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1.
Identification of insulin receptors on the insulin-independent variant 1246-3A cell line 总被引:1,自引:0,他引:1
1246-3A cell line is an insulin-independent variant isolated from the adipogenic cell line 1246 which can proliferate in the absence of insulin, has lost the ability to differentiate, and secretes an insulin-related factor called IRF similar to pancreatic insulin and different from IGFs. In contrast, the parent adipogenic cell line 1246 is dependent on the presence of insulin to proliferate and differentiate in defined medium. In the present paper, we examined if the loss of response to insulin observed for 1246-3A cells was accompanied by alterations in the insulin receptor properties. Insulin binding and tyrosine kinase activity of insulin receptors isolated from 1246-3A cells and from the parent cell line 1246 were measured; 125I-insulin binding to intact cells was 75% lower for the 1246-3A cells than for the 1246 cells. This was due to a decrease in receptor number without major change in receptor affinity. However, when the cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed for both cell lines. Down regulation of insulin receptors by insulin occurred in a dose-dependent fashion, which was similar for both cell lines. Phosphorylation experiments were performed by incubation of the partially purified insulin receptor with insulin and [gamma-32P]ATP. They indicated that insulin stimulated phosphorylation of the 95-kDa molecular weight beta subunit of the receptor, in a similar fashion for both cell types. These data suggest that the insulin-independent cell line 1246-3A does not possess a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties and that the loss of response to insulin can be attributed to the fact the 1246-3A cells secrete IRF which bind to cell surface receptors and stimulate their proliferation. 相似文献
2.
The teratoma-derived insulin-independent cell line 1246-3A produces and secretes polypeptide mitogens in its culture serum-free medium. Mitogenic activities were separated by Sephadex G-50 chromatography into two fractions eluted, one in the void volume region, another one with an apparent Mr of 6 kDa. Only the 6 kDa mitogen presents properties similar to pancreatic insulin as estimated by radioimmunoassay, radio-receptor assay, and biochemical characterization. As a consequence, this factor is called insulin-related factor (IRF). Evidence presented in this paper indicate that ectopic IRF binds to insulin receptors on the producer cells, 1246-3A and acts in an autostimulatory manner. 相似文献
3.
Purification and biochemical characterization of a human autocrine growth factor produced by Epstein-Barr virus-transformed B cells 总被引:7,自引:0,他引:7
J Buck U H?mmerling M K Hoffmann E Levi K Welte 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(9):2923-2928
Autocrine growth factor for Epstein-Barr virus-transformed human B cells (aBGF), a protein that is constitutively produced by the human EBV-transformed B cell line 5/2, has been purified from serum-free conditioned medium. The purification involved sequential ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and reversed-phase high performance liquid chromatography. The purified protein has a m.w. of 16,000 in NaDodSO4/polyacrylamide gel electrophoresis and an isoelectric point between 7.0 and 8.0. The relative molecular mass 16,000 form exists in equilibrium with dimeric and tetrameric forms. aBGF supports the growth of EBV-transformed B cells, which have been deprived of their own conditioned medium. The purified aBGF is fully effective at 0.5 ng/ml and has no interleukin 1 activity in the lymphocyte activation factor assay. Because several randomly selected lines of EBV-transformed cells and one EBV-negative lymphoma cell line both produce aBGF activity and show growth dependency on aBGF and because stimulation of normal B cells with anti-immunoglobulin M is increased by aBGF, we propose that aBGF has general significance for growth control of human B cells. 相似文献
4.
The 1246-3A cell line is an insulin-independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246-3A cell line releases in its culture medium two types of transforming growth factors, TGF-alpha- and TGF-beta-like polypeptides, and a growth inhibitor. TGF-alpha like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high-molecular-weight TGF-alpha-like factors competed with 125I-EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF-alpha antibody, not by incubation with anti-EGF antibody. Both fractions promoted anchorage-independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c-3T3 cells in a fashion similar to EGF and synthetic TGF-alpha. In addition to secreting TGF-alpha-like polypeptides, 1246-3A cells produce TGF-beta. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage-independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5-11 kDa. This fraction was different from TGF-beta and TGF-alpha as determined by specific radioreceptor competition assays. TGF-alpha and TGF-beta-like polypeptides could represent autocrine growth stimulators for the insulin-independent 1246-3A cells and act in synergy with insulin-related factor (IRF) for an optimal stimulation of 1246-3A cell proliferation in serum-free medium. 相似文献
5.
H Gascan A Godard C Ferenz J Naulet V Praloran M A Peyrat R Hewick Y Jacques J F Moreau J P Soulillou 《The Journal of biological chemistry》1989,264(36):21509-21515
Six human T lymphomas and an NK-like cell line were tested for their ability to produce HILDA, one of the two human growth promoting activities for the DA1.a cells. Among them, the HSB2 cell line turned out to be the only one secreting significant HILDA activity (200-400 units/ml) after activation with 50 nM phorbol myristate acetate. Subclones of the HSB2 cell line were obtained by limiting dilution experiments. One of them (2B3) was found to secrete 1,000-5,000 units/ml of HILDA after phorbol myristate acetate activation in the presence of 10% fetal calf serum and 200-500 units/ml in serum-free conditions. 2B3-HILDA was purified from serum-free conditioned medium by a four-step procedure including fast flow cationic exchange at pH 6, concanavalin A chromatography, reverse-phase high performance liquid chromatography and gel-filtration high performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified radiolabeled cytokine revealed a single band of Mr 43,000, which co-electrophoresed with the biological activity. The NH2-terminal amino acid sequence of the first residues of the protein were determined and found to be similar to the equivalent residues deduced from the molecularly cloned cytokine. Isoelectric point determination revealed some charge heterogeneity of HILDA, which focused to pH 8.5-9 after neuraminidase treatment. Carbohydrate content of the cytokine was studied by deglycosylation experiments which showed that the O-linked oligosaccharides represented 2,000-3,000 and that the N-linked sugars account for half of the apparent molecular weight of HILDA. 相似文献
6.
Imaginal disc growth factor (IDGF) is a soluble polypeptide growth factor that was first identified from the conditioned medium of Drosophilia imaginal disc C1.8+ cells. Working with insulin, IDGF stimulated the growth of cultured imaginal disk cells, which suggested that IDGF might function as a cofactor of Drosophila insulin or insulin like peptide. Here we report a new member of the IDGF family, named MbIDGF, from the cabbage armyworm, Mamestra brassicae. Using a cloned cDNA of MbIDGF, recombinant MbIDGF protein was expressed in baculovirus-infected Sf9 cells and purified. Without insulin, the recombinant MbIDGF protein stimulated cell growth of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from the fat bodies and hemocytes of M. brassicae, in a dose-dependent manner. The saturation of growth stimulation by MbIDGF was attained for the two types of cells at 80 ng/ml (0.8 nM) and 300 ng/ml (6 nM), respectively. The results suggest that MbIDGF may stimulate the growth of lepidopteran cells by a new mechanism without associating with the insulin pathway. 相似文献
7.
Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptor 总被引:1,自引:0,他引:1
The conditioned medium from Sertoli cells contains a potent mitogen(s) that can markedly stimulate the proliferation of 4 different cell lines of endoderm or mesoderm origin in the presence or absence of serum. With A431 cells, conditioned medium produced in a dose-dependent manner up to a 5.2-fold increase in cell number after 5 days in culture. Addition of follicle-stimulating hormone (FSH), testosterone, retinol, and insulin to the Sertoli cells increased the secretion of the mitogenic activity. The ability of Sertoli cell conditioned medium (SCCM) to displace 125I-labeled epidermal growth factor (125I-EGF) from formalin-fixed A431 cells was also examined. The SCCM from Sertoli cells incubated with insulin contained 1.42 ng eq of EGF/ml; testosterone, retinol, and FSH (in the presence of insulin) further increased the secretion of this EGF competing activity to 2.09, 2.56, and 3.22 ng eq/ml, respectively. The amount of EGF competing activity was positively correlated with mitogenic activity. Separation of SCCM by gel filtration on Bio-Gel P-10 produced three major peaks of EGF-competing activity at apparent Mr = 1800-2100, 3800-4200, and 8000-9500. Chromatographing SCCM (in the presence of protease inhibitors) on size exclusion high performance liquid chromatography revealed two peaks of EGF competing activity at Mr about 8000 and 2000 coincident with and proportional to peaks of mitogenic activity. This activity was heat-sensitive and resistant to reducing agents, and addition of an equivalent amount of EGF as that present in SCCM produced an inhibition in growth of the A431 cells compared to a 3-fold stimulation with SCCM. Thus, the Sertoli cells secrete a potent mitogen that is distinct from EGF and alpha TGF. This factor that we have termed Sertoli cell-secreted growth factor is hormonally regulated by FSH, testosterone, and retinol and may play an important role in controlling spermatogenesis. 相似文献
8.
The mouse C3H teratoma-derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin-independent cell line called 1246-3A was isolated. It was found that, in contrast to 1246 cells, 1246-3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 10(6) cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor alpha- and beta-like polypeptides which stimulate their proliferation. Highly tumorigenic insulin-independent cell lines able to give rise to tumor when injected at a density of 10(4) cells/mouse were isolated by using an in vitro-in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF-beta 1. The binding of TGF-beta 1 to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF-beta 1 to the cells. However, the decreased number of cell surface TGF-beta 1 binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF-beta protein by the tumorigenic cells, as all of TGF-beta produced by the cells was in a latent form. Affinity cross-linking experiments indicated that the 1246 cell line displayed several TGF-beta cross-linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross-linked bands, particularly of the 92 and 70 kDa species. 相似文献
9.
Stimulation of embryonic mouse limb bud mesenchymal cell growth by peptide growth factors 总被引:1,自引:0,他引:1
The possible role of peptide growth factors in mammalian intrauterine cell growth has been investigated using primary cultures of undifferentiated mesenchymal cells from 11-day mouse embryo limb buds. When grown as monolayer cultures, proliferation is greatly favored by high cell densities. In medium containing 0.2% serum, purified epidermal growth factor (EGF), fibroblast growth factor (FGF), multiplication stimulating activity (MSA), insulin, and somatomedin-C (Sm-C) do not increase cell growth, but a 30-40,000 molecular weight component of mouse fetal liver conditioned medium is stimulatory. On the other hand, when limb bud cells are grown as high density or micromass cultures, a method which better approximates in vivo growth conditions, all of the purified growth factors tested stimulate cell growth significantly. These growth factors have additive effects when used in combination, the best stimulation being observed with liver medium (10% v/v), EGF (10 ng/ml), FGF (200 ng/ml), and either insulin (1 microgram/ml) or Sm-C (20 ng/ml). We conclude that the response of limb bud cells to growth stimulation is influenced by the manner in which the cells are cultured and that at least four different growth factors are required for optimal in vitro proliferation. One of these, the active component of liver medium, appears to be a previously uncharacterized growth factor. 相似文献
10.
Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification. 总被引:1,自引:0,他引:1
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A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg. 相似文献
11.
J W Groelke J J Coalson J B Baseman 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1985,179(3):309-317
In mass cell culture conditions, protease dissociated ferret tracheal epithelial cells (FTE) proliferated in growth factor-supplemented F12 medium to high cell densities (0.5 X 10(5) cells/cm2) with an average population doubling time of 24 hr. The growth factor constituents of the F12 medium included epidermal growth factor (25 ng/ml), insulin (1 microgram/ml), transferrin (10 micrograms/ml), hydrocortisone (18 ng/ml), hypothalamus extract (30-100 micrograms/ml), and conditioned medium from mouse 3T3 fibroblasts. Growth of these cells under clonal conditions was achieved by the partial replacement of F12 medium with M199 medium which was attributed, in part, to the presence of vitamin A in M199 medium. Serum did not stimulate the growth of FTE cells. The epithelial cell nature of these cells in culture was confirmed by ultrastructural features and by immunofluorescent staining for fibronectin. 相似文献
12.
Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells 总被引:13,自引:0,他引:13
A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages. 相似文献
13.
C Monet-Kuntz F Guillou I Fontaine Y Combarnous 《Journal of reproduction and fertility》1992,94(1):189-201
Ovine transferrin (o-transferrin) was purified from sheep serum by fractionated precipitation with ammonium sulphate, ion-exchange chromatography on DEAE trisacryl and finally by affinity chromatography on Affigel blue to remove albumin. Ovine transferrin was identified by its apparent molecular weight in sodium dodecyl sulphate polyacrylamide gel electrophoresis and by its N-terminal amino-acid sequence. The procedure presented in this report permits the preparation of highly purified o-transferrin with a good recovery (52% of initial total immunoactivity). An antiserum against o-transferrin was then raised in rabbits, using this highly purified preparation. A specific radioimmunoassay was set up using 125I-labelled o-transferrin. Its detection threshold (4 ng/ml) was low enough to measure o-transferrin in spent culture media of ovine Sertoli cells, which ranged between 15 and 600 ng/ml. Sheep seminiferous tubule cells, containing approximately 80% Sertoli cells, were cultured at a high density (1.5 x 10(6) cells/cm2) on a thin layer of reconstituted basement membrane. Kinetic studies showed that basal daily secretion of o-transferrin was reduced by half (-49%) between Day 1 and Day 2 of culture, and progressively decreased thereafter. Under FIRT (500 ng ovine follicle-stimulating hormone (FSH)/ml + 10 micrograms insulin/ml + 500 ng retinol/ml + 5 x 10(-7) mol/l testosterone) stimulation, the ratio of stimulated to basal secretions increased 11-fold between Day 1 (1.1) and Day 6 (12). When 10% fetal calf serum was added, mean o-transferrin secretion was a third of that in serum-free medium, suggesting that fetal calf serum contains factors that inhibit secretion of ovine Sertoli cell transferrin. In the presence of serum, the ratio of FIRT-stimulated to basal secretions doubled between Day 1 (1.0) and Day 4-6 (2.0). Between Days 2 and 4 of culture, insulin had a slight stimulatory effect on o-transferrin secretion (128% of control at 10 micrograms insulin/ml), as well as epidermal growth factor (124% of control at 50 ng/ml). Testosterone at up to 5 x 10(-7) mol/l had no effect; 500 ng retinol/ml doubled o-transferrin secretion (218% of control) as did 500 ng FSH/ml (220% of control). A combination of retinol and FSH increased the secretion 4-fold, indicating that maximal stimulation of o-transferrin secretion by ovine Sertoli cells requires the combined actions of mechanisms dependent and independent of cAMP. 相似文献
14.
15.
Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor 总被引:40,自引:0,他引:40
N A Nicola D Metcalf M Matsumoto G R Johnson 《The Journal of biological chemistry》1983,258(14):9017-9023
A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M. 相似文献
16.
Purification of decidual prolactin-releasing factor, a placental protein that stimulates prolactin release from human decidual tissue 总被引:1,自引:0,他引:1
S Handwerger D Capel G Korner R Richards 《Biochemical and biophysical research communications》1987,147(1):452-459
Decidual prolactin-releasing factor (PRL-RF), a placental protein that stimulates the release of prolactin from human decidual tissue, has been purified from conditioned medium of human placental explants. The purification scheme consisted of ethanol extraction, anion exchange chromatography on DEAE-cellulose, size exclusion chromatography on Spherogel TSK-3000, and either a) immunoaffinity chromatography using an antiserum to a partially purified PRL-RF preparation or b) acetic acid-urea/SDS 2-dimensional PAGE. The apparent molecular weight of the purified releasing factor, estimated by SDS-PAGE, was 23,500 Da; and the half-maximal dose for the acute stimulation of prolactin release from human decidual cells was 0.05-0.1 ug/ml (2.2-4.4 nM). 相似文献
17.
人胰腺细胞培养及胰岛素的分泌王石泉,汤国枝,张鹤云,李敏意,金以丰(南京大学生物化学系,南京210093)胰岛β细胞的体外培养获得胰岛素已有报道,但大多采用新生大鼠胰腺,且β细胞成活率低,分泌量少,还处在研究阶段[1-4].本实验采用人胰腺细胞做较大... 相似文献
18.
Frank D. Bhmer Wolfram Lehmann H. Eberhard Schmidt Peter Langen Richard Grosse 《Experimental cell research》1984,150(2)
A growth inhibitor for Ehrlich ascites mammary carcinoma cells in vitro has been purified from bovine mammary gland. The purification procedure involving homogenization and differential centrifugation under hypotonic conditions, ammonium sulfate precipitation, ultrafiltration, gel chromatography and preparative polyacrylamide gel electrophoresis (PAGE) yielded an inhibitor showing half-maximal inhibition of cell proliferation in concentrations of 1–3 ng protein per ml. Upon 125I labelling and analysis by SDS gel electrophoresis, most purified preparations revealed a single band of 12–14 kD, likely to be representative for the inhibitory protein. The inhibitor was shown to affect resumption of proliferation of stationary cells; however, it was inactive towards cells stimulated by incubation with medium before adding the inhibitor. The inhibitor is heat-labile, does not act by exhausting essential components of culture medium, and its action is antagonized by insulin. 相似文献
19.
Isolation and amino-terminal sequence of a novel cellular growth inhibitor (inhibitory diffusible factor 45) secreted by 3T3 fibroblasts 总被引:1,自引:0,他引:1
C Blat P Bohlen J Villaudy G Chatelain A Golde L Harel 《The Journal of biological chemistry》1989,264(11):6021-6024
A growth inhibitory protein named inhibitory diffusible factor 45 (IDF45) has been purified to homogeneity from medium conditioned by dense cultures of mouse 3T3 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fast protein liquid chromatography-purified bioactive material showed a single band corresponding to a protein of 45 kDa. The molecule is a monomer. The pI of the molecule, as determined by isoelectric focusing, is about 6.5. The amino-terminal sequence of the protein was established as Ser-Ala-Gly-Ala-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. The protein inhibits DNA synthesis in chicken embryo fibroblasts in a dose-dependent manner (ED50: 40 ng/ml, approximately 1 nM). The chemical properties of IDF45, i.e. molecular weight and amino-terminal sequence, clearly distinguish it from other known growth inhibitory proteins. 相似文献
20.
Insulin-like growth factor binding protein secretion by muscle cells: effect of cellular differentiation and proliferation 总被引:6,自引:0,他引:6
Several cell types have been shown to secrete insulin-like growth factor binding proteins (IGF-BP) in vitro. Since IGF-BP influences cell responsiveness to IGF, three muscle cell types were investigated to determine if they produced IGF-BP and to identify factors that regulate IGF-BP secretion. Porcine smooth muscle cells (pSMC), rat L6 skeletal muscle cells, and mouse BC3H-1 myocytes were used. IGF-BP activity in serum-free conditioned media was quantitated with a polyethylene glycol precipitation method. All three cell types secreted IGF-BP activity into the medium. Insulin was a potent stimulant of IGF-BP secretion for each cell type. Specifically, 1 microgram/ml insulin increased the IGF-BP concentration in conditioned media from 10.5 +/- 1.3 to 15.0 +/- 1.5 ng/ml in confluent L6 myotubes, from 42.5 +/- 11.1 to 90.5 +/- 9.8 ng/ml in confluent BC3H-1 cells, and from 2.1 +/- 0.1 to 3.8 +/- 0.1 ng/ml in confluent pSMC. L6 myotubes required more insulin (8 micrograms/ml) to achieve a half-maximal stimulation of IGF-BP secretion than confluent pSMC, differentiation deficient L6.DD cells or BC3H-1 cells, where half-maximal stimulation occurred between 125 and 300 ng/ml. L6 myoblasts were 40-fold more sensitive to insulin stimulation of IGF-BP secretion than L6 myotubes. IGF-I, although it interferes with the assay and thereby lowers the amount of detectable IGF-BP, stimulated the secretion of IGF-BP from all three cell types. Dexamethasone, (10(-7) M) decreased IGF-BP secretion into the media by approximately 50% for all three cell types. Affinity cross-linking and ligand blotting of 125I-IGF-I to conditioned media from each cell type showed (IGF-BP)-(IGF-I) complexes with molecular weights ranging 32-40 kDa (24-32 kDa for IGF-BP and 7.5 kDa for IGF-I). Insulin stimulated cell proliferation for both L6 myoblasts and BC3H-1 myocytes. This cell proliferative response was associated with an increase in IGF-BP secretion/cell in response to insulin. In contrast dexamethasone decreased L6 myoblast proliferation and decreased IGF-BP secretion/cell. We conclude that IGF-BP is secreted by each muscle cell type and that the state of cellular differentiation or quiescence influences its basal and insulin-stimulated secretion. Insulin and IGF-I are stimulators of IGF-BP secretion, whereas dexamethasone inhibits IGF-BP secretion. Because these hormones control muscle cell growth and differentiation, the IGF-BP may play an important regulatory role in these processes. 相似文献