Evidence against a role for insulin-like growth factor II in the autonomous growth of rat 18,54-SF cells.

The 18,54-SF rat cell line multiplies in serum-free medium and has been reported to produce insulin-like growth factor II (IGF-II) and to possess IGF-II/mannose 6-phosphate (Man-6-P) receptors, raising the possibility of autocrine growth stimulation by IGF-II acting through this receptor. When serum-free medium was changed every 24 h the 18,54-SF cells multiplied at the same rate as when the medium was not changed. An antibody (No. 3637) which blocks the binding of IGF-II to the IGF-II/Man-6-P receptor did not decrease or increase the growth rate of the 18,54-SF cells when medium was changed every 24 h. When the medium was changed every 12 h the rate of accumulation of cells in the culture was decreased. Addition of IGF-I or IGF-II at 10, 50, and 100 ng/ml every 12 h did not correct this decrease in cell number. When the medium was replaced every 12 h, cells in the periphery of the cultures gradually became nonviable as assessed by trypan blue uptake. However, the percentage of cells synthesizing DNA in the center of the cultures (approximately 50% during a 3 h pulse with tritiated thymidine) was the same whether the medium was changed every 12 h or left unchanged. Addition of IGF-I or IGF-II to the fresh medium change every 12 h did not increase the percentage of cells synthesizing DNA.     

Expression of insulin-like growth factor II and receptors for insulin-like growth factor II, insulin-like growth factor I and insulin in isolated and cultured rat hepatocytes.

Insulin-like growth factor II is believed to play an important role in fetal growth and development. The insulin-like growth factor II gene expression is tissue specific and developmentally regulated. We have previously shown an enhanced level of insulin-like growth factor II messenger RNA and protein in human hepatocellular carcinomas. This led to the suggestion that hepatocytes might be involved in insulin-like growth factor II expression. However, previous studies based on in situ hybridization only showed insulin-like growth factor II messenger RNA in liver sinusoidal cells. This paper reports on the analysis of the expression of insulin-like growth factor II and insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs in vivo in isolated rat hepatocytes at various stages of development and in vitro in adult rat hepatocytes primary culture. Our study indicates that isolated rat hepatocytes synthesize insulin-like growth factor II messenger RNA with a switch between fetal and adult messenger RNA profiles occurring 21 days after birth. In addition, adult rat hepatocytes in culture expressed insulin-like growth factor II messenger RNA and protein. Insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs were also detected. Therefore these results are consistent with the hypothesis that insulin-like growth factor II acts as an autocrine growth factor for hepatocytes in addition to having a paracrine effect. They also indicate that primary culture of hepatocytes is a good model for further studies on insulin-like growth factor II gene regulation.     

Metabolic actions of insulin-like growth factor II in cultured adult rat hepatocytes are not mediated through the insulin-like growth factor II receptor

Summary Short- and long-term regulation of hepatic carbohydrate metabolism by insulin-like growth factor II was studied in primary cultures of adult rat hepatocytes and compared to the metabolic potency of insulin. Insulin-like growth factor II stimulated glycogen synthesis from [¹⁴C]glucose, uptake of [³H]aminoisobutyric acid and [¹⁴C]lactate formation from [¹⁴C]glucose up to three-fold. Basal glycogenolysis was inhibited to about 10%, and glucagon-activated glycogenolysis was blocked completely. The enzymatic activity of glucokinase and pyruvate kinase was induced two-fold, the glucagon-dependent induction of phosphoenolpyruvate carboxykinase was antagonized. Compared to insulin, half-maximal responses required up to 50 times higher insulin-like growth factor II concentrations ranging from 10–20 nmol/l. A similar difference was observed for binding affinity of insulin-like growth factor II to the insulin receptor. The interaction with the insulin-like growth factor II/man-nose 6-phosphate (IGF-II/Man-6-P) receptor was examined by studying ¹²⁵I-insulin-like growth factor II binding and uptake of lysosomal enzymes. The affinity of insulin-like growth factor II to the IGF-II/Man-6-P receptor was considerably higher than for the insulin receptor. Antibodies against the IGF-II/Man-6-P receptor did not affect metabolic responses to insulin-like growth factor II, while binding to its receptor and the receptor-mediated endocytosis of arylsulphatase A were strongly inhibited. Thus, in adult rat liver insulin-like growth factor II appeared to exert metabolic actions not via interaction with its own receptor but through low affinity binding to hepatic insulin receptors.     

Identification of a novel family of growth hormone-related proteins secreted by rat placenta

Mid-to-late gestation rat placenta synthesizes a number of proteins related to prolactin, including rat placental lactogen II (rPL-II), rat prolactin-like protein A (rPLP-A) and rat prolactin-like protein B (rPLP-B). This study identifies a new family of proteins synthesized and secreted by gestation day 15 placental explants which exhibit amino acid homology to growth hormone precursors from several species. Placental explant medium was fractionated by ammonium sulfate precipitation and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis to isolate four distinct proteins with Mr values of 28,000, 23,000, 25,000 and 30,000 and pI values of 5.7, 5.7, 5.4 and 5.3, respectively. These proteins represent a major fraction of secretory proteins with Mr in the 20,000 to 30,000 range. Immunoblot analysis showed that none of the four proteins crossreacted with antipeptide antisera against rPL-II, rPLP-A, or rPLP-B. These proteins were electrophoretically transferred from two-dimensional SDS-polyacrylamide gels onto an Immobilon PVDF membrane and N-terminal amino acid microsequencing carried out with a gas phase sequencer. N-terminal sequences of 45, 37, 37 and 32 amino acid residues were identified for proteins 1, 2, 3 and 4, respectively. The four proteins exhibit 76% to 97% homology. Computer analysis further revealed a 28% identity in a 32 amino acid overlap which begins at residue 14 of the 28,000 Mr protein (protein 1) and at residue 31 of growth hormone precursors of rat, mouse and human. The 32 amino acid overlap is 78% homologous if conservative amino acid replacements are included.     

Characterization of insulin-like growth factor binding proteins secreted by cultured bovine theca and granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) secreted by bovine granulosa and theca interna cells cultured in the presence of different luteinizing factors--insulin (2 micrograms/ml), forskolin (10 microM), or a combination of the two were examined and characterized. Direct binding of [125I]IGF to the conditioned media was compared to progesterone production under these different treatments. In theca cells, maximal secretion of IGFBPs was achieved using forskolin alone, whereas maximal progesterone production was induced by the insulin+forskolin treatment. In contrast, maximal secretion of both IGFBPs and progesterone in granulosa cells was achieved using forskolin alone. IGFBP species secreted by the two cell types under the different treatments were detected by ligand blotting. Conditioned media from theca cells in serum-free medium collected on the seventh day of culture exhibited three bands of 34, 40 and 44 kDa when treated with insulin or forskolin. The intensity of the 40-44 kDa complex was enhanced and a 21 kDa band appeared when cells were treated with a combination of insulin plus forskolin. Conditioned media of granulosa cells stimulated with insulin or forskolin exhibited 21, 27, 29, 34 and 40-44 kDa bands. Treatment with insulin+forskolin greatly increased the intensity of a 40-44 kDa complex. A similar shift towards high molecular weight binding proteins was observed when these media were analyzed by high-performance liquid chromatography gel filtration. These findings substantiate the secretion of IGFBPs by bovine theca and granulosa cells and show it to be dependent on culture treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of uterine insulin-like growth factor I mRNA and insulin-like growth factor II mRNA by estrogen in the rat

IGF-I and IGF-II are peptides with mitogenic properties. In this study mRNA for IGF-I and IGF-II was analysed in rat uterine tissue after different endocrine manipulations and the possibility of an estrogenic regulation of IGF expression was investigated. Both IGF-I and IGF-II mRNA were present in uterine tissue. The level of IGF-I mRNA, but not IGF-II mRNA, was reduced following ovariectomy. Administration of estradiol (2.5 micrograms/day for 4 days) to ovariectomized rats increased IGF-I mRNA 8-fold to levels seen in intact animals. In adult animals hepatic IGF-I mRNA did not appear to be increased by estrogen treatment. Low levels of IGF-II mRNA were detected in the uterus, but showed no dependence on estrogen. The inductive effect of estrogen on uterine IGF-I mRNA could not be substituted for by growth hormone administration (0.5 mg/100 g, ip for 6 h). The present results suggest IGF-I as a potential candidate for a mediator of estrogen-induced growth. Both estrogen and GH induce IGF-I mRNA and a tissue specificity for these hormones is indicated where GH regulates hepatic and estrogen uterine IGF-I mRNA.     

Identification of a type II insulin-like growth factor receptor in fish embryos

To determine whether fish have an insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6-P) receptor similar to that of mammals, we have performed binding, cross-linking, and immunoprecipitation experiments with wheat-germ-agglutinin- and mannose 6-phosphate (M6-P)-affinity-purified receptor preparations from fish embryos. In both receptor preparations, IGF-II binding was specific, because labeled IGF-II could only be completely displaced by cold IGF-II but not by IGF-I or insulin. Labeled IGF-II bound to a protein with a molecular mass of approximately 250 kDa, which could be immunoprecipitated with an antibody against the rat IGF-II receptor. IGF-II stimulated tyrosine kinase activity in wheat germ agglutinin preparations and was more potent than insulin or IGF-I, but neither peptide stimulated tyrosine kinase activity in M6-P preparations. Two fish cell lines (CHSE-214 and EPC) were used to confirm the IGF-II binding data obtained in the receptor preparations, revealing the presence of highly specific IGF-II binding and the absence of insulin binding. Furthermore, a decrease of the IGF-I receptors on the cell surface did not alter IGF-II binding in EPC cells. In conclusion, we have detected the presence of IGF-II/M6-P receptors in fish embryos that are similar in structure and specificity for their ligand to those found in mammals.     

Effects of alpha-interferon on insulin-like growth factor-I, insulin-like growth factor-II and insulin-like growth factor binding protein-3 secretion by a human lung cancer cell line in vitro

The aim of our study was to evaluate the effect of alpha-interferon (alpha-IFN) on cell growth and on the different IGF system components in a human non-small cell lung cancer line (Calu-6) in vitro. Our results confirm the release of IGF-I and IGF-II by these cells. The amount of IGF-II in conditioned media (10.25 +/- 3.95 nM/10(6) cells, mean +/- SE) was more than 10-fold higher than that of IGF-I. alpha-IFN treatment reduced IGF-II levels in the media, with a maximal effect between 1 and 10 U/ml (delta% of control: -31 and -55%, respectively, p < 0.05). IGF-I was significantly reduced at 0.5 U/ml (p < 0.01). No difference, however, was observed in IGF mRNA expression between untreated and alpha-IFN treated cells. An increase in IGF-I and IGF-II intracellular levels in alpha-IFN treated cultures was observed, suggesting that alpha-IFN can regulate the transfer of these peptides into the cells. Furthermore, IGF type-I and particularly type-lI receptor expression was increased after alpha-IFN treatment. IGFBP-3 was detected only in trace amounts in the conditioned media; however, it showed an increase after alpha-IFN treatment (+110% at 1 U/ml). IGFBP-3 mRNA expression showed a slight increase after treatment with 1 and 10 U/ml. alpha-IFN (1-10 U/ml) reduced the stimulatory effect of IGF-I on cell replication (p < 0.01), inhibited (p < 0.01) cell replication in untreated and in fetal calf serum (FCS)-stimulated cells, and increased apoptosis in Calu-6 cells. Our data suggest that alpha-IFN may exert its effects at the cellular level in part through modification of the local IGF system.     

Distribution of insulin-like growth factor II receptor immunoreactivity in rat tissues

Antibodies specific for the insulin-like growth factor II (IGF-II) receptor were used to study its distribution in a number of rat tissues and cell lines in order to determine which cells might be responsive to local or circulating IGF-II. In cultured 18-54,SF and B104 neuroblastoma cells, plasma membrane and cytoplasmic staining corresponding to Golgi apparatus could be seen, consistent with the glycoprotein nature of this receptor. Antibody binding was also seen in the central nervous system, confined primarily to the choroid plexus, and the vascular and ependymal elements. Some staining was seen in the parenchyma of the brain, in addition to binding around nerve sheaths and axon bundles. There were high levels of immunoreactivity in all three lobes of the pituitary, including vascular and cellular elements. In liver, highest levels of immunoreactivity occurred in the sinusoidal cells. In lung, IGF-II receptor immunostaining was seen in the alveoli and around the bronchioles. Staining in kidney was observed in glomeruli, tubules, and Bowman's capsules. Lower levels of immunostaining were seen in skeletal muscle, located primarily around the muscle sheaths. Localization of IGF-II receptor to cells of known function in different tissues will help elucidate the role of this ligand-receptor system in regulating growth and metabolism.     

Regulation of insulin-like growth factor II receptors by growth hormone and insulin in rat adipocytes.

The acute and long-term effects of growth hormone (GH) on the binding of insulin-like growth factor II (IGF-II) were evaluated in adipose cells from hypophysectomized rats given replacement therapy with thyroxine and hydrocortisone and in cells from their sham-operated littermates. After the cells were incubated with insulin and/or GH, the recycling of IGF-II receptors was metabolically inhibited by treating the cells with KCN. IGF-II binding was 100 +/- 20% higher in cells from GH-deficient animals when compared with sham-operated controls. These GH-deficient cells also showed an increased sensitivity for insulin as compared with control cells (the EC50 for insulin was 0.06 ng/ml in GH-deficient cells and 0.3 ng/ml in control cells). However, the maximal incremental effect of insulin on IGF-II binding was reduced approximately 27% by hypophysectomy. GH added to the incubation medium increased the number of IGF-II binding sites by 100 +/- 18% in cells from hypophysectomized animals. This increase was rapidly induced (t1/2, approximately 10 min), but the time course was slower than that for the stimulatory effect of insulin. Half-maximal effect of GH on IGF-II binding was obtained at approximately equal to 10 ng/ml. Thus, GH added in vitro exerted a rapid insulin-like effect on the number of IGF-II receptors. GH also appears to play a regulating role for maintaining the cellular number of IGF-II receptors and, in addition, modulates the stimulatory effect of insulin on IGF-II binding.     

Characterization of type I and type II insulin-like growth factor receptors in an intestinal epithelial cell line

Insulin-like growth factors (IGFs) and insulin stimulate DNA and protein synthesis in IEC-6 cells (an intestinal epithelial cell line) grown in a chemically defined medium. IGF-I stimulates proliferation of IEC-6 cells at a lower concentration (ED50 = 1.6 nM) than either insulin or IGF-II. To gain insight into the mechanisms by which IGFs stimulate IEC-6 cell growth, we have examined the characteristics of specific IGF receptors on IEC-6 cells. Binding of 125I-IGF-I and 125I-IGF-II to IEC-6 monolayers was analyzed by incubation with various concentrations (0.2 nM to 0.5 microM) of radiolabeled IGFs for 16 h at 3 C. Scatchard plots of 125I-IGF-I binding were linear, suggesting a single class of binding sites with KD = 3.1 +/- 0.35 nM and Bmax = 50.7 +/- 6 fmol/10(6) cells. IGF-II was potent in displacing 125I-IGF-I (KI = 8.1 +/- 0.85 nM), but insulin had little effect. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed three bands with Mr of 270,000, 245,000, and 133,000, and the major band was the 133,000 species. Labeling of the 133,000 and 270,000 bands was greater than or equal to 80% inhibited by 10(-7) M unlabeled IGF-I, less potently inhibited by IGF-II and not at all by insulin. These results suggest that the 133,000 band represents the alpha-subunit of the type I IGF receptor. Scatchard plots of 125I-IGF-II binding to IEC-6 cell monolayers were curvilinear, suggesting two classes of binding sites: high affinity, low capacity sites, KD = 0.87 +/- 0.08 nM and Bmax = 28 +/- 2.5 fmol/10(6) cells; low affinity, high capacity sites, KD = 60 = +/- 8.8 nM and Bmax = 1780 +/- 230 fmol/10(6) cells. Neither IGF-I nor insulin was effective in inhibiting 125I-IGF-II binding. Affinity cross-linking with 125I-IGF-II labeled predominantly a 245,000 band, suggesting that this species is the type II receptor. A band with Mr 131,000 was barely detectable with 125I-insulin. These results indicate that IEC-6 cells have abundant quantities of the type I and II IGF receptors and few insulin receptors, suggesting that the mitogenic effect of IGFs is mediated through the type I IGF receptor.     

Demonstration that a human hepatoma cell line produces a specific insulin-like growth factor carrier protein

Insulin-like growth factors (IGF's) circulate in blood complexed to specific carrier proteins. The BRL 3A and BRL 3A2 rat liver cell lines secrete a 30,000-50,000 mol wt IGF carrier protein. Since the liver parenchymal cell is a likely source of IGF carrier protein synthesis, we have evaluated medium conditioned by three human hepatocellular carcinoma- or hepatoblastoma-derived cell lines for the presence of IGF carrier protein. The HEP G2 and the HEP 3B, but not the PLC/PRF/5, cell lines secrete a specific IGF carrier protein(s) into serum-free medium. This carrier protein is specific for IGF molecules. Multiplication-stimulating activity, IGF I, and IGF II were equipotent in competing for the binding of [125I]multiplication-stimulating activity to HEP G2 medium. The HEP G2 IGF carrier protein is trypsin sensitive, acid stabile, and does not contain a glycoprotein moiety. It has a molecular size of 30,000-50,000, as assessed by Sephadex G-200 gel filtration. These studies suggest that the HEP G2 cell line is a useful model to study the synthesis and secretion of human IGF carrier protein.     

The bovine placenta: a specific radioreceptor assay for both insulin-like growth factor I and insulin-like growth factor II

Binding of labelled IGF-I and IGF-II was studied to bovine, ovine and human placental cell membranes. The data show a preponderance of type I receptors in human placental membranes, and of type II receptors in ovine placental membranes, confirming reported data. In contrast, bovine placental membranes are rich in both type I and type II receptors. Therefore, the bovine placenta offers a good model for measuring specifically IGF-I (cross-reactivity with IGF-II 7%) and IGF-II (cross-reactivity with IGF-I 4%). By Scatchard analysis the apparent Kd (1-1.36 nmol/l) for the high affinity binding sites of the type I receptor is similar in all three preparations. Total binding capacity in ovine placental membranes is, however, 4 times lower. The affinity for the type II receptor is lower than for type I, whereas total binding capacity is higher. Affinity cross-linking confirms the competition experiments, showing binding of IGF-I to typical type I and of IGF-II to type II receptors.     

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.     

Comparison of receptors for insulin-like growth factor II from various rat tissues

Receptors for insulin-like growth factor II (IGF-II) have been compared in solubilized microsomal membranes from rat lung, brain, kidney, heart, epididymal and subcutaneous fat, ovary, testis and adrenals. Highest binding/microgram protein was seen with testicular membranes. Receptors from all tissues showed high affinity for human IGF-II (mean association constant = 65 litres/nmol) and a high degree of specificity (mean IGF-I cross-reactivity 0.3%; no cross-reactivity with insulin). Affinity labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed binding was only to a type-II IGF receptor, with a major band seen at a molecular weight of about 230,000 in lung, brain, kidney and testis, and 240,000 in heart, fat and adrenal gland. All tissues showed broad or bimodal pH dependence of binding, with optima seen at about pH 6 and pH 9. Mild stimulation of IGF-II binding by low calcium concentrations (1-2 mmol/l) was seen in all tissues, although higher concentrations were inhibitory in the brain. It was concluded that IGF-II receptors from different rat tissues, when studied under uniform conditions, show similar binding affinities but differences in size and regulation which might be missed if receptors are examined in separate studies.     

Characterization and regulation of a specific cell membrane receptor for somatomedin-C/insulin-like growth factor I in cultured rat granulosa cells

The ovarian granulosa cell has recently been found to be a site of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) production, reception, and action, thereby raising the prospect of a novel autocrine control mechanism concerned with granulosa cell ontogeny. It is the objective of the in vitro studies reported herein to explore the characteristics of the murine granulosa cell membrane Sm-C/IGF-I receptor and its regulation by gonadotropic, lactogenic, and beta 2-adrenergic signalling. Provision of FSH (150 ng/ml) to granulosa cells from immature rats cultured for 72 h under serum-free conditions resulted in a 3.1-fold increase over control values in specific cell-bound [125I]iodo-Sm-C/IGF-I. Binding to FSH-primed cells proved time, temperature, and pH dependent; optimal steady state conditions were achieved after an 8-h incubation at 15 C and a pH of 8.0. Although subject to regulation by the cellular density of plating, the binding of [125I]iodo-Sm-C/IGF-I to its receptor proved saturable (apparent Kd = 3.3 X 10(-9) M) as well as reversible; complete or partial tracer displacement was effected by competitive inhibition and dilution, respectively. Specificity studies revealed the competition for [125I]iodo-Sm-C/IGF-I binding to follow a relative rank order of potency of Sm-C/IGF-I much greater than multiplication-stimulating activity greater than insulin, but disclosed limited or no displacement by a series of chemically related and unrelated polypeptides. By Scatchard and Hill analysis, both control and FSH-treated cells displayed a single class of noninteracting binding sites; the FSH-enhanced binding represented largely increased binding capacity, rather than affinity. Significantly, up-regulation of granulosa cell Sm-C/IGF-I binding was not limited to FSH; qualitatively comparable increments in [125I]iodo-Sm-C/IGF-I binding were obtained after treatment with luteotropic, and beta 2-adrenergic (but not lactogenic) granulosa cell agonists. Taken together, these studies provide further evidence for the existence of high affinity, low capacity, specific cell membrane receptors for Sm-C/IGF-I in cultured rat granulosa cells. Our findings further indicate that the ability of FSH to enhance granulosa cell Sm-C/IGF-I binding largely reflects increased binding capacity rather than affinity and that this heterologous up-regulatory phenomenon may not be limited to FSH. As such, our observations of comparable up-regulation after luteotropic and beta 2-adrenergic (but not lactogenic) stimulation are in keeping with the view that cAMP may play an intermediary role in the regulation of granulosa cell type I IGF receptors.     

Regulation of insulin-like growth factor I messenger ribonucleic acid levels by serum in cultured rat fibroblasts.

Fibroblasts represent one of the in vivo sites of extrahepatic insulin-like growth factor I (IGF-I) production. In this study, cultured fibroblasts prepared from the skin of neonatal rats were used as a model to assess the role of serum in regulating IGF-I messenger RNA (mRNA) levels. IGF-I mRNA, as demonstrated by Northern blot analysis, was present in the cultured fibroblasts, and serum free media which was conditioned by fibroblasts for 20 h contained 108 pg/ml of immunoreactive IGF-I. Fetal calf serum (FCS) decreased steady state IGF-I mRNA levels, as measured by solution hybridization/RNase protection assay, in fibroblasts in a time- and dose-dependent fashion. Incubation of fibroblasts for 18 h in the presence of 0.3%, 0.6%, or 1% FCS decreased IGF-I mRNA levels to 76%, 56%, and 46% of the levels present in control cells which were maintained in serum free media with 0.25% BSA. Maximal inhibition to approximately 20% of control levels was seen with 4-10% FCS. In contrast, basic fibroblast growth factor and beta-actin mRNA levels increased 2- and 4-fold, respectively, with increasing concentrations of FCS. Treatment of the cells with 10 micrograms/ml cycloheximide resulted in partial abrogation of the inhibitory effect of FCS while protein synthesis in the cells was decreased to 6% of control levels. The addition of 2 micrograms/ml of insulin or 15-100 ng/ml of IGF-I to the fibroblasts did not reproduce the inhibitory effect of FCS. Finally, the inhibitory factor(s) present in the FCS was partially removed/inactivated by charcoal stripping or heat inactivating the serum, but delipidation of the FCS by chloroform extraction had no effect on the inhibitory effect of FCS. In summary, FCS contains a factor(s) that decreases IGF-I mRNA levels in cultured fibroblasts in a time- and dose-dependent fashion. The partial abrogation of the inhibitory effect of FCS with cycloheximide treatment suggests that this effect is at least partially dependent upon new protein synthesis. Furthermore, the studies using delipidated, heat-inactivated, and charcoal-stripped serum suggest that the inhibitory factor(s) is a peptide.     

Somatomedin-C/insulin-like growth factor I as an enhancer of androgen biosynthesis by cultured rat ovarian cells

The ovarian granulosa cell has recently been shown to be a site of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) production, reception, and action. These observations have generally been interpreted to suggest the existence of an autocrine loop concerned with granulosa cell physiology. It is the objective of the in vitro studies reported herein to extend these observations by evaluating the interaction of Sm-C/IGF-I with the adjacent thecal-interstitial cell. Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) thecal-interstitial cells from immature rats with Sm-C/IGF-I (50 ng/ml) or hCG (1 ng/ml), resulted in 2.1- and 4.0-fold increments in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstane-17-one), the main androgenic steroid identified in culture media. However, combined treatment with both agents unmasked a synergistic interaction producing a 3.3-fold increase in the hCG-stimulated accumulation of androsterone, an effect consequent to enhanced androgen biosynthesis rather than diminished degradation. Unaccounted for by an increase in viable ovarian cell numbers and independent of the hCG dose (0.1-10 ng/ml) used, the Sm-C/IGF-I effect proved time and dose dependent, with a projected minimal effective dose of 3 ng/ml and a minimal time requirement of 72 h. [125I]Iodo-Sm-C/IGF-I binding to untreated highly enriched thecal-interstitial cells proved saturable, with a single class (Hill coefficient = 0.98 +/- 0.01) of high affinity (Kd = 3.0 nM), low capacity (maximum binding = 10,840 +/- 2,108 sites/cell) binding sites. Limited specificity studies using related peptides produced a rank order of competitive potency of: Sm-C/IGF-I greater than multiplication stimulating activity greater than insulin, a pattern compatible with the presence of type I IGF receptors. Other related peptides, such as porcine proinsulin and porcine desoctapeptide insulin, proved weakly effective in inhibiting Sm-C/IGF-I binding to its receptor; unrelated peptides such as porcine relaxin and erythropoietin were without effect. Taken together, these findings suggest that 1) the thecal-interstitial cell, like the granulosa cell, may be a site of Sm-C/IGF-I reception and action, and 2) the ability of high dose insulin to stimulate ovarian androgen biosynthesis may be due to its capacity to act as a Sm-C/IGF-I surrogate, its high dose requirements reflecting cross-interaction with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)