Structural and biological characterization of bovine insulin-like growth factor binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) purified from bovine serum shares 19 of 25 amino-terminal amino acid residues with IGFBP-3 purified from human, rat, and porcine sources. A newly characterized bovine fibroblast model was used to investigate the biological effects of purified bovine IGFBP-3 (bIGFBP-3). Coincubation of insulin-like growth factor I (IGF-I) with increasing concentrations of bIGFBP-3 produced a dose-dependent inhibition of IGF-I-stimulated [3H]aminoisobutyric acid (AIB) uptake in cultured bovine fibroblasts. Inhibition was complete at equimolar concentrations of IGF-I and bIGFBP-3. Inhibition of IGF-I-stimulated [3H]AIB uptake paralleled the ability of bIGFBP-3 to prevent [125I]IGF-I cell surface binding. In contrast, preincubation with bIGFBP-3 resulted in a dose-dependent enhancement of IGF-I-stimulated [3H]AIB uptake; a 32-86% increase in IGF-I bioactivity was seen after a 24 h preexposure to 10 nM bIGFBP-3, and a 2- to 6-fold potentiation was seen after a 72 h preincubation. Preincubation with bIGFBP-3 increased both the sensitivity and maximal responsiveness of the cells to IGF-I. The potentiating effects of bIGFBP-3 were associated with increased [125I]IGF-I binding to cultured bovine fibroblasts. Affinity cross-linking experiments indicated that the increase in IGF-I binding was due to increased membrane-associated bIGFBP-3 rather than to a bIGFBP-3-induced increase in type I IGF receptors. bIGFBP-3 had no effect on insulin stimulation of [3H]AIB uptake under either experimental condition. These data suggest that soluble bIGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I receptor binding, whereas surface-associated bIGFBP-3 enhances the growth-promoting effects of IGF-I in bovine fibroblasts. We propose that IGFBP-3 serves a dual function in modulating IGF action in vivo.     

Receptor-mediated endocytosis and lysosomal processing of insulin-like growth factor I by mitogenically responsive cells

The processing of insulin-like growth factor I (IGF-I) by MG-63, an IGF-I-responsive human osteosarcoma cell line, has been investigated. At 37 C, the binding of [125I] IGF-I to monolayers of MG-63 cells reaches a maximum after about 1 h and slowly declines thereafter. The addition of the lysosomotropic agents methylamine, chloroquine, and monensin to the binding medium prevents the decline in [125I]IGF-I binding observed in the untreated cells and causes a 1.5- to 3-fold increase in cell-associated radioactivity after 4 h. Leupeptin, an inhibitor of lysosomal proteases, and colchicine, an inhibitor of endosomal transport, also increase cell-associated [125I]IGF-I. Three observations indicate that the increased radioactivity associated with the treated cells is the result of intracellular accumulation of the ligands and not the result of an increase in cell surface IGF binding. First, no increase in [125I]IGF-I binding is observed in cells preincubated with methylamine at 37 C but transferred to 4 C (where endocytosis is inhibited) before the addition of the radiolabeled ligands. Second, the increased radioactivity bound by methylamine-treated cells is not removed by washing the cells with dilute acid, a treatment that removes surface-bound [125I]IGF-I. Third, in leupeptin-treated cells [125I]IGF-I accumulates in a subcellular fraction with properties characteristic of lysosomes. Both alpha IR-3 (100 nM), an antibody that specifically inhibits binding to the type I IGF receptor, and high concentrations of insulin (900 nM) inhibit the accumulation of [125I]IGF-I by methylamine-treated cells, indicating that internalization of IGF-I occurs through the type I IGF receptor and not through the type II IGF receptor or the IGF-binding protein(s) that is also present on these cells. These results demonstrate that in MG-63 cells IGF-I is endocytosed via the type I IGF receptor and that the endocytosed hormone is degraded, at least in part, in lysosomes. These findings are similar to those described for the processing of insulin and other growth factors by their target cells and extend further the homology between IGF-I and these other agents.     

Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor I, platelet-derived growth factor, and transforming growth factor beta

Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H]proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21-day-old fetal rat calvariae. IGF I, PDGF, and TGF beta increased bone matrix apposition in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF beta was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF beta were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF beta are capable of stimulating bone formation in vitro.     

Interaction of insulin-like growth factor I with porcine thyroid cells cultured in monolayer

The interaction of insulin-like growth factor I (IGF-I) with porcine thyroid cells cultured in monolayer was studied. Specific binding of [125I]iodo-IGF-I to thyroid cells was a reversible process dependent on the time and temperature of incubation. A steady state was achieved in 18 h at 4 C and averaged 14.2 +/- 2% (mean +/- SD)/10(6) cells. Binding of [125I]iodo-IGF-I was inhibited by unlabeled IGF-I; half-maximal inhibition occurred at concentrations of 2-5 ng/ml. Multiplication-stimulating activity (rat IGF-II) and pork insulin had relative potencies of 1:20 and 1:300 compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with a Ka of 4.3 X 10(10) M-1, 49,000 binding sites were estimated per cell. Affinity cross-linking and autoradiography demonstrated the presence of type I IGF receptors. Thyroid cells also had specific receptors for insulin, but specific binding of [125I]iodoinsulin (2.03 +/- 0.03%/10(6) cells) was much lower than that of [125I]iodo-IGF-I. Preincubation of thyroid cells with IGF-I or insulin caused a concentration-dependent decrease in [125I]iodo-IGF-I binding due to an apparent loss of receptors. Preincubation with epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, or TSH did not alter subsequent binding of [125I]iodo-IGF-I. Low concentrations of IGF-I stimulated DNA synthesis and proliferation of thyroid cells and acted synergistically with epidermal growth factor. Multiplication-stimulating activity and insulin had relative potencies in stimulating DNA synthesis comparable to their abilities to inhibit the binding of [125I]iodo-IGF-I to thyroid cells, suggesting that their effects are mediated primarily by IGF-I receptors. Preincubation with IGF-I did not alter cAMP responsiveness to TSH. We, thus, demonstrated the presence of functional and regulated IGF-I receptors on porcine thyroid cells.     

Structural and immunological characterization of insulin-like growth factor II binding to IM-9 cells

The structural and immunological properties of the insulin-like growth factor II (IGF-II) receptor on IM-9 lymphoblasts were studied using a combination of competitive binding and affinity cross-linking techniques as well as with a panel of polyclonal and monoclonal antireceptor antibodies. Unlike IGF-II binding to the classical type II IGF receptor, [125I] IGF-II binding to IM-9 cells was potently inhibited not only by unlabeled IGF-II, but also by insulin (50% inhibition of binding at 2.5 and 1.5 nM, respectively). Affinity cross-linking of [125I] IGF-II to intact cells, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated that the overwhelming majority of IGF-II binding was to a type I receptor (apparent mol wt, greater than 300,000 unreduced and 135,000 reduced), with minimal binding to a type II receptor (apparent mol wt, 220,000 unreduced and 240,000 reduced). After preincubation with five different antireceptor antibodies, inhibition of [125I] IGF-II binding was comparable to inhibition of [125I]insulin binding. These studies demonstrate that in IM-9 cells, the majority of IGF-II binding is to a type I receptor with high affinity for both insulin and IGF-II. Whether this is an atypical insulin receptor or a unique type I receptor remains to be established.     

Receptors for insulin-like growth factors and growth effects of multiplication-stimulating activity (rat insulin-like growth factor II) in rat embryo fibroblasts

Levels of multiplication-stimulating activity (MSA) in fetal rat serum are high (2-4 micrograms/ml), suggesting that MSA may have a role in fetal growth. We now demonstrate that fibroblasts derived from rat embryos (REFs) have specific MSA receptors and respond to MSA with increased DNA synthesis. Two types of insulin-like growth factor (IGF) receptors were demonstrated by competitive binding of radioiodinated MSA, IGF-I, and IGF-II and by chemical cross-linking of [125I]iodo-MSA and [125I]iodo-IGF-I to REFs. One type of receptor (mol wt, 260,000 under reducing conditions) did not interact with insulin, and another type of receptor (mol wt, 130,000, under reducing conditions) was recognized by insulin. Scatchard analysis of [125I]iodo-MSA binding data was consistent with one class of noninteracting binding sites. A biological response of MSA, increased DNA synthesis, was demonstrated with autoradiography in REFs. During a 16-hr incubation, DNA synthesis was stimulated by normal rat serum, and platelet-poor plasma plus platelet-derived growth factor (PDGF), but not by serum from hypophysectomized (hypox) rats or hypophysectomized (hypox) platelet-poor plasma plus PDGF. However, when MSA was added to hypox serum or to hypox platelet-poor plasma plus PDGF, DNA synthesis was stimulated to the level achieved by normal rat serum. By contrast, during a longer cell multiplication experiment, REFs grew equally well in normal or hypox rat serum, raising the possibility that REFs may produce a MSA-like factor.     

Sensitization of human fibroblasts to insulin-like growth factor I by serum deprivation and dexamethasone pretreatment

Pretreatment of confluent human fibroblast cultures for two days in dexamethasone, serum free medium increased 10-20 fold the sensitivity of the cells to insulin-like growth factor I (IGF I) stimulation of amino acid uptake using the amino acid analog alpha-aminoisobutyric acid (AIB). This increased sensitivity resulted both from the use of serum free medium and the addition of dexamethasone to the serum free media. Pretreatment of the cells for 1, 2, or 3 days before assay showed that the maximum increase in sensitivity was obtained after a two day pretreatment. Pretreatment of the cells also increased their sensitivity to insulin and bovine insulin-like growth factor II stimulation of AIB uptake similar to that seen for IGF I. No consistent effect of the pretreatment was observed on either the basal level of AIB uptake or the maximal hormonal stimulation of AIB uptake. Nor was any change noted in the shape of the dose response curves. Addition of IGF I to the pretreatment medium greatly reduced the sensitivity of pretreated cells. [125I]IGF I binding studies done on suspended fibroblasts indicated that there was up to a two fold increase in the number of receptors with no increase in their affinity for IGF I. Thus, pretreatment of fibroblasts with dexamethasone and serum free medium greatly enhances their sensitivity to IGF I stimulation of AIB uptake and makes this an excellent in vitro bioassay system for IGF I.     

Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3E. coli) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3CHO) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3E. coli or hIGFBP-3CHO produced a dose-dependent inhibition of IGF-I but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGAFBP-3E. coli or hIGFBP-3CHO potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3E. coli for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3H]AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3E. coli with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 Mr hIGFBP-3E. coli seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3E. coli forms of 12,000-27,000 Mr. Cell-associated IGFBP-3E. coli (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3E. coli in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.     

Characterization of functional type I insulin-like growth factor receptors from human choriocarcinoma cells

Specific receptors for insulin-like growth factor I (IGF-I) on cultured human choriocarcinoma cells (JEG-3 and BeWo) were characterized. The binding of 125I-labeled recombinant (Thr59)IGF-I to the cells was reversible and time, temperature, and pH dependent. Steady state of binding occurred after 16 h at 4 C, pH 7.4. Natural human IGF-I (hIGF-I), hIGF-II, recombinant (N-Met)IGF-I, rat multiplication-stimulating activity, and insulin were 200%, 37%, 37%, 1.6%, and 0.1% as potent as (Thr59)IGF-I in inhibiting the binding of [125I]iodo-(Thr59)IGF-I to JEG-3 cells, respectively. Epidermal growth factor was ineffective. The half-maximal displacement of [125I]iodo-(Thr59)IGF-I by unlabeled (Thr59)IGF-I occurred at 11 +/- 2 ng/ml (mean +/- SEM) in both JEG-3 and BeWo cells. Scatchard analysis of the competitive binding data revealed linear plots indicating a single species of binding sites with an association constant of 0.8 X 10(9) M-1 for the binding of [125I]iodo-(Thr59)IGF-I to both cell lines. The binding capacity was 30,000 and 20,000 sites/cell for JEG-3 and BeWo cells, respectively. Chemical cross-linking of [125I]iodo-(Thr59)IGF-I to JEG-3 cells revealed two receptor complexes of 130K and 260K. Their formation was completely inhibited by an excess of unlabeled (Thr59)IGF-I or hIGF-II. Increasing amounts of insulin affected both labeled bands equally, suggesting that the 130K and 260K bands represent the monomer and dimer forms, respectively, of the ligand-binding alpha-subunit of type I IGF receptor. (Thr59)IGF-I, in a dose-dependent manner, stimulated uptake of nonmetabolizable alpha-[3H]aminoisobutyric acid by JEG-3 cells, showing that the receptor is biologically active. Our results demonstrate that choriocarcinoma cells possess functional high affinity type I IGF receptors and suggest that IGF-I is involved in the growth-regulating processes of JEG-3 and BeWo cells. These cells may provide a useful model to study the role of IGFs in trophoblast physiology.     

Insulin-like growth factor receptors and binding protein in rat neuroblastoma cells

B104, an established rat neuroblastoma cell line exhibiting specific neuronal qualities, was chosen as a model to study insulin-like growth factor (IGF) binding and action in the central nervous system. Specific binding of [125I]IGF-II to B104 membranes averaged 12.2 +/- 4.0% (mean +/- SD)/100 micrograms/ml protein compared with [125I]IGF-I binding of 10.1 +/- 2.9%. In competitive binding studies employing [125I]IGF-II as the radioligand, high affinity for IGF-II was demonstrated (50% displacement at 2.7 ng/ml), with none for IGF-I or insulin. Upon cross-linking [125I]IGF-I to membranes under reducing conditions, two prominent bands were observed, migrating with apparent mol wt (Mr) of 135,000 and 280,000. Both bands were inhibited by IGFs and insulin, but not by R-II-PABI, a polyclonal antibody to the type 2 receptor. These bands presumably represent the alpha-subunit and an incompletely reduced alpha-alpha-dimer of the type 1 IGF receptor. When cross-linking [125I]IGF-II to membranes under reducing conditions, the primary labeled bands migrated with apparent Mr of 260,000 and 280,000. These bands were inhibited by IGF-II and R-II-PABI, but not by insulin, and probably represent the monomeric type 2 receptor. In addition, we observed a minor band at apparent Mr 35,000, which was inhibited by IGF but not by insulin. By a modified cross-linking technique, we confirmed the existence of a small IGF-binding protein in the serum-free conditioned medium of B104 cultures, migrating as two bands with apparent Mr of 33,000-39,000. These proteins demonstrated high affinity for IGF-I and IGF-II, but none for insulin. In summary, this study demonstrates the presence in B104 rat neuroblastoma cells of 1) abundant classical type 1 and type 2 IGF receptors, and 2) a secreted and membrane-associated small IGF-binding protein.     

Regulation of receptors for insulin-like growth factors in cultured arterial smooth muscle cells by thyroid hormone

The effect of tri-iodothyronine (T3) on the binding of 125I-labelled insulin-like growth factors (IGF) to cultured arterial smooth muscle cells was investigated. When cells which were grown to confluency were incubated with 125I-labelled IGF and different concentrations of T3, low concentrations of T3 (0.1-10 nmol/l) increased the binding of 125I-labelled IGF. High concentrations of T3 (1 mumol/l) could not induce this effect. Scatchard analysis of the binding of 125I-labelled IGF in the presence of different concentrations of unlabelled IGF showed a high-affinity, low-capacity binding system and a second lower affinity, high-capacity binding system. In the presence of T3 (1 nmol/l) Scatchard analysis indicated that the affinity of 125I-labelled IGF to its binding sites was altered. After a 2-h preincubation of the cell layers with Dulbecco's Modified Eagle's Medium containing T3 (1-100 nmol/l), the 125I-labelled IGF binding was enhanced in a dose-dependent manner by T3. Scatchard analysis showed a significant increase in the number of IGF binding sites.     

Ovarian granulosa cell-derived insulin-like growth factor (IGF) binding proteins: release of low molecular weight, high-affinity IGF-selective species.

The ovarian granulosa cell has previously been shown to be a site of insulin-like growth factor (IGF) I production, reception, and action. It is the objective of this study to characterize in greater detail the soluble IGF binding activity released by this cell type. To this end, use was made of granulosa cells from immature diethylstilbestrol-treated rats. Serum-free media conditioned for 72 h by cultured untreated cells acquired polyethylene glycol (PEG)-precipitable [125I]IGF-I binding activity. The latter proved cell density-dependent, displaying a minimal inoculum requirement of less than or equal to 3 x 10(5) cells/culture. The daily elaboration of IGF-I binding activity appeared constant throughout the 72 h experimental period, the overall time-dependent accumulation of binding activity (over the same time period) proving virtually additive. Scatchard analysis of detailed competition studies with IGF-I suggests that the latter ligand binds to granulosa cell-derived IGF binding protein(s) (IGFBPs) with an apparent affinity of 3 x 10(-10) M. Qualitatively similar results were obtained when using [125I]IGF-II suggesting that the IGFBPs in question are not IGF-I-selective. In fact, specificity studies using either [125I]IGF-I or [125I]IGF-II revealed a rank order of competitive potencies compatible with that observed in other tissues so studied (IGF-II greater than IGF-I much greater than insulin). The proteinacious nature of the acid-stable IGF binding activity under study was indicated by its sensitivity to relatively low concentrations of cycloheximide, its apparent deactivation following repeated cycles of freezing and thawing, and its virtual elimination when subjected to boiling or trypsin treatment. Cycloheximide-induced blockade of protein biosynthesis also revealed that the IGF binding activity is subject to measurable turnover thereby suggesting that its accumulation represents the balance struck between synthetic and degradative processes. Western ligand blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated media revealed a non-glycosylated major band doublet of 28-29 kDa. A single minor IGFBP species represented by a 23 kDa band was also appreciated in some but not all experiments. Taken together, these findings document the ability of ovarian granulosa cells to secrete a heterogenous mix of low molecular weight, high-affinity IGF-selective binding species. As such, these observations are in keeping with the concept of a complete intraovarian IGF system replete with ligands, receptors, and soluble binding activity.     

Regulation of insulin receptors in the human ovary: in vitro studies

The association between insulin resistance and ovarian hyperstimulation has led to a hypothesis that insulin stimulates ovarian steroidogenesis. This possible effect of insulin on the ovary could be mediated by either the insulin receptor or the type I insulin-like growth factor (IGF) receptor, both of which have been described in the human ovary. We examined the in vitro regulation of insulin receptors by LH, FSH, multiplication-stimulating activity (MSA), and insulin in ovarian stromal fragments from 24 women. [125I]Insulin binding was measured in the presence and absence of increasing concentrations of insulin, IGF-I, LH, and FSH. Neither LH nor FSH competed with [125I]insulin for binding sites, but preincubation with LH or FSH reduced [125I]insulin binding by 19-38%. Preincubation with MSA reduced [125I]insulin binding by 34-48%. The affinity of the insulin receptors, determined by Scatchard analysis, did not change (Ka = 3.3 X 10(8) mol-1). A concentration of 10 ng/mL insulin in the preincubation medium reduced [125I]insulin binding by 40%, whereas an insulin concentration of 50 or 500 ng/mL completely obliterated specific [125I]insulin binding. [125I]Insulin binding fully recovered, however, 4 h after termination of tissue exposure to insulin. The specificity of [125I] insulin binding was confirmed by studies with IGF-I. We conclude that of the hormones examined, insulin is the most potent regulator of human ovarian insulin receptors in vitro. Down-regulation of insulin receptors by insulin was reversed within 4 h after withdrawal of insulin. MSA, FSH, and LH also down-regulated the number of human ovarian insulin receptors in vitro, but were less potent. These phenomena, if also present in vivo, could be important factors in the regulation of ovarian function by insulin in normal and pathological states.     

Different insulin-like growth factor binding species in human placenta and decidua

Previous studies demonstrated that human decidua secretes a 34K insulin-like growth factor binding protein (34K IGF-BP) earlier designated placental protein 12, whereas placenta contains IGF receptors and IGF mRNA. We studied binding of [125I]IGF-I to paired placental and decidual tissues using competitive binding, gel filtration, RIA, and cross-linking techniques, and compared the binding characteristics of these two tissues which are located in close proximity in vivo. The effect of decidual cytosols on [125I]IGF-I binding to placental membranes also was studied. Consistent with previous data the dominating binding species in placental membranes were IGF receptors. In contrast, the binding of [125I]IGF-I to decidual membranes was mainly due to binding species with mol wts of 34K and 39K. The 34K IGF-BP was more readily detected in decidual cytosols than in decidual membranes and little was detected in placental cytosols. Purified 34K IGF-BP as well as decidual cytosols inhibited [125I]IGF-I binding to placental receptors. The inhibitory effect of decidual cytosol on IGF receptor binding was linearly correlated to the decidual content of 34K IGF-BP. The results suggest that the decidual 34K IGF-BP might act as a local modulator of the IGF action at the interface between the decidua and the placenta.     

Hormonal regulation of type I insulin-like growth factor receptors of Leydig cells in hypophysectomized rats

The effects of hCG and various pituitary hormones on type I insulin-like growth factor (IGF) receptors of purified Leydig cells of hypophysectomized rats were studied. The number of type I IGF receptors of Leydig cells obtained from hypophysectomized rats (18.0 +/- 1.5 fmol/10(6) cells) was lower than that in normal rats (54.6 +/- 5.3 fmol/10(6) cells; P less than 0.05). After a single administration of hCG (10 U, ip), specific binding of [125I]IGF-I to purified Leydig cells increased 3-fold. Scatchard analyses of the binding data suggested that increased binding was the result of an increase in receptor number, whereas binding affinity remained unaltered. Type I IGF receptor increased within 12 h and remained persistently elevated 96 h after hCG treatment. Administration of hCG (10 U, ip) daily for 5 days increased type I IGF receptor levels to 73.2 +/- 8 fmol/10(6) cells (P less than 0.001). FSH caused a small but significant increase in type I IGF receptors. Concomitant administration of FSH and hCG further enhanced IGF-I-binding capacity. IGF-I-binding affinity of Leydig cells treated with FSH or FSH plus LH was not significantly different from that in the control hypophysectomized rats. Daily administration of GH for 5 days also upregulated type I IGF receptors, whereas PRL had no effect. FSH, GH, and PRL administration had no effect on serum testosterone levels. Serum testosterone levels increased to 3.99 +/- 0.35 ng/ml after 5 days of treatment with hCG. Concomitant administration of FSH and hCG caused a further increased in serum testosterone levels (6.13 +/- 0.46 ng/ml; P less than 0.01). The present study suggests that type I IGF receptors of Leydig cells can be up-regulated by LH, FSH, and GH. However, hCG/LH seems to be the most important factor in maintaining and regulating type I IGF receptors of Leydig cells. Steroidogenic and growth-promoting effects of hCG and pituitary hormones on Leydig cells may be mediated by increased type I IGF receptors.     

Characterization of insulin-like growth factor I receptor on human erythrocytes

[125I]Insulin-like growth factor I (IGF-I) specifically bound to erythrocytes; the binding was saturable, and time and temperature dependent. Steady state binding was reached at 16 h at 4 C, and specific binding averaged 14.3 +/- 0.7% (+/- SEM) at a concentration of 3.6 X 10(9) cells/ml in seven normal subjects. [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose-dependent manner. Scatchard analysis indicated a linear plot, and Ka and number of binding sites/cell were 1.43 +/- 0.07 X 10(9) M-1 and 20.7 +/- 2.2, respectively. Compared to IGF-I, the relative potencies of multiplication-stimulating activity and insulin for displacing [125I]IGF-I binding were 20% and 1%, respectively. [125I]IGF-I binding to erythrocytes from patients with acromegaly was lower than binding to cells from pituitary dwarfs. An inverse correlation between plasma IGF-I level and the number of IGF-I-binding sites per cell was found (r = -0.75; P less than 0.005). These results demonstrate that [125I]IGF-I binding to erythrocytes can be used for clinical measurement of the IGF-I receptor.     

Regulation of low molecular weight insulin-like growth factor binding proteins in experimental diabetes mellitus.

Circulating insulin-like growth factor (IGF) bioactivity is reduced in animals and patients with diabetes mellitus. We sought to determine whether the availability and levels of specific IGF binding proteins (BPs) are altered in animals with experimental diabetes, and might contribute to changes in circulating IGF bioactivity in experimental diabetes. Female Sprague-Dawley rats were administered streptozotocin or citrate buffer iv, and then killed either 3 days later, or else after 4-day insulin treatment (7.5 U/kg human NPH twice daily), or 2 days after insulin was discontinued. Serum [125I]IGF-I binding activity was markedly increased in diabetic animals compared to controls when analyzed by Sephacryl S-200 chromatography, dot blot, and affinity labeling techniques, due to increased binding to low mol wt BPs (81 +/- 4% of ligand eluting with low mol wt BPs in diabetic serum vs. 22 +/- 3% in control, P less than 0.001). In contrast, activated charcoal removed ligand from these BPs and underestimated the availability of BPs in diabetes. Serum binding activity fell toward control levels during insulin therapy, then rose again after insulin was withdrawn, corresponding to changes in metabolic status. To distinguish changes in specific BPs, serum proteins were separated by 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to nitrocellulose. Ligand blotting with [125I]IGF-I demonstrated that serum levels of a 32 K mol wt IGF BP are markedly increased in diabetic rats and decline during insulin therapy. Levels of this 32 K IGF BP rose again after insulin was discontinued, demonstrating regulation in accordance with changes in insulin and metabolic status. Western analysis and affinity labeling with immunoprecipitation revealed that this 32 K protein is distinct from the 34 K fetal rat BP, and is immunologically related to the type 1 human IGF BP. We conclude that circulating [125I]IGF-I binding activity is markedly increased in animals with acute streptozotocin-induced diabetes, due to changes in low mol wt proteins, including a 32 K type 1 IGF BP that is regulated by changes in insulin and/or metabolic status. Regulation of low mol wt IGF BPs by insulin, and perhaps other factors, may play an important role in the modulation of tissue growth factor bioactivity in metabolic disease.     

Evidence for the presence of type I insulin-like growth factor receptors on rat pancreatic A and B cells

Purified rat pancreatic islet cells express somatomedin receptors which are identified by their affinity for insulin-like growth factor (IGF)-I, IGF-II, and insulin. Binding of [125I]IGF-I to islet A cells was half-maximally inhibited by 7.10(-10) M IGF-I, while IGF-II, insulin, and proinsulin were respectively 10-, 500-, and 10,000-fold less potent displacers of IGF-I binding. Unrelated hormones such as glucagon or GH did not compete with [125I]IGF-I binding to A cells. The concentration of IGF-I receptors on A cells was estimated at 5000 IGF-I binding sites per cell with affinity constant (Ka) of 2 X 10(9) M-1. Islet B cells were found to exhibit a reversible time- and temperature-dependent binding with [125I]IGF-I. Specificity and affinity of IGF-I binding sites were identical for islet A and B cells. Linear Scatchard plots of competitive binding data on B cells suggest 1 single class of IGF-I receptors in a concentration of 12,000 sites per cell. The presence of high affinity receptors for IGF-I on adult islet A and B cells provides a molecular basis for this growth factor to influence growth, survival, and/or function of these endocrine cell types. Their low affinity for insulin should be considered as a potential mechanism for this hormone to influence, at high concentration, the function of islet A and B cells.