Structural origins of the functional divergence of human insulin-like growth factor-I and insulin

Human insulin-like growth factors I and II (hIGF-I, hIGF-II) are potent stimulators of cell and growth processes. They display high sequence similarity to both the A and B chains of insulin but contain an additional connecting C-domain, which reflects their secretion without specific packaging or precursor conversion. IGFs also have an extension at the C-terminus known as the D-domain. This paper describes four homologous hIGF-1 structures, obtained from crystals grown in the presence of the detergent SB12, which reveal additional detail in the C- and D-domains. Two different detergent binding modes observed in the crystals may reflect different hIGF-I biological properties such as the interaction with IGF binding proteins and self-aggregation. While the helical core of hIGF-I is very similar to that in insulin, there are distinct differences in the region of hIGF-I corresponding to the insulin B chain C-terminus, residues B25-B30. In hIGF-I, these residues (24-29) and the following C-domain form an extensive loop protruding 20 A from the core, which results in a substantially different conformation for the receptor binding epitope in hIGF-I compared to insulin. One notable feature of the structures presented here is demonstration of peptide-bond cleavage between Ser35 and Arg36 resulting in an apparent gap between residues 35 and 39. The equivalent region of proinsulin is involved in hormone processing demanding a reassessment of the structural integrity of hIGF-I in relation to its biological function.     

Altered placental insulin and insulin-like growth factor-I receptors in diabetes

We have compared the characteristics of IGF-I and insulin receptors in placentas of normals and insulin dependent diabetic patients. Specific binding of both IGF-I and insulin in placental membranes from patients with good glycemic control (as reflected by blood hemoglobin content) was unaltered while that in the placental membranes from the patients with poor glycemic control was increased to approximately 20% of the normals. This observed small but significant (p less than 0.05) increase in binding of IGF-I and insulin to placental membranes from diabetic patients with poor glycemic control was further magnified, approximately twice (p less than 0.001) the normal, when the membrane receptors were purified by lectin chromatography. The kinetic analysis of IGF-I and insulin binding in both membranes and lectin purified receptors revealed that the increased binding of insulin and IGF-I to the placentas from diabetic patients with poor glycemic control was due to an approximately 2 fold increase (p less than 0.001-0.05) in the receptor numbers without any significant changes of the affinities. The molecular characteristics of the receptors in these diabetic patients, as revealed by the cross-linking studies, did not reveal any changes when compared to the normals. The parallel changes of IGF-I and insulin receptors, shown here, are in accordance with the homologous nature of these two receptors. The increased receptor numbers of these two interrelated hormones in placentas of diabetics with poor glycemic control may be relevant to the altered placental functions in diabetic pregnancy.     

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.     

Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells.

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.     

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.     

Dissection of the growth versus metabolic effects of insulin and insulin-like growth factor-I in transfected cells expressing kinase-defective human insulin receptors

We have recently reported that the expression of an in vitro mutated, kinase-defective insulin receptor (A/K1018) leads to cellular insulin resistance when expressed in Rat 1 fibroblasts. That is, despite the presence of normal numbers of activatable native insulin receptors in the host cell, the A/K1018 receptors prevent the normal receptors from phosphorylating endogenous substrates and from signalling insulin action, perhaps by competing for limiting amounts of these substrates. We report here that insulin-like growth factor I-stimulated phosphorylation of two endogenous substrate proteins, pp220 and pp170, is also inhibited in cells expressing A/K1018 receptors. Because insulin-like growth factor I stimulation of glucose uptake is not inhibited in cells with A/K1018 receptors while pp220 and pp170 phosphorylation is inhibited, it is unlikely that either pp220 or pp170 are involved in mediating the stimulation of glucose transport. In contrast, insulin-like growth factor I-mediated stimulation of mitogenesis is inhibited in cells with A/K1018 receptors. Thus, pp170 or pp220 could be involved in mitogenic signalling. We also report that both H2O2 and tetradecanoylphorbolacetate stimulate glucose transport normally in cells with A/K1018 receptors. Phorbol esters also lead to the phosphorylation of both normal and A/K1018 receptors on serine and/or threonine. This argues that phorbol esters or H2O2 bypass the normal proximal steps in signalling insulin action.     

Binding and production of insulin-like growth factor-I in rat mammary gland.

1. Mammary tissue from pregnant rat presents low and high affinity IGF-I functional receptors. 2. Mammary explants from pregnant and lactating rats secrete IGF-I and its production was related to the developmental stage of the gland. 3. An inverse relationship between IGF-I production and tissue binding capacity was observed.     

In vitro effect of insulin and insulin-like growth factor-I on cell multiplication and adrenocorticotropin responsiveness of fetal adrenal cells

The present study examined the effects of both insulin and insulin-like growth factor-I (IGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ml IGF-I was as potent as 10 micrograms/ml insulin. The acute cyclic adenosine 3',5'-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1-24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1-24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1-24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (congruent to 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 microgram/ml. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat insulin-like growth factor-I and -II mRNAs are unchanged during compensatory adrenal growth but decrease during ACTH-induced adrenal growth

The insulin-like growth factors (IGFs) may be important autocrine and paracrine mediators of organ growth. We used solution-hybridization/ribonuclease protection assays to examine IGF-I and IGF-II mRNA abundance during hypertrophy or the rat adrenal gland induced by unilateral adrenalectomy or by adrenocorticotropic hormone (ACTH) infusion. Adrenal IGF-I mRNA did not change during the period of rapid organ growth at 18 or 66 h after unilateral adrenalectomy. ACTH infusion induced a time- and dose-dependent decrease in adrenal IGF-I mRNA despite significant increases in gland size. IGF-II mRNA also remained unchanged after unilateral adrenalectomy and decreased after ACTH infusion, to a greater extent than IGF-I mRNA. Liver IGF-I mRNA did not change with ACTH exposure, indicating an effect specific to the adrenal. We also measured adrenal P450scc mRNA as a marker of steroidogenic capacity. P450scc mRNA was unchanged after unilateral adrenalectomy and increased with ACTH infusion. Thus IGF-I and IGF-II mRNAs respond in parallel, but in different fashions with different stimuli for adrenal growth. The decrease in IGF mRNA after exposure to ACTH may be a factor in the ACTH-induced inhibition of compensatory hypertrophy after unilateral adrenalectomy.     

Receptors for insulin-like growth factor-I (IGF-I) in myocardial capillary endothelium of the intact perfused heart

Isolated intact, beating hearts were perfused with HPLC-pure [125]-IGF-I (1 ng/ml) alone or [125]-IGF-I (1 ng/ml) plus varying concentrations of unlabeled IGF-I (10-3,000 ng/ml) or unlabeled insulin (1,000-100,000 ng/ml). After 1 min of perfusion with peptides, the hearts were rapidly fixed, sectioned and analyzed for radioautographic [125I] grain counts. Greater than 90% of [125I] grains were shown to represent intact [125I]-IGF. Maximal grain counts over capillaries occurred after perfusion with [125I]-IGF-I alone and decreased in a dose-dependent manner when unlabeled IGF-I was coperfused. Coperfusion of [125I]-IGF-I with unlabeled insulin also decreased 125I grains over capillaries but less potently than unlabeled IGF-I. EM radioautography demonstrated that [125I]-IGF-I grains were localized over capillary endothelial cells. Thus, specific IGF-I receptors are present in the capillary endothelium of the intact heart and have properties similar to IGF-I receptors in cultured capillary endothelial cells.     

Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-II receptor: comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I

Although insulin-like growth factors (IGF) I and II bind with high affinity to structurally discrete receptors, they bind with a lesser affinity to each other's receptor. We have evaluated the affinity of five different IGF-I preparations (three natural IGF-I preparations, one synthetic preparation, and one recombinant DNA-derived) for the IGF-II receptor in rat placental membranes, 18-54,SF cells and BRL-3A cells. In all tissues tested, the natural IGF-I preparations demonstrated an affinity for the IGF-II receptor which was 10-20% that of IGF-II. However, the recombinant and synthetic IGF-I preparations exhibited substantially lower affinities than natural IGF-I for this receptor, with only 10-25% reduction in (125-I)iodo IGF-II binding at peptide concentrations up to 400 ng/ml. Radioimmunoassay of the natural IGF-I preparations with an antibody directed against the unique C-peptide region of IGF-II demonstrated that contamination of IGF-I preparations with immunoreactive IGF-II could not exceed 5%. These results demonstrate that IGF-I purified from human plasma has a different affinity for the IGF-II receptor than does synthetic or recombinant IGF-I. Furthermore, these data are consistent with the hypothesis that IGF-I, itself, may be heterogeneous, and that subforms may vary in their affinities for the IGF receptors. Alternatively, IGF-I preparations which have been considered to be pure may be contaminated with small amounts of IGF-II, resulting in overestimation of the affinity of IGF-I for the type II IGF receptor.     

Reciprocal modulation of insulin and insulin-like growth factor-I receptor affinity by calcium

In contrast to its stimulation of insulin binding to human placental membranes, calcium inhibited the binding of insulin-like growth factor-I. The effects on receptors for both peptides were half-maximal at 2 mM calcium, and were entirely due to alterations in high affinity binding sites for the respective ligands. Calcium decreased the affinity of insulin-like growth factor-I sites, while stimulating the expression of high affinity insulin sites. Competition by each peptide at the receptor for the other peptide was enhanced by calcium. Modulation by calcium might provide a mechanism to amplify functional differences between the two structurally similar receptors.     

Pharmacodynamic considerations with recombinant human insulin-like growth factor-I in children

AIM: To report effects of weight-based recombinant human insulin-like growth factor-I (rhIGF-I) on IGF axis parameters in children with hyperinsulinism. METHODS: Open label trial with subcutaneous rhIGF-I (40 microg/kg/dose). Patients studied were children (1 month to 11 years) with diffuse hyperinsulinism (n = 7). Serial serum IGF and insulin-like growth factor binding protein (IGFBP) concentrations were measured by RIA and analyzed by linear Pearson regression. RESULTS: Following the initial rhIGF-I dose, total insulin-like growth factor-I (IGF-I) rose by 56% at 30 min (p < 0.01) and 85% at 120 min (p < 0.02). Serum IGF-II, IGFBP-2, and IGFBP-3 levels did not change. Peak serum IGF-I levels within 12 h of the initial rhIGF-I dose were 167-700 mg/ml. The variable peak IGF-I response is attributable in part to IGFBP-3 differences across this pediatric age range. Models of rhIGF-I dosing based upon body surface area (BSA) or initial IGFBP-3 resulted in predictable peak serum IGF-I levels (r = 0.78; p < 0.03). Recalculating rhIGF-I dosing based upon the BSA . IGFBP-3 product correlated closely with peak IGF-I level (r = 0.85; p < 0.007). CONCLUSIONS: Weight-based IGF-I dosing in this cohort resulted in variable IGF-I levels. Considering BSA and serum IGFBP-3 concentration in children is appropriate for subcutaneous IGF-I administration. A combination of these values may yield predictable individualization of rhIGF-I dosing.     

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.     

Regulation of the aromatase activity of human placental cytotrophoblasts by insulin, insulin-like growth factor-I, and -II

Studies to be reviewed were stimulated by the clinical observation, albeit controversial, that diabetic pregnancy may be associated with lower serum oestrogen levels than nondiabetic pregnancy. Pregnant diabetic women are usually intensively treated with insulin to maintain euglycemia, frequently resulting in peripheral hyperinsulinemia. The placenta, which is the primary source of oestrogens during pregnancy, would be exposed to this elevation in circulating insulin levels. Similarly, insulin-like growth factors (IGFs), which are synthesized and secreted by placental tissues and could influence placental function in an autocrine or paracrine fashion, may be elevated in diabetic pregnancy. We will review studies, which show that (i) insulin, insulin-like growth factor-I (IGF-I), and -II inhibit the aromatase activity of human cytotrophoblasts, (ii) these peptides can inhibit aromatase by activation of their respective receptors, and (iii) the potency of IGF-II in suppressing aromatase greatly exceeds that of either insulin or IGF-I. Finally, evidence will be reviewed, which suggests that inositolglycan mediators (‘second messengers’) serve as the signal transduction system for insulin's inhibition of aromatase activity. Hence, placental exposure to increased concentrations of insulin and/or IGFs in the pregnant diabetic woman may result in inhibition of aromatase activity and decreased serum oestrogen levels.     

Receptor autoradiographic localization of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands

We report here the first evidence of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands, obtained at autopsy. Sections of tissue were incubated with 0.1 nM [125I]IGF-I and analyzed using [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry. Specific binding sites of [125I]IGF-I were found to be localized in the definitive zone, fetal zone, and fetal medulla of the fetal adrenal glands. In the adult adrenal glands, the entire cortex and medulla were specifically labeled with [125I]IGF-I. Specific binding obtained at a concentration of 0.1 nM [125I]IGF-I to areas in the fetal and adult human adrenal glands was competitively displaced by unlabeled IGF-I, with an IC50 value of 0.34-2.54 nM, and 0.38-0.73 nM, respectively, whereas insulin was much less potent in displacing the binding. Acquisition of this knowledge will aid in studies on cell growth and steroid-catecholamines biosynthesis of the human adrenal gland.     

The effects of insulin-like growth factor-I and insulin on placental lactogen production by human term placental explants

The effects of insulin-like growth factor (IGF)-I and insulin on placental lactogen production (hPL) by term human placental explants were studied. The hPL content in medium and explant decreased rapidly after first 24 hours of culture. The decrease thereafter was gradual and reached a plateau by day 4 of culture. The decrease of HPL content in placental culture has previously been suggested being due to the depletion of a rapidly secreting preformed pool of hPL. Addition of IGF-I (0.1-10 micrograms/ml) and insulin (1-20 micrograms/ml) stimulated the decreased level of hPL in tissue and medium after 24 hours in culture. IGF-I was 10 times more potent than insulin in stimulating hPL. These findings suggest that IGF-I and insulin effects the production of hPL by placenta. The lower potency of insulin may indicate that the effect of insulin on hPL production is via IGF-I receptor.     

Increased autophosphorylation of insulin-like growth factor-I and insulin receptors in placentas of diabetic women

Autophosphorylation of insulin and insulin-like growth factor (IGF)-I receptors were measured in lectin purified receptor preparations from placentas of normal and diabetic patients. The basal and insulin or IGF-I stimulated phosphorylation of the approximately 94 kD protein, corresponding to beta-subunit of the insulin and IGF-I receptors, were approximately 2 times greater (p less than 0.05) in placentas from diabetic patients with poor glycemic control (as judged by their serum HbA1c level) compared to the normals. The magnitude of IGF-I or insulin stimulation of the phosphorylation of the 94 kD protein was comparable in placentas from both diabetic and normal patients. Immunoprecipitation and immunodepletion of IGF-I receptor by alpha-IR3, a monoclonal antibody to IGF-I receptor, revealed the increased basal phosphorylation of the approximately 94 kD protein in placentas of diabetic patients to be associated with IGF-I and insulin receptors. The magnitude of IGF-I and insulin stimulated phosphorylation of the immunoprecipitated and immunodepleted IGF-I receptor, respectively, was the same in both normal and diabetic patients. These results suggested that the increased basal phosphorylation of the 94 kD protein in placentas from diabetic patients may be intrinsic to IGF-I and insulin receptor, however, the regulatory mechanisms effecting the increase may not be dependent on IGF-I or insulin.