Evidence from monoclonal antibody studies that insulin stimulates deoxyribonucleic acid synthesis through the type I somatomedin receptor

Somatomedin-C/insulin-like growth factor-I (Sm-C/IGF-I) and insulin stimulate DNA synthesis and cell replication in cultured human fibroblasts. It has been postulated that the growth-promoting actions of both peptides are mediated through the type I Sm-C/IGF-I receptor. This study tests this hypothesis using two recently developed monoclonal antibodies. The antibody designated sm 1.2 is directed to Sm-C, whereas the antibody designated alpha IR-3 is directed against the type I receptor for Sm-C/IGF-I. Radiolabeled monoclonal antibody alpha IR-3 was bound to human foreskin fibroblasts in a reversible time-dependent fashion, with 90% of the specific binding complete after 6 h of incubation at 15 C. Binding of [125I]alpha IR-3 was completely inhibited by excess unlabeled antibody, but not by 50 nM Sm-C or 1000 nM insulin. Specific binding of [125I]Sm-C fell to 27% of the control value in the presence of 50 nM alpha IR-3, and this concentration of antibody significantly reduced the mitogenic response to both Sm-C and insulin. Antibody sm 1.2 blocked the mitogenic response to exogenous Sm-C, but did not block the response to insulin; indeed, in some experiments, sm 1.2 enhanced the response to insulin. We postulate that this enhancement is the result of neutralizing endogenously produced Sm-like substances. This study provides further evidence that the growth-promoting effects of insulin in this cell type are the result of interaction with the Sm-C/IGF-I receptor.     

The type II insulin-like growth factor receptor does not mediate deoxyribonucleic acid synthesis in human fibroblasts

Two insulin-like growth factor (IGF) receptors, the type I and type II IGF receptors, have been described. While substantial evidence indicates that the type I receptor is involved in the regulation of cell division, it is uncertain if the type II receptor also mediates this response. Similarly, the role of the insulin receptor in mediating DNA synthesis remains controversial. To address these questions, we used a monoclonal antibody (alpha IR-3) to specifically inhibit type I IGF receptor activity and examined the effects of this inhibition on IGF- and insulin-stimulated DNA synthesis in human fibroblasts. WI-38 human embryonic lung fibroblasts have both type I and type II IGF receptors, as determined by cross-linking [125I] IGF-I and [125I]IGF-II to monolayers of these cells. In serum-free medium both IGF-I and IGF-II stimulate DNA synthesis in WI-38 fibroblasts, with half-maximal effects occurring at 1.5 +/- 0.3 (+/- SD) and 3.4 +/- 1.4 nM, respectively. At maximally effective concentrations, however, both hormones stimulate DNA synthesis to equal levels. alpha IR-3 binds to the type I, but not the type II, IGF receptor on WI-38 cells. It also inhibits IGF binding to the type I receptor on these cells. alpha IR-3 competitively inhibited both IGF-I- and IGF-II-stimulated DNA synthesis in WI-38 cells, but had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated DNA synthesis. These results indicate that in WI-38 fibroblasts the mitogenic effects of both IGF-I and IGF-II are mediated through the type I receptor and that the type II IGF receptor is not directly involved in this response. To define the role of the insulin receptor in mediating DNA synthesis we compared the effects of alpha IR-3 on insulin-stimulated DNA synthesis in a variety of human cell lines under identical experimental conditions. With WI-38 and HEL, another human embryonic lung fibroblast cell line, alpha IR-3 competitively inhibited the mitogenic effect of insulin. However, in two other fibroblast cell lines (GM498 and HES) and an osteogenic sarcoma cell line (MG63), alpha IR-3 inhibited IGF, but not insulin-stimulated DNA synthesis. These results indicate that human cell lines differ in the receptor type through which insulin stimulates DNA synthesis and that these differences are intrinsic properties of the cell lines and are not artifacts resulting from differences in experimental conditions.     

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.     

Rat C6 glial cells synthesize insulin-like growth factor I (IGF-I) and express IGF-I receptors and IGF-II/mannose 6-phosphate receptors

We have used the rat C6 glial cell line as a model system to study the role of insulin-like growth factors (IGF) in neuroglial cells of the central nervous system (CNS). Northern blot analysis of C6 RNA demonstrated the presence of IGF-I mRNA and undetectable IGF-II mRNA. IGF-I and IGF-binding protein(s), but not IGF-II, were detected in C6 glial cell-conditioned medium. The level of IGF-I was 1-4 ng/ml in conditioned medium based on a human IGF-I standard. The immunoreactive IGF-I inhibited [125I]IGF-I binding to the IGF-I receptor on chick embryo fibroblasts and stimulated [3H]thymidine incorporation into chick embryo fibroblast DNA. Competitive binding and affinity cross-linking experiments using [125]IGF-I and [125I]IGF-II demonstrated the presence of IGF-I receptors (type I) and IGF-II/mannose 6-phosphate receptors (type II) on C6 glial cell membranes. An immunoglobulin (no. 3637) directed against the rat IGF-II receptor blocked the degradation of [125I]IGF-II added to C6 glial cells, presumably by blocking receptor-mediated internalization. We were unable to demonstrate an autocrine role for IGF in the C6 glial cell line, since [3H]thymidine incorporation into DNA was stimulated equally well by IGF-I-deficient rat serum and normal serum, and added IGF did not stimulate [3H]thymidine incorporation into DNA when tested alone or when added to IGF-I-deficient serum. We propose that neuroglial cell-derived IGF-I may serve as a paracrine growth stimulus in the central nervous system.     

Insulin stimulation of aminoisobutyric acid transport in human skin fibroblasts is mediated through both insulin and type I insulin-like growth factor receptors

A monoclonal antibody to the human type I insulin-like growth factor (IGF-I) receptor (alpha IR-3) was used to distinguish actions of insulin and IGF-I that are mediated through insulin as opposed to IGF-I receptors on human skin fibroblasts. Both insulin and IGF-I stimulate uptake of the nonmetabolized alanine analog alpha-aminoisobutyric acid (AIB) in these cells. alpha IR-3 inhibited AIB uptake stimulated by both of these hormones in a dose-dependent manner. However, the pattern of hormone action in the presence of alpha IR-3 differed for the two hormones. In the case of IGF-I, alpha IR-3 potently inhibited AIB uptake at low hormone concentrations, but this inhibition was overcome by high hormone concentrations, consistent with impairment of IGF-I action through the IGF-I receptor. In the case of insulin, the action of low concentrations (i.e. 10 ng/ml) was not inhibited, but that of higher insulin concentrations was, suggesting a dual receptor mechanism of cell stimulation by insulin. alpha IR-3 will be an important tool in further studies of the biology of the IGF-I receptor in normal and abnormal human cells.     

Insulin and insulin-like growth factor I regulate the same biological functions in HEP-G2 cells via their own specific receptors

The receptors for insulin and insulin-like growth factor I (IGF-I) are closely related molecules, with an extracellular binding domain and an intracellular tyrosine kinase domain. The interaction of insulin and IGF-I with their respective receptors activates the receptor kinase domain, leading to the biological actions of the hormones. Since insulin generally regulates metabolic events and IGF-I generally regulates growth events, it is believed that structural differences in the tyrosine kinase domains of the two respective receptors may elicit different biological responses via different transmembrane signaling mechanisms. We studied the regulation of glycogen metabolism and amino acid uptake in human cultured HEP-G2 hepatoma cells, which have distinct receptors for both insulin and IGF-I. The receptor specificity of these responses was probed with specific monoclonal antibodies to both the insulin and IGF-I receptors. Stimulation of both [3H]glucose incorporation into glycogen and alpha-[3H]aminoisobutyric acid uptake by insulin was half-maximal at concentrations of 1-5 nmol/L. These effects were blocked by the insulin receptor monoclonal antibody MA-10, but not by the IGF-I receptor antibody alpha IR-3. Stimulation of both functions by IGF-I was half-maximal at concentrations of 1-5 nmol/L, and these effects were inhibited by alpha IR-3, but not by MA-10. These studies indicate that in HEP-G2 cells both insulin and IGF-I, via their own receptors, stimulate the same biological responses.     

Acromegaloid patients with type A insulin resistance: parallel defects in insulin and insulin-like growth factor-I receptors and biological responses in cultured fibroblasts

A subset of patients with the syndrome of acanthosis nigricans and insulin resistance type A is characterized by acromegaloid features in addition to hyperinsulinemia, hyperandrogenemia, and an inherent defect in insulin receptor function. It has been proposed that the acromegaloid features result from the interaction of insulin at concentrations encountered in vivo, with a functionally intact insulin-like growth factor-I (IGF-I) receptor closely related to the insulin receptor. We investigated this possibility by examining binding and hormone-stimulated [14C]glucose uptake, [3H]thymidine uptake, and receptor autophosphorylation by both insulin and IGF-I in cultured fibroblasts from two affected patients. In comparison to normal fibroblasts, [125I]insulin binding, insulin-stimulated [14C]glucose, and [3H]thymidine uptake, and insulin-stimulated autophosphorylation were each reduced by approximately 50-60% of the absolute values in controls. In contrast to expectation, each of these apparent defects in insulin binding and action were mirrored by a parallel decrease in IGF-I binding and action. Thus, [125I]IGF binding was approximately 50%, IGF-I stimulated [3H]thymidine uptake was approximately 40% and 60% of the control value, and IGF-I-stimulated receptor autophosphorylation was reduced by 40%. Incubation of fibroblasts with insulin at 25 ng/mL reduced subsequent binding of [125I]IGF-I by approximately 20% and did not enhance maximal stimulation of [3H]thymidine incorporation. We conclude that in some patients with acanthosis nigricans and acromegaloid features, IGF-I receptors of cultured fibroblasts may share the inherent defects of insulin receptor function. These in vitro data do not explain the acromegaloid features observed in vivo, suggesting that acromegaloid features are mediated by other mechanisms.     

Growth-stimulatory monoclonal antibodies against human insulin-like growth factor I receptor.

Monoclonal antibodies (mAbs) against purified human placental insulin-like growth factor I (IGF-I) receptors were prepared and characterized. Three IgG mAbs were specific for the human IGF-I receptor and displayed negligible crossreactivity with the human insulin receptor. They stimulated 125I-labeled IGF-I (125I-IGF-I) or 125I-IGF-II binding to purified human placental IGF-I receptors and to IGF-I receptors expressed in NIH 3T3 cells in contrast to the well-studied mAb alpha IR-3, which inhibits 125I-IGF-I or 125I-IGF-II binding to both forms of IGF-I receptors. The mAbs introduced in this study stimulated DNA synthesis in NIH 3T3 cells expressing human IGF-I receptors approximately 1.5-fold above the basal level and the IGF-I- or IGF-II-stimulated level. In contrast, alpha IR-3 inhibited both basal and IGF-I or IGF-II-stimulated DNA synthesis by approximately 30%. Inhibition of IGF-II-stimulated DNA synthesis by alpha IR-3 was as potent as its inhibition of IGF-I-stimulated DNA synthesis, although IGF-II binding to the IGF-I receptors was not inhibited by IGF-II as potently as was IGF-I. With the purified IGF-I receptors, both inhibitory and stimulatory mAbs were shown to activate autophosphorylation of the IGF-I receptor beta subunit and to induce microaggregation of the receptors. These results suggest that conformational changes resulting from receptor dimerization in the presence of either type of mAb may affect the signal-transducing function of the IGF-I receptor differently. These additional mAbs and alpha IR-3 immunoprecipitated nearly 90% of IGF-I binding activity from Triton X-100-solubilized human placental membranes, indicating that IGF-I receptor reactive with these mAbs is the major form of the IGF-I receptor in human placenta.     

Multiple factors influence insulin-like growth factor-I binding to human skin fibroblasts

The binding of [125I]insulin-like growth factor-I ([125I]IGF-I) to human skin fibroblasts (HSF) is regulated by multiple factors. In monolayers of HSF, IGF-I binds to both the type I IGF receptor and IGF-binding proteins (BPs) associated with the cell surface. [125I]IGF-I binding to both of these proteins depends markedly on the sodium chloride concentration of the binding buffer. In monolayers of HSF, replacing 120 mM NaCl with isoosmotic concentrations of sucrose increases binding of [125I]IGF-I by 2- to 6-fold. Enhancement of [125I]IGF-I binding in the absence of sodium chloride is also seen in HSF in suspension, in human erythrocytes, in monolayers of HEP G2 cells and FRTL5 cells, and in membranes prepared from human placentae. Kinetic analysis of [125I]IGF-I binding to HSF monolayers reveals that association rates are increased and dissociation rates are decreased in the absence of sodium chloride. The binding of [125I]alpha IR-3, a monoclonal antibody to the human type I IGF receptor, to monolayers and suspensions of HSF also depends on the sodium ion concentration; it is 5- to 7-fold higher in the absence of sodium chloride. Binding of [125I]IGF-I to monolayers of HSF also depends on NaCl under conditions where alpha IR-3 saturates the type I IGF receptor but does not affect IGF-BPs. These findings demonstrate that sodium chloride has a marked effect on the interaction of IGF-I with the type I IGF receptor in the plasma membrane and with BPs associated with the surface of intact HSFs. Since an effect is also evident in membranes prepared from intact tissues (human placenta), occurs at 4 C, and occurs with cells devoid of BPs, a mechanism involving receptor or BP translocation seems unlikely, at least as the sole explanation for these findings. Sodium ions (and other ions) may induce a conformational change in the receptor and BPs and cause decreased availability of both the IGF-I-binding site and the alpha IR-3 epitope on the receptor and the IGF-binding site on the BP.     

Interaction of the monoclonal antibodies alpha IR-1 and alpha IR-3 with insulin and somatomedin-C receptors

alpha IR-3, a monoclonal antibody that interacts with the somatomedin-C receptor, inhibited the binding of somatomedin-C, but not of insulin, to human placental membranes and intact IM-9 cells. alpha IR-1, a monoclonal antibody that interacts with the insulin receptor, did not inhibit the binding of either hormone. Inhibition of somatomedin-C binding by alpha IR-3 was mainly due to a decrease in its affinity. 125I-Labeled alpha IR-3 bound specifically to placental membranes and intact IM-9 cells and was inhibited by concentrations of unlabeled alpha IR-3 that were lower than those required to inhibit somatomedin-C binding. [125I]alpha IR-3 binding was also inhibited by somatomedin-C and insulin, but only at very high concentrations. A410, a rabbit antiserum that reacts with both receptors for insulin and somatomedin-C, also inhibited labeled alpha IR-3 binding. alpha IR-I did not. These results help to define the epitopes with which these antibodies interact.     

Normal somatomedin-C/insulin-like growth factor I binding and action in cultured human fibroblasts from Turner syndrome

Growth retardation is a major manifestation of Turner syndrome (TS). Since plasma growth hormone and somatomedin-C/insulin-like growth factor I (SM-C/IGF-I) levels are generally normal, growth failure has been ascribed to peripheral defects in SM-C/IGF-I receptors or action. We have measured the binding of [125I]SM-C/IGF-I to cultured fibroblast monolayers derived from patients with Turner syndrome, and have evaluated SM-C/IGF-I stimulation of both [3H]thymidine incorporation and cell replication. When compared to fibroblasts from normal adults, newborns, and age-matched children, no significant differences were observed in specific binding of [125I]SM-C/IGF-I to fibroblast monolayers, and displacement curves demonstrated similar receptor affinities for all groups. Similarly, equivalent SM-C/IGF-I stimulation of [3H]thymidine incorporation was seen in both Turner and control fibroblasts. In the absence of serum, SM-C/IGF-I, at a concentration of 10-25 ng/ml, stimulated thymidine incorporation 3.7-11.8-fold in Turner fibroblasts and 1.9-9.8-fold in control cells. In combination with 1.0% human hypopituitary serum (HHS), SM-C/IGF-I stimulated thymidine incorporation 20-70-fold in all cell lines. Cell replication in both TS and control cells was increased 90% by the combination of SM-C/IGF-I + 0.5% HHS, and 140% by SM-C/IGF-I + 0.5% HHS + dexamethasone. We conclude that TS fibroblasts have normal SM-C/IGF-I receptors and sensitivity, and are capable of enhanced DNA synthesis and replication following SM-C/IGF-I stimulation.     

Influence of growth factors on an insulin-producing cell line (RINm5F)

The influence of IGF-I, insulin and epidermal growth factor (EGF) as well as glucose as control was studied on both growth and function of RINm5F cells, an insulin-producing cell line derived from rat insulinoma tissue. [3H]thymidine incorporation and DNA content (fluorometrically) were measured for estimating cell growth, insulin release and biosynthesis [( 3H]leucine incorporation) as parameters for cell function. There was a significant increase in both [3H] thymidine incorporation and DNA content into RIMm5F cells by glucose already at very low concentrations. IGF-I and high concentrations of insulin too were able to stimulate cell growth (insulin also in additional experiments with isolated rat islets). EGF, however, was without growth effect on this cell line (in contrast to previous own results in isolated islets). Insulin secretion and biosynthesis were also increased by very low glucose concentrations. IGF-I and EGF were ineffective regarding these functional parameters in RINm5F cells. Besides for glucose, our results demonstrate a role for IGF-I and insulin, but not for EGF, in regulating growth of these cells.     

Somatomedin C/insulin-like growth factor I binding and action in human fibroblasts aged in culture: impaired synergism with dexamethasone

Somatomedin C/insulin-like growth factor I (SM-C/IGF-I) binding and action were compared in human fibroblasts serially passaged in vitro. Binding of [125I]SM-C/IGF-I was identical in early and late passage cells, with 50% displacement at 4 ng/ml SM-C/IGF-I. Maximal concentrations of SM-C/IGF-I (100 ng/ml) stimulated [3H]thymidine incorporation equally in both early and late passage cells. Furthermore, dose response curves indicated that there was no alteration in the sensitivity of cells to SM-C/IGF-I with age. Preincubation with dexamethasone (10(-7) M) potentiated SM-C/IGF-I's mitogenic effect in early passage cells. In contrast, dexamethasone had no effect on SM-C/IGF-I stimulation of [3H]thymidine incorporation or replication of late passage cells. These data indicate that fibroblasts passaged in vitro have normal SM-C/IGF-I binding and are capable of responding to the mitogenic effect of SM-C/IGF-I. The synergism between SM-C/IGF-I and dexamethasone in stimulating DNA synthesis and replication of early passage fibroblasts is lost, however, in late passage cells.     

Characterization and regulations of the receptor for insulin-like growth factor-I in the FRTL-5 rat thyroid follicular cell line

In previous studies we have shown that insulin-like growth factor I (IGF-I) has a mitogenic effect in a line of rat thyroid follicular cells, the FRTL-5. In view of this effect, we undertook studies to identify and characterize some physicochemical and binding properties of the receptor for IGF-I in these cells and to determine what role it plays in the mitogenic activity of insulin and insulin-like growth factors in the FRTL-5 cell. Binding of 125I-labeled IGF-I (biosynthetic Thr59-IGF-I) to FRTL-5 was a function of time, temperature, and pH and was completely inhibited by high concentrations of unlabeled IGF-I. Scatchard plots of four saturation studies revealed a single apparent binding site with an average Ka of 4.2 +/- 0.6 X 10(9) M-1 (mean +/- SD) and an average maximum binding capacity of 20 +/- 2 pm/100 micrograms cellular protein. Rat IGF-II (rIGF-II) and insulin were far less potent that IGF-I in inhibiting the binding of [125I] IGF-I, and bovine TSH was without effect. 125I-Labeled IGF-II also bound to FRTL-5 cells. Binding was completely inhibited by unlabeled rIGF-II and, with lesser potency, by IGF-I. Even at high concentrations, insulin failed to inhibit the binding of [125I]IGF-II. Disuccinimidyl suberate cross-linked [125I]IGF-I to a moiety in FRTL-5 that had an apparent mol wt of approximately 135,000, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Cross-linking of [125I]IGF-I was inhibited in a dose-dependent manner by unlabeled IGF-I and, with far lower potency, by rIGF-II and insulin. All three peptides stimulated the incorporation of [3H]thymidine into the DNA of FRTL-5 cells, IGF-I being the most potent, followed in decreasing order of potency of rIGF-II and insulin. The mitogenic activities of these polypeptides correlated well with their abilities to inhibit the binding of [125I]IGF-I. These data indicate that the FRTL-5 cell possesses a receptor for IGF-I that resembles in its binding and physicochemical properties the receptor for IGF-I in other tissues (type I IGF receptor) and that mediates the mitogenic response to IGF-I and insulin in these cells. FRTL-5 cells also contain a receptor for IGF-II (type II IGF receptor), but its role vis-à-vis that of the type I IGF receptor in relation to the mitogenic effect of IGF-II in these cells is uncertain.     

Inability of a mouse cell line transformed to produce biologically active recombinant human insulin-like growth factor I (IGF-I) to respond to exogenously added IGF-I

A plasmid expression vector encoding human insulin-like growth factor I (hIGF-I) in the form of a 97-amino acid precursor protein containing the first 27 amino acids of prebovine GH and the 70 amino acids of hIGF-I has been used to transform mouse L cells. A stably transformed mouse L cell clone has been isolated which expresses and secretes hIGF-I. The secreted peptide comprises 3% of the protein in conditioned medium. IGF-I can be purified to homogeneity in 2 chromatographic steps. One liter of conditioned medium yields approximately 200 micrograms purified peptide. Amino-terminal sequence analysis confirms that the signal peptide has been proteolytically hydrolyzed from the precursor protein before secretion to form [Ala0]hIGF-I. The recombinant peptide and serum-derived hIGF-I are equipotent as inhibitors of the binding of [125I]IGF-I to the type 1 receptor of human placenta and to a crude preparation of acid-stable human serum binding proteins. The peptides are equipotent in 2 in vitro assays, the stimulation of the rate of 2-[1,2-N-3H]deoxyglucose transport in BC3H1 cells and the stimulation of [methyl-3-3H]thymidine incorporation into DNA in A10 cells. In contrast to a control mouse L cell line, DNA synthesis in the [Ala0]IGF-I-secreting line is completely unresponsive to [Thr59]IGF-I, while it responds normally to calf serum (10%). Thus, the [Ala0]IGF-I-secreting line is selectively desensitized to IGF-I. The binding of [125I]IGF-I to both lines is identical, indicating that the loss of responsiveness to IGF-I is not due to a loss of cell surface receptor. The ability to render mouse L cells unresponsive to IGF-I is transferred in the conditioned medium of the [Ala0]IGF-I-secreting cell line. In addition, pretreatment of control cells with [Thr59]IGF-I (10 nM) results in attenuation of the response to a subsequent dose of IGF-I. These data indicate that prolonged exposure to high levels of IGF-I may cause a postreceptor-mediated desensitization to IGF-I. Alternatively, IGF-I may promote secretion of an inhibitor of IGF-mediated DNA synthesis.     

Identification of a receptor for somatomedin-like polypeptides in human fibroblasts.

We have demonstrated a specific receptor for somatomedin-like growth polypeptides in human fibroblasts in culture using the closely related polypeptide, multiplication stimulating activity (MSA), as the radioligand. Polypeptides purified from human plasma, somatomedin A and acid soluble nonsuppressible insulin-like activity (NSILA-s), competed potently for 125I-MSA binding, as did unlabeled MSA. Although insulin and proinsulin also strongly inhibited MSA binding, the properties of the growth peptide receptor differed from those of the human fibroblasts insulin receptor. Somatomedin A, NSILA-s, MSA, insulin and proinsulin all stimulated the incorporation of [3H]thymidine into DNA in human fibroblasts. We propose that these polypeptides induce DNA synthesis through their interaction with the growth peptide receptor.     

Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts.

Insulin-like growth factor-I (IGF-I) initiates its diverse biological effects by binding to type I IGF receptors on cells. In addition, IGF-I associates with distinct proteins that can modulate its actions. One of these IGF-binding proteins, IGFBP-3, is the major circulating form in adults and is produced by many cells in culture. We investigated the effect of purified bovine IGFBP-3 on IGF-I binding and IGF-I stimulation of amino acid uptake and DNA synthesis in cultured bovine fibroblasts, a cell culture system highly suitable for these types of studies. Incubation of cells with IGF-I resulted in time- and dose-dependent decreases in [125I]IGF-I binding and IGF-I stimulated [3H]aminoisobutyric acid uptake and [3H]thymidine incorporation. Preincubation with 4 nM IGF-I resulted in a 50-60% decrease in IGF-I receptor binding, accompanied by marked decreases in IGF-I-stimulated [3H]aminoisobutyric acid uptake (50-60%) and [3H]thymidine incorporation (80-90%). Preincubation with the IGF-I analog [QAYL]IGF-I (4 nM) or with 100 nM insulin, growth factors that bind and activate type I IGF receptor signalling but have little or no affinity for IGFBP-3, had effects comparable to IGF-I, decreasing both IGF-I binding and action 50-95%. The addition of IGFBP-3 during the preincubation period with IGF-I blocked the decrease in receptor availability and prevented the cells from becoming desensitized. IGFBP-3 did not prevent the [QAYL]IGF-I- or insulin-induced receptor loss and cellular resistance to IGF-I. These data indicate that IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I. Thus, cell-derived IGFBP-3 may function in a buffering capacity to restrict IGF-I and target cell interaction, thereby modulating the biological response to changes in local IGF-I levels.     

Response of chondrocytes isolated from human foetal cartilage to plasma somatomedin activity

Plasma somatomedin activity enhanced the incorporation of [3H]thymidine into chondrocytes isolated from human foetal cartilage during weeks 13-21 of gestation. Human growth hormone (0.1-20 muu./ml), human placental lactogen (0.1-5 microgram/ml) and insulin (0.25-10 muu./ml) had no direct effects on the synthesis of DNA in these chondrocytes, although insulin at concentrations of 2.5-100 mu./ml increased [3H]thymidine incorporation by up to 400%.     

A role for insulin-like growth factor-I in the regulation of human thyroid cell growth by thyrotrophin

Human thyroid epithelial cells in monolayer culture were found to release radioimmunoassayable insulin-like growth factor-I (IGF-I) over a 48-h culture period in serum-free medium. In the presence of supraphysiological concentrations of TSH (1-100 mU/ml) known to be inhibitory to DNA synthesis by human thyroid cells, the release of IGF-I was found to be inhibited in six thyroid cultures studied. In only one out of the six was IGF-I release increased in the presence of physiological mitogenic concentrations of TSH (0.1-100 microU/ml). Human thyroid fibroblasts, established by long-term culture of thyroid epithelial cells under fibroblast-selective conditions, also secreted IGF-I which was unaffected by the presence of TSH at both low and high concentrations. Using a monoclonal antibody against human IGF-I, monolayer cultures of both human thyroid epithelial cells and human thyroid fibroblasts showed positive immunocytochemical staining for IGF peptide. However, fixed sections of intact thyroid tissue only showed positive staining for IGF peptide associated with the fibrous layers surrounding the thyroid follicle, with no staining of the follicular epithelial cells. The growth of human thyroid epithelial cells was also found to be increased by IGF-I (25-100 ng/ml) added in medium plus 1% fetal calf serum as assessed by the incorporation of [3H]thymidine into DNA. In the presence of a monoclonal antibody to IGF-I the increase in [3H]thymidine uptake in response to IGF-I was abolished as was that seen in response to TSH.(ABSTRACT TRUNCATED AT 250 WORDS)