Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells.

The effects of insulin-like growth factor-II (IGF-II) on the proliferation and differentiation of ovarian granulosa cells were studied in cultured human and porcine granulosa cells. IGF-II significantly increased basal progesterone secretion in granulosa cells at concentrations of 1-100 ng/ml. A stimulatory effect was also observed in gonadotropin-stimulated porcine granulosa cells treated with IGF-II. The secretion of estradiol by basal and gonadotropin-stimulated porcine granulosa cells was also significantly increased by adding IGF-II. IGF-II led to dose-dependent increases in [3H]thymidine incorporation into DNA and in the number of granulosa cells. To further characterize the cellular mechanisms underlying the stimulatory effects of IGF-II on the proliferation and differentiation of granulosa cells, we investigated the intermediary roles of cyclic AMP and intracellular Ca2+ concentration ([Ca2+]i). Treatment with 100 ng/ml IGF-II produced a significant increase in the basal accumulation of cyclic AMP in porcine granulosa cells. However, no change of [Ca2+]i by IGF-II was noted. IGF-II produced effects in accumulation that were similar to those of IGF-I. Our findings suggest that IGF-II may be a general stimulator in the proliferation and differentiation of granulosa cells, and that cyclic AMP may be a second messenger for the effects of IGF-II in ovarian granulosa cells.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

The receptor for insulin-like growth factor II mediates an insulin-like response.

Insulin-like growth factor II (IGF-II) shares sequence homology and predicted three-dimensional structure with insulin and IGF-I. IGF-II can bind, therefore, to a limited extent with the receptors for these two other hormones, as well as to a distinct receptor for IGF-II. Previous studies have been unable to attribute a particular response of IGF-II through its own receptor. In the present studies, the IGF-II receptor is shown to mediate the stimulation of glycogen synthesis in human hepatoma cells since: (i) IGF-II is found to be capable of stimulating a response at concentrations in which it would primarily interact with its own receptor; (ii) the response to IGF-II was not blocked by monoclonal antibodies which inhibit the responses of cells through the insulin and IGF-I receptors; and (iii) polyclonal antibodies to the IGF-II receptor were found to mimic the ability of IGF-II to stimulate glycogen synthesis. These results indicate that the IGF-II receptor mediates a particular biological response--stimulation of glycogen synthesis in hepatoma cells. Furthermore, a monovalent Fab fragment of the polyclonal antibody to the IGF-II receptor was also shown to stimulate glycogen synthesis in these cells. These data indicate that clustering of the IGF-II receptor is not required to stimulate a biological response.     

The interaction between the adenylate cyclase system and insulin-stimulated glucose transport. Evidence for the importance of both cyclic-AMP-dependent and -independent mechanisms.

The counter-regulatory effect of adenosine, isoprenaline and selected cyclic AMP analogues on insulin-stimulated 3-O-methylglucose transport and insulin binding were studied in rat fat-cells. Isoprenaline alone had no consistent effect on glucose transport in the presence of maximally effective insulin concentrations. However, it decreased insulin binding by approx. 20% and increased EC50 (concn. giving 50% of maximal stimulation) for insulin from 8 +/- 1 to 17 +/- 2 mu units/ml. Adenosine deaminase (ADA) alone only exerted a slight effect, whereas isoprenaline and ADA in combination consistently decreased the maximal effect of insulin on glucose transport, decreased insulin binding by approx. 30% and markedly decreased insulin-sensitivity (EC50 61 +/- 8 mu units/ml). In cells from pertussis-toxin-treated animals, isoprenaline alone decreased the insulin response by approx. 75%, decreased insulin binding by approx. 45% and caused a marked rightward shift in the dose-response curve for insulin (EC50 103 +/- 34 mu units/ml). The importance of cyclic AMP for these effects was evaluated with the analogue N6-monobutyryl cyclic AMP, which is resistant to hydrolysis by the phosphodiesterase. The importance of phosphodiesterase activation by insulin was studied with 8-bromo cyclic AMP, which is an excellent substrate for this enzyme. N6-Monobutyryl cyclic AMP, in contrast with 8-bromo cyclic AMP, markedly impaired insulin-sensitivity (EC50 approx. 100 mu units/ml). However, the maximal effect of insulin was only slightly attenuated. In conclusion: (1) beta-adrenergic stimulation and cyclic AMP markedly alter insulin-sensitivity, but not responsiveness, mainly through post-receptor perturbations; (2) when cyclic AMP is increased phosphodiesterase activation by insulin is a critical step to elicit insulin action; (3) adenosine modulates the insulin-antagonistic effect of beta-adrenergic stimulation via Ni (inhibitory nucleotide-binding protein) through both cyclic-AMP-dependent and -independent mechanisms.     

Characterization of the stimulatory action of insulin on insulin-like growth factor II binding to rat adipose cells. Differences in the mechanism of insulin action on insulin-like growth factor II receptors and glucose transporters

Insulin is known to increase the number of cell surface insulin-like growth factor II (IGF-II) receptors in isolated rat adipose cells through a subcellular redistribution mechanism similar to that for the glucose transporter. The effects of insulin on these two processes, therefore, have now been directly compared in the same cell preparations. 1) Insulin increases the steady state number of cell surface IGF-II receptors by 7-13-fold without affecting receptor affinity; however, insulin stimulates glucose transport activity by 25-40-fold. 2) The insulin concentration required for half-maximal stimulation of cell surface IGF-II receptor number is approximately 30% lower than that for the stimulation of glucose transport activity. 3) The half-time for the achievement of insulin's maximal effect at 37 degrees C is much shorter for IGF-II receptor number (approximately 0.8 min) than for glucose transport activity (approximately 2.6 min). 4) Reversal of insulin's action at 37 degrees C occurs more rapidly for cell surface IGF-II receptors (t1/2 congruent to 2.9 min) than for glucose transport activity (t1/2 congruent to 4.9 min). 5) When the relative subcellular distribution of IGF-II receptors is examined in basal cells, less than 10% of the receptors are localized to the plasma membrane fraction indicating that most of the receptors, like glucose transporters, are localized to an intracellular compartment. However, in response to insulin, the number of plasma membrane IGF-II receptors increases only approximately 1.4-fold while the number of glucose transporters increases approximately 4.5-fold. Thus, while the stimulatory actions of insulin on cell surface IGF-II receptors and glucose transport activity are qualitatively similar, marked quantitative differences suggest that the subcellular cycling of these two integral membrane proteins occurs by distinct processes.     

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.     

Insulin regulates the expression of the insulin-like growth factor binding protein 2 mRNA in rat hepatocytes.

The goal of this study was to find out whether GH or insulin regulate the mRNA expression of the fetal binding protein of insulin-like growth factor (IGFBP-2). Primary hepatocytes from adult rats were used as a test system. IGFBP-2 mRNA was abundant in cells cultured in the absence of hormones and markedly reduced in cultures containing insulin. Addition of GH had no effect on IGFBP-2 mRNA levels although the cells are responsive to GH as demonstrated by a GH mediated elevation of IGF l mRNA levels. Half-maximal down-regulation of IGFBP-2 mRNA levels occurred at an insulin concentration of 1 to 2 x 10(-10) M. The finding that insulin is a potent negative regulator of hepatic IGFBP-2 mRNA levels suggests a physiologically important regulatory link between the two hormones insulin and IGF l.     

Growth hormone dependence of somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II messenger ribonucleic acids

The GH dependence of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) and insulin like growth factor II (IGF-II) mRNAs was investigated by Northern blot hybridizations of polyadenylated RNAs from liver, pancreas, and brain of normal rats, untreated hypophysectomized rats, and hypophysectomized rats 4 h or 8 h after an ip injection of human GH (hGH). Using a 32P-labeled human Sm-C/IGF-I cDNA as probe, four Sm-C/IGF-I mRNAs of 7.5, 4.7, 1.7, and 1.2 kilobases (kb) were detected in rat liver and pancreas but were not detectable in brain. In both liver and pancreas, the abundance of these Sm-C/IGF-I mRNAs was 8- to 10-fold lower in hypophysectomized rats than in normal rats. Within 4 h after injection of hGH into hypophysectomized animals, the abundance of liver and pancreatic Sm-C/IGF-I mRNAs was restored to normal. A human IGF-II cDNA was used as a probe for rat IGF-II mRNAs which were found to be very low in abundance in rat liver and showed no evidence of regulation by GH status. In pancreas, IGF-II mRNA abundance was below the detection limit of the hybridization procedures. The brain contained two IGF-II mRNAs of 4.7 and 3.9 kb that were 5-fold lower in abundance in hypophysectomized rats than in normal rats. These brain IGF-II mRNAs were not, however, restored to normal abundance at 4 or 8 h after ip hGH injection into hypophysectomized animals. To investigate further, the effect of GH status on abundance of Sm-C/IGF-I and IGF-II mRNAs in rat brain, a second experiment was performed that differed from the first in that hypophysectomized rats were given an injection of hGH into the lateral ventricle (intracerebroventricular injection) and a rat Sm-C/IGF-I genomic probe was used to analyze Sm-C/IGF-I mRNAs. In this experiment, a 7.5 kb Sm-C/IGF-I mRNA was detected in brain polyadenylated RNAs. The abundance of the 7.5 kb mRNA was 4-fold lower in hypophysectomized rats than in normal rats and was increased to 80% of normal within 4 h after icv administration of hGH to hypophysectomized animals. As in the first experiment, the abundance of the 4.7 and 3.9 kb brain IGF-II mRNAs was lower than normal in hypophysectomized rats. Brain IGF-II mRNAs were increased to 50% of normal in hypophysectomized rats given an icv injection of hGH but within 8 h after the injection rather than at 4 h as with Sm-C/IGF-I mRNAs.     

Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors

Insulin-like growth factor (IGF) I (greater than or equal to 10(-10)M, insulin-like growth factor II (greater than or equal to 10(-9) M), insulin (greater than or equal to 10(-9) M, and epidermal growth factor (EGF, greater than or equal to 10(-11) M) caused rapid membrane ruffling in KB cells. The morphological change was observed within 1 min after the addition of these growth factors and was accompanied by microfilament reorganization, but not by microtubule reorganization. IGF-I, IGF-II, and insulin induced morphologically very similar or identical membrane ruffles with the order of potency IGF-I greater than IGF-II greater than insulin, whereas EGF-induced membrane ruffles were morphologically different. KB cells possessed EGF receptors, type I IGF receptors, and insulin receptors, but few or no type II IGF receptors. Monoclonal antibody against type I IGF receptors, which completely inhibited the binding of 125I-IGF-I to the cells but did not inhibit the binding of 125I-insulin, caused marked inhibition of IGF-I (10(-8) M)-stimulated membrane ruffling. IGF-II (10(-8) M)-stimulated membrane ruffling was partially inhibited in the presence of this antibody, but insulin (10(-7) M)-stimulated membrane ruffling was only slightly inhibited. In contrast, monoclonal antibody against insulin receptors blocked insulin (10(-7) M) stimulation, but not IGF-I (10(-8) M) stimulation, of membrane ruffling. Thus, this study provides evidence that IGF-I and insulin act mostly through their own (homologous) receptors and that IGF-II acts by cross-reacting with both type I IGF and insulin (heterologous) receptors in causing rapid alterations in cytoskeletal structure.     

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

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

Inhibition of growth hormone-stimulated lipolysis by somatostatin, insulin, and insulin-like growth factors (somatomedins) in vitro

The effects of somatostatin, insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II)/MSA on growth hormone (GH) (1 microgram/ml)-induced lipolysis were examined employing chicken adipose tissue in vitro. Basal and GH-stimulated glycerol release were inhibited by somatostatin (1 ng/ml) and by IGF-II/MSA (10 and 100 ng/ml). Insulin and IGF-I (10 and 100 ng/ml) completely inhibited the lipolytic response to GH without affecting basal glycerol release. Insulin and IGF-I were equipotent in inhibiting GH-induced lipolysis while IGF-II is only 16% as potent as insulin.     

beta-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes

Incubation of intact rat adipocytes with physiological concentrations of catecholamines inhibits the specific binding of 125I-insulin and 125I-epidermal growth factor (EGF) by 40 to 70%. Affinity labeling of the alpha subunit of the insulin receptor demonstrates that the inhibition of hormone binding is directly reflective of a specific decrease in the degree of receptor occupancy. The stereospecificity and dose dependency of the binding inhibitions are typical of a classic beta 1-adrenergic receptor response with half-maximal inhibition occurring at 10 nM R-(-)-isoproterenol. Specific alpha-adrenergic receptor agonists and beta-adrenergic receptor antagonists have no effect, while beta-adrenergic receptor antagonists block the inhibition of 125I-insulin and 125I-EGF binding to receptors induced by beta-adrenergic receptor agonists. Further, these effects are mimicked by incubation of adipocytes with dibutyryl cyclic AMP or with 3-isobutyl-1-methylxanthine. The beta-adrenergic inhibition of both 125I-insulin and 125I-EGF binding is very rapid, requiring only 10 min of isoproterenol pretreatment at 37 degrees C for a maximal effect. Removal of isoproterenol by washing the cells in the presence of alprenolol leads to complete reversal of these effects. The inhibition of 125I-EGF binding is temperature dependent whereas the inhibition of 125I-insulin binding is relatively insensitive to the temperature of isoproterenol pretreatment. Scatchard analysis of 125I-insulin and 125I-EGF binding demonstrated that the decrease of insulin receptor-binding activity may be due to a decrease in the apparent number of insulin receptors while the inhibition of EGF receptor binding can be accounted for by a decrease in apparent EGF receptor affinity. The decrease in the insulin receptor-binding activity is physiologically expressed as a dose-dependent decrease of insulin responsiveness in the adipocyte with respect to two known responses, stimulation of insulin-like growth factor II receptor binding and activation of the glucose-transport system. These results demonstrate a beta-adrenergic receptor-mediated cyclic AMP-dependent mechanism for the regulation of insulin and EGF receptors in the rat adipocyte.     

Loss of insulin-like growth factor II receptor expression promotes growth in cancer by increasing intracellular signaling from both IGF-I and insulin receptors

The insulin-like growth factor-II receptor (IGF-IIR) is frequently mutated or deleted in some malignant human tumors, suggesting that the IGF-IIR is a tumor suppressor. However, the exact mechanism by which IGF-IIR suppresses growth in tumors has not been definitively established. We demonstrate that IGF-IIR-deficient murine L cells (D9) have higher growth rates than IGF-IIR-positive L cells (Cc2) in response to IGF-II. IGF-II levels are higher in growth-conditioned medium from D9 versus Cc2 cells. Receptor neutralization studies and measurements of insulin receptor substrate 1 phosphorylation confirm that the enhanced growth of D9 cells is due to increased stimulation of the IGF-I and insulin receptors by IGF-II. In contrast, the levels of secreted latent and active transforming growth factor beta (TGF-beta) are similar for both D9 and Cc2 cells, indicating that the slower growth of Cc2 cells is not due to activation of latent TGF-beta by IGF-IIR and growth inhibition. The results directly demonstrate that down regulation of the IGF-IIR promotes the growth of transformed D9 cells by sustaining IGF-II, which binds to and activates IGF-IR and insulin receptor to increase intracellular growth signals.     

Expression and function of insulin-like growth factor receptors on anti-CD3-activated human T lymphocytes.

The pattern of expression of receptors for insulin-like growth factors (IGF-I and IGF-II) and insulin was studied on monocyte-depleted human peripheral blood T cells activated via anti-CD3. Binding assays demonstrated the sequential appearance of receptors for IGF-I, IGF-II, and insulin on activated T cells. IGF-IR appeared early, their expression reaching maximum levels at or before the peak of cellular proliferation. IGF-IIR expression generally followed that of the IGF-IR and was more transient, with increases and decreases in expression paralleling the rise and decline of cellular proliferation. Insulin receptor expression remained low throughout the activation time course. The identity of the IGFR on anti-CD3-activated T cells was confirmed in affinity cross-linking experiments. These data demonstrated a 135,000 Mr peptide that specifically binds radiolabeled IGF-I and corresponds to the alpha subunit of the type I IGF-IR, and a 260,000 Mr peptide that specifically binds radiolabeled IGF-II and corresponds to the type II IGFR. We have additionally found that IGF-I and IGF-II (in nanomolar concentrations) produce as much as a threefold enhancement of T cell proliferation early in the activation process, correlating with the early appearance of IGF-IR. The effect of both IGF appeared to be mediated through the type I receptor, since an antibody (alpha IR3), which blocks binding to the alpha subunit of this receptor, inhibited enhancement by up to 83%. Furthermore, we have found expression of IGF-IR on T cells after activation to be associated with both CD4+ and CD8+ T cell subpopulations. These observations provide a foundation for investigating the contribution of IGF in regulating T cell proliferation, differentiation, and effector function.     

Factors regulating insulin-like growth factor binding protein-3 secretion from human hepatoma (HepG2) cells

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is a growth hormone (GH) dependent carrier of the IGFs in human serum. Apart from GH regulation the hormonal control of IGFBP-3 production is not well established and although the liver is considered to be the main source of circulating IGFBP-3, there are no in vitro studies of the effect of both insulin and IGFs on the IGFBP-3 produced in human hepatoma cells. The effect of sex hormones as well as cortisol has not been studied. To elucidate this we performed cell culture studies on HepG2 cells in the presence of various effectors. Insulin, IGF-I and IGF-II brought about a 1.5-2-fold enhancement of IGFBP-3 release at 7.5-30 nM concentrations. In contrast, cortisol decreased IGFBP-3 secretion by 30-40% whereas estradiol, tamoxifen and testosterone had no effect at physiological concentrations. We conclude that, in addition to GH, also insulin, IGF-I and IGF-II and glucocorticoids can modulate IGFBP-3 secretion by human hepatoma cells.     

Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process

The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.     

Role of growth-hormone and insulin-like growth-factor-binding proteins.

Some peptide hormones are associated with specific, high-affinity plasma proteins. The major binding protein (BP) for growth hormone (GH) in humans is a circulating fragment of the GH membrane receptor, consisting of the hydrophilic, extracellular portion of that transmembrane glycoprotein. The circulating levels of GH-BP mirror the levels of GH receptors. There are 4 well-characterized insulin-like growth factor (IGF)-BPs. One IGF-binding component in plasma is a fragment of the extracellular portion of the IGF-II/mannose-6-phosphate receptor, analogous to the GH-BP. The 3 other cloned IGF-BPs form a homologous family of proteins with differences in structure, glycosylation and hormonal control that suggest differences in function. The GH- and IGF-BPs play a major role in the metabolism and biological action of these peptide hormones.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

Presumptive mediators of growth hormone action on insulin-like growth factor I release by porcine ovarian granulosa cells

The role of cAMP/protein kinase A (PKA)- and tyrosine kinase (TK)-dependent intracellular mechanisms in mediating the action of porcine growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion by porcine ovarian granulosa cells was studied. It was observed that GH-induced stimulation of IGF-I secretion was accompanied by an increase in cAMP production. The stimulation of PKA by the addition of either a cAMP agonist or a phosphodiesterase inhibitor to the medium increased IGF-I release by the cells, indicating a direct stimulation of IGF-I release by cyclic nucleotides. Moreover, the stimulatory effect of GH on IGF-I was completely suppressed by the addition of the PKA blocker Rp-cAMPS. Neither TK blocker altered the basal IGF-I level, but both strongly suppressed the GH-induced increase in IGF-I accumulation. Taken together, these findings suggest that cAMP/PKA- and/or TK-dependent pathways may be involved in the mediation of GH action on IGF-I release by porcine granulosa cells.