I-sensitive tyrosine kinase activity. Type II IGF receptors are monomers (Mr 250,000) which bind IGF II with three times higher affinity than IGF I. Both types of IGF receptor recognize insulin weakly. DNA synthesis measured by cellular thymidine incorporation was stimulated 2-fold by IGF I and IGF II. IGF I was more potent than IGF II, and both were significantly more potent than insulin. Our findings suggest that IGF I is synthesized in fetal rat astrocytes and acts as a growth promoter for the same cells by activation of the type I IGF receptor tyrosine kinase. We propose that IGF I acts through autocrine or paracrine mechanisms to stimulate astroglial cell growth during normal brain development.     

Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (IGF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with K(D) of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with K(D) of 0.14 nM and for the high-affinity insulin binding sites with K(D) of 10 nM. Dissociation rate constants of insulin and IGF1 were determined to be 0.015 min(-1) and 0.013 min(-1), respectively, allowing estimation of ligand association rate constants. Combined, our results suggest that, in addition to IR and IGF1R homodimers, substantial numbers of hybrid IR-IGF1R heterodimers are present on the surface of these cells.     

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.     

Hybrid molecules containing the A-domain of insulin-like growth factor-I and the B-chain of insulin have increased mitogenic activity relative to insulin

Two synthetic insulin-like compounds consisting of the B-chain of insulin linked via disulfide bonds to A chains corresponding to the A-domain or the A- and D-domains of insulin-like growth factor I (IGF-I) have been evaluated for mitogenic activity and for binding to IGF receptors and IGF carrier proteins. Both compounds are 3- to 5-fold more potent mitogens than insulin, and have a comparably increased affinity for the type I IGF receptor that mediates these mitogenic effects in chick embryo fibroblasts. Neither compound interacts with IGF carrier proteins. These results indicate that the A-domain of IGF-I is importantly involved in its growth-promoting properties.     

Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig.

125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15-19 of pregnancy. Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days. The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones. Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types. The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors. Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000. The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst.     

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.     

Mitogen-potentiating action and binding characteristics of insulin and insulin-like growth factors in Chinese hamster fibroblasts

alpha-Thrombin alone is able to stimulate DNA synthesis reinitiation of G0-arrested Chinese hamster lung fibroblasts (CC139) as well as continued growth of these cells in serum-free medium. Although insulin at high concentrations (1-10 micrograms/ml) is not intrinsically mitogenic for these cells, it potently enhances the growth-promoting action of thrombin. The generation time of CC139 cells in the defined medium, transferrin, alpha-thrombin, insulin, is around 15 h. To determine whether this effect of insulin is mediated via putative receptors for the insulin-like growth factors (IGFs) on these cells, we examined the abilities of two IGFs, Multiplication-Stimulating Activity (MSA) and IGF-I, to potentiate the thrombin-induced reinitiation of DNA synthesis. Both IGFs were found to be as effective as insulin for this biological effect; however, much lower concentrations were required to elicit half-maximal response, 100 ng/ml of MSA and 30 ng/ml of IGF-I. Detailed binding studies using 125I-labelled insulin, MSA, and IGF-I revealed that CC139 cells specifically bind all three polypeptides with IC50 values for the corresponding ligands of 1-2 ng/ml, 80-100 ng/ml, and 30-40 ng/ml, respectively. 125I-MSA binding was insulin-insensitive, whereas insulin did compete with 125I-IGF-I for binding to CC139 cells. These results indicate that CC139 cells possess at least two types of IGF receptors, an insulin-insensitive IGF receptor with high affinity for MSA which apparently mediates its biological effect, and an insulin-sensitive IGF-I receptor. Insulin appears to exert its mitogen-potentiating activity in CC139 fibroblasts by interacting with the IGF-I receptor.     

Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes

Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.     

Type I insulin-like growth factor receptors in human ovarian stroma

Hyperandrogenism observed in a variety of hyperinsulinemic states is thought to be due to an effect of insulin mediated through the type I insulin-like growth factor (IGF) receptors. These receptors, however, have not yet been demonstrated in normal human ovarian cells capable of androgen production. We now report the presence of type I IGF receptors in membrane preparations of human ovarian stroma. The ovarian stromal tissue was obtained from women undergoing indicated oophorectomy. Stromal plasma membranes were prepared. Specific 125I-IGF-I binding was 6.6 +/- 0.2%/100 micrograms protein. The affinity constant estimated by Scatchard analysis was 4.6 X 10(-9) M. 50% inhibition of 125I-IGF-1 binding was observed at 5 ng/ml of IGF-1. Specificity of the 125I-IGF-I-binding sites was confirmed by analogue specificity studies and in experiments utilizing monoclonal antibody to the IGF-I receptor, alpha-IR-3. IGF-II and insulin competed with 125I-IGF-I for the binding sites, but with an affinity significantly lower than that of IGF-I: 50% inhibition was observed at approximately 60 ng/ml of IGF-II or insulin. alpha-IR-3, a monoclonal antibody with high specificity for the type I IGF receptor, effectively inhibited 125I-IGF-I binding in a dose-dependent manner, confirming that the 125I-IGF-I binding was indeed to the type I IGF receptor. We conclude that type I IGF receptors are present in human ovarian stroma. These receptors may mediate effects of insulin on the ovary in hyperinsulinemic insulin-resistant states.     

Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: Relation to human somatomedins and insulin

The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component ot adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.     

Insulin and Insulin-like Growth Factor-1 Receptors Act as Ligand-specific Amplitude Modulators of a Common Pathway Regulating Gene Transcription

Insulin and insulin-like growth factor-1 (IGF-1) act on highly homologous receptors, yet in vivo elicit distinct effects on metabolism and growth. To investigate how the insulin and IGF-1 receptors exert specificity in their biological responses, we assessed their role in the regulation of gene expression using three experimental paradigms: 1) preadipocytes before and after differentiation into adipocytes that express both receptors, but at different ratios; 2) insulin receptor (IR) or IGF1R knock-out preadipocytes that only express the complimentary receptor; and 3) IR/IGF1R double knock-out (DKO) cells reconstituted with the IR, IGF1R, or both. In wild-type preadipocytes, which express predominantly IGF1R, microarray analysis revealed ∼500 IGF-1 regulated genes (p < 0.05). The largest of these were confirmed by quantitative PCR, which also revealed that insulin produced a similar effect, but with a smaller magnitude of response. After differentiation, when IR levels increase and IGF1R decrease, insulin became the dominant regulator of each of these genes. Measurement of the 50 most highly regulated genes by quantitative PCR did not reveal a single gene regulated uniquely via the IR or IGF1R using cells expressing exclusively IGF-1 or insulin receptors. Insulin and IGF-1 dose responses from 1 to 100 nm in WT, IRKO, IGFRKO, and DKO cells re-expressing IR, IGF1R, or both showed that insulin and IGF-1 produced effects in proportion to the concentration of ligand and the specific receptor on which they act. Thus, IR and IGF1R act as identical portals to the regulation of gene expression, with differences between insulin and IGF-1 effects due to a modulation of the amplitude of the signal created by the specific ligand-receptor interaction.     

Binding of insulin-like growth Factor ii and multiplication-stimulating activity-stimulated phosphorylation in basolateral membranes from dog kidney

Biologic actions of insulin and insulin-like growth factors (IGFs) are thought to be initiated by binding of peptides to tissues, followed by phosphorylation of specific hormone receptors. Both insulin and IGF bind to renal membranes, suggesting functional roles for these peptides in kidney. The present studies further characterize the interaction of multiplication-stimulating activity (MSA)/IGF II with its renal receptor. Specific binding of 125I-IGF II was measured in basolateral membranes isolated from proximal tubular cells of dog kidney. Binding was half-maximal at 10(-9) M MSA and was not inhibited by human growth hormone, IGF I, insulin, or anti-insulin receptor antibodies. Concentration-dependent MSA-stimulated phosphorylation of a Mr 135,000 protein band was demonstrated in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels from basolateral membrane suspensions. Insulin increased phosphorylation of this band only in the presence of MSA, while a Mr 92,000 band was consistently phosphorylated with insulin alone. The phosphorylated Mr 135,000 band which had been solubilized with detergent from basolateral membranes was immunoprecipitated using serum from a patient with anti-insulin receptor antibodies suggesting that the band is the alpha subunit of the insulin receptor. This was supported by the demonstration of covalent cross-linkage of 125I-insulin to the Mr 135,000 band. We conclude that receptor-mediated MSA-stimulated phosphorylation of isolated basolateral membranes may reflect a process by which biological actions of IGF II are mediated in vivo. Our data suggest that insulin and IGF II may interact by regulating protein phosphorylation.     

Insulin and IGF receptors are developmentally regulated in the chick embryo eye lens

We have previously reported that insulin-like growth factor (IGF) receptors appear to predominate over insulin receptors in early stages of embryogenesis in the chick (days 2-3 whole embryo membranes). Overall, [125I]IGF I and II binding to specific receptors was maximal when the rate of brain growth is highest. In the present study we used the embryonic chick lens, a well-defined tissue composed of a single type of cell, to analyse whether changes of insulin and IGF I binding are correlated with changes in growth rate and differentiation state of the cells. We show that both insulin receptors and IGF receptors are present in the lens epithelial cells, and that each type is distinctly regulated throughout development. While there is a direct correlation between IGF-binding capability and growth rate of the cells, there is less relation to differentiation status and embryo age. Insulin receptors, by contrast, appear to be mostly related to the differentiated state of cells, decreasing sharply in fibers, irrespective of their developmental age.     

Human teratocarcinoma cells express functional insulin-like growth factor I receptors

Using iodinated insulin-like growth factors (IGFs) we have detected receptors for IGF-I at the cell surface of the clonally derived human embryonal carcinoma cell line Tera 2 clone 13. Affinity crosslinking of IGFs to Tera 2 clone 13-derived membrane preparations revealed the presence of proteins with features of both type-I and type-II IGF receptors. Treatment of Tera 2 clone 13 cells with retinoic acid to induce differentiation results in an increased number of cell surface receptors, apparently without altering the ratio of type-I and type-II receptors. In addition, Tera 2 clone 13 IGF-I receptors catalyze (auto)phosphorylation at tyrosine upon IGF-I and insulin binding. These findings suggest that type-I IGF receptors might be involved in mediating the effects of IGFs and insulin upon the proliferation of Tera 2 clone 13 cells.     

A novel fetal insulin-like growth factor (IGF) I receptor. Mechanism for increased IGF I- and insulin-stimulated tyrosine kinase activity in fetal muscle

Insulin and insulin-like growth factor (IGF) I receptors from fetal and adult rat skeletal muscle were compared in order to gain insight into the evolving functions of the hormones during development. Basal, insulin-stimulated, and IGF I-stimulated receptor phosphorylation and tyrosine kinase activity are severalfold higher in partially purified receptor preparations from fetal muscle in comparison with equal numbers of receptors from adult muscle. There are distinct insulin and IGF I receptors with Mr 95,000 beta subunits in adult muscle, as evidenced by hormone dose-response curves, immunoprecipitation with specific antibodies, binding to insulin and IGF I affinity columns, and analysis of tryptic phosphopeptides. In addition to these two receptor species, fetal muscle contains a receptor with a Mr 105,000 beta subunit. The fetal receptor is structurally more closely related to the IGF-I receptor than the insulin receptor on the basis of its precipitation with specific antibodies, binding to an IGF I affinity column, and tryptic phosphopeptide map. The fetal receptor does not appear to bind insulin but, unlike the IGF-I receptor, its phosphorylation is stimulated by low physiological concentrations of both insulin and IGF I. This could be explained by the cross-phosphorylation of fetal receptors by activated insulin receptors. Expression of the fetal receptor is highest in the fetus and decreases markedly during the first 2 weeks of postnatal life. The fetal receptor appears to account for the high tyrosine kinase activity of fetal muscle and may be an important mediator of responses to both insulin and IGF I early in development.     

In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-14C]glucose to 14CO2, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace 125I-insulin from its receptors on adipocytes but did competitively inhibit 125I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.     

Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9).

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.     

Cholecystokinin and insulin regulate insulin-like growth factor II binding to pancreatic receptors; Evidence of a role for intracellular calcium

The binding of ¹²⁵I-labeled insulin-like growth factor II (¹²⁵I-IGF II) to mouse pancreatic acini was stimulated (45%) by insulin and inhibited (30%) by cholecystokinin octapeptide (CCK₈). When CCK₈ and insulin were added together, the effect on IGF II binding was similar to that seen when CCK₈ was added alone. Two lines of evidence suggest that this effect of cholecystokinin on basal and insulin-stimulated ¹²⁵I-IGF II binding was mediated via a change in intracellular calcium: (1) the cholinergic agent carbachol inhibited IGF II binding to its receptors; (2) addition of the Ca²⁺ ionophore A23187 mimicked the effects of CCK₈ and carbachol. In contrast to its effects on IGF II binding to acini, CCK₈ had only small effects on IGF I binding and no effects on insulin binding.