Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography

We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.     

Free-ligand accelerated dissociation of insulin-like growth factor 1 (IGF-1) from the type I IGF receptor is reduced by insulin-like growth factor binding protein 3

Insulin-like growth factor binding proteins (IGFBP) can inhibit or accentuate the mitogenic activities of insulin-like growth factor 1 (IGF-1) depending upon the experimental model employed. Inhibitory effects may be attributed to sequestration of IGF-1 onto IGFBP rather than the type I IGF receptor. We have demonstrated that the presence of IGFBP in a simple equilibrium binding assay significantly reduces the total amount of IGF-1 bound to the type I IGF receptor and increases the IC₅₀ for IGF-1 binding. On the basis of such an experiment, performed at equilibrium, IGFBP should reduce the mitogenic activity of IGF-1. Recent work has demonstrated an inverse correlation between the dissociation rate of insulin-like molecules from their receptors and their mitogenic activity. It has also been suggested that the increased rate of dissociation of insulin and IGF-1 from their receptors at increased ligand concentrations serves as a ‘dampening’ mechanism to decrease mitogenic signalling. We have demonstrated increased rates of dissociation of IGF-1 from the type I IGF receptor with increasing concentrations of IGF-1. Furthermore, IGFBP-3 inhibits the acceleration of dissociation rates due to increased IGF-1 levels. Thus, under receptor saturating conditions IGFBP-3 may act to increase mitogenesis by increasing the residence time of individual molecules of IGF-1 upon the type I IGF receptor.     

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

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Polyubiquitination of insulin-like growth factor I receptor (IGF-IR) activation loop promotes antibody-induced receptor internalization and down-regulation

Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.     

The structural basis for insulin-like growth factor I receptor high affinity binding

We have recently identified high and low affinity insulin-like growth factor I (IGF I) binding sites in solubilized human placental membranes and purified the high affinity IGF I receptor by IGF I affinity chromatography (Tollefsen, S. E., Thompson, K., and Petersen, D. J. (1987) J. Biol. Chem. 262, 16461-16469). To define the structural basis for high affinity IGF I binding, we have examined the effect of disulfide bond reduction on the binding parameters of the high affinity IGF I receptor. We find that the disulfide bonds linking the two alpha beta dimers of the IGF I receptor heterotetramer are reduced by incubation at pH 8.75 with 2 mM dithiothreitol (DTT) for 5 min at room temperature. Gel filtration chromatography on a Superose 12 fast protein liquid chromatography column indicates that the alpha beta dimers do not remain associated by noncovalent interactions after reduction. Scatchard plots of IGF I binding to the IGF I receptor incubated at pH 8.75 with or without DTT indicate that the IGF I receptor alpha beta dimers have a 6.1 +/- 1.6 (mean +/- S.D.) times lower affinity than the heterotetramer for IGF I. The total binding capacity of the IGF I receptor treated with DTT is 1.6 +/- 0.3 (mean +/- S.D.) times higher than that of an equal amount of receptor treated without DTT. These results are consistent with a model in which the heterotetramer binds a single IGF I molecule with high affinity, whereas each of the two alpha beta dimers binds an IGF I molecule with lower affinity after dissociation. We conclude that association of two alpha beta dimers is required for formation of an IGF I receptor with high affinity for its ligand.     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1)

The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.     

Insulin/insulin-like growth factor I hybrid receptors have different biological characteristics depending on the insulin receptor isoform involved

The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.     

An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes

We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).     

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.     

Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors.

The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.     

Differences in time course of internalization of receptors of insulin and insulin-like growth factor (IGF-1) in isolated rat hepatocytes

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is a most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones at isolated rat hepatocytes, the internalization time course of ¹²⁵I-insulin and ¹²⁵I-IGF-I are traced at 37 and 12°C. There are established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37°C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. But essential differences in the internalization course of these two related peptides were obvious at the temperature of 12°C. The internalization level of insulin receptors at 12°C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocyte plasma membrane. At 12°C a slight decrease of the proportion of intracellular ¹²⁵I-IGF-I correlated with a decrease in the ¹²⁵I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12°C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar “inhibition mechanism” of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to action of cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.     

The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis

Several type-1 membrane proteins undergo regulated intramembrane proteolysis resulting in the generation of biologically active protein fragments. Presenilin-dependant gamma-secretase activity is central to this event and includes amyloid precursor protein (APP), Notch and ErbB4 as substrates. Here we show that the insulin-like growth factor 1 receptor (IGF-IR) undergoes regulated intramembrane proteolysis. A metalloprotease-dependant ectodomain-shedding event generates a approximately 52 kDa IGF-IR-carboxyl terminal domain (CTD). The IGF-IR-CTD is consequentially a substrate for gamma-secretase cleavage, liberating a approximately 50 kDa intracellular domain (ICD) that can be inhibited by a specific gamma-secretase inhibitor. This study suggests that the IGF-IR is a substrate for gamma-secretase and may mediate a function independent of its role as a receptor tyrosine kinase.     

Signaling pathway of insulin and insulin-like growth factor 1 (IGF-1) as a potential regulator of lifespan

The experimental material accumulated for two decades allows concluding that regulation of lifespan has hormonal control based on the evolutionary conservative insulin/IGF-1 receptor signaling pathway. Data obtained on the commonly accepted models of longevity — nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster, and rodents — demonstrate that reduction of the insulin/IGF-1 signaling pathway results in an increase of the lifespan. There is shown involvement in the longevity mechanism of a large group of genes whose products perform control of metabolism, feeding behavior, reproduction, and resistance to oxidative stress. Discussed in this review are current concepts of the insulin/IGF-1 signaling system as a regulatory “longevity module” and of its possible role in prolongation of life in the higher vertebrates, including human.     

Signal pathway of insulin and insulin-like growth factor 1 (IGF-1) as a potential regulator of lifespan

The experimental material accumulated for two decades allows concluding that regulation of lifespan has hormonal control based on the evolutionary conservative insulin/IGF-1 receptor signal pathway. Data obtained on the commonly accepted models of longevity - nematode Caenorhabditis elegans, Drosophila Drosophila melanogaster, and rodents - demonstrate that reduction of the insulin/IGF- 1 signal pathway leads to an increase of the lifespan. There is shown involvement of the longevity mechanism of a large group of genes whose products perform control of metabolism, alimentary behavior, reproduction, resistance to oxidative stress. Discussed in this review are current concepts of the insulin/IGF-1 signal system as a regulatory "longevity module" and of its possible role in prolongation of life in the higher vertebrates, including human.     

The C region of human insulin-like growth factor (IGF) I is required for high affinity binding to the type 1 IGF receptor

We have produced and characterized the binding properties of three structural analogs of human insulin-like growth factor I (hIGF-I). These analogs are [1-62]hIGF-I, an analog lacking the carboxyl-terminal 8-amino acid D region of hIGF-I; [1-27, Gly4, 38-70]hIGF-I, an analog in which residues 28-37 of the C region of hIGF-I are replaced by a 4-reside glycine bridge; and [1-27,Gly4,38-62]hIGF-I, an analog with the C region glycine replacement and a D region deletion. The removal of the D region of hIGF-I has little effect on binding to the type 1 and type 2 insulin-like growth factor (IGF) receptors. [1-62]hIGF-I has 2-fold higher affinity for the insulin receptor and 4-fold higher affinity for IGF serum-binding proteins. The replacement of the C region of hIGF-I with a four-glycine span results in a 30-fold loss of affinity for the type 1 IGF receptor. However this analog has near normal affinity for the type 2 IGF receptor, the insulin receptor, and IGF serum-binding proteins. Incorporating the C region glycine replacement and the D region deletion into one analog does not affect binding to either the type 2 receptor or to IGF serum-binding proteins. As predicted from the single deletion analogs [1-27,Gly4,38-62]hIGF-I has reduced affinity for the type 1 IGF receptor (approximately 40-fold) and increased affinity for the insulin receptor (5-fold). These data indicate that determinants in the C region of hIGF-I are involved in maintaining high affinity binding to the type 1 IGF receptor and that neither the C region nor the D region are required for high affinity binding to the type 2 IGF receptor or to IGF serum-binding proteins.     

Differential activation of insulin receptor substrates 1 and 2 by insulin-like growth factor-activated insulin receptors

The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.     

Distribution of receptors for insulin and insulin-like growth factor I (somatomedin C) in the adrenal gland

Rat adrenal glands contain cell surface high-affinity receptors for several peptide hormones. Receptors for IGF-I were abundant in this tissue, but receptors for insulin were relatively scarce. The behavior of adrenal membrane IGF-I receptors in radioligand binding assays was similar to the behavior of IGF-I receptors from other tissues, with a KD congruent to 6.2 x 10(-9) M. Covalent cross-linking studies with [125I]IGF-I revealed an IGF-I receptor alpha-subunit with Mr congruent to 135,000 on dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, as well as a smaller radiolabeled peptide, Mr = 116,000. In contrast, little binding of [125I]insulin to adrenal membranes was observed and no labeling occurred in cross-linking studies using [125I]insulin. These results contrast with the findings of whole-body autoradiographic studies that indicated substantial binding of [125I]insulin to adrenal glands and suggest that IGF-I, rather than insulin, may play a critical role in the growth and development of the adrenal gland.