Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: Relation to human somatomedins and insulin

The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component ot adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.     

Insulin Promotes the Hydrolysis of a Glycosyl Phosphatidylinositol in Cultured Rat Astroglial Cells

Abstract: Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol-specific phospholipase C and glycosyl phosphatidylinositol-specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 n M insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1–2 min. In addition, an inverse concentration-dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C-mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin-like growth factor (IGF)-I and/or IGF-II receptors, as demonstrated by affinity cross-linking studies. Also, the effects of 5 n M IGF-I or 5 n M IGF-II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacyl-glycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF-I, and especially with IGF-II, for 10 min. Despite the remarkable homology among insulin, IGF-I, and IGF-II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.     

Insulin-like growth factor-II induces hypertrophy of adult cardiomyocytes via two alternative pathways

Insulin-like growth factor (IGF)-II is known to induce hypertrophy of isolated adult rat ventricular cardiomyocytes cultured in the absence of serum. However, it is not known how the growth factor exerts this hypertrophic effect. We show here that IGF-II induces hypertrophy of the cultured cardiomyocytes via two alternative pathways: (1) an IGF-I receptor-dependent pathway, or (2) a lysosome-dependent pathway when the IGF-I receptor-dependent pathway is blocked.     

Evidence that somatomedin is synthesized by multiple tissues in the fetus

Production of somatomedin-C, a growth hormone-dependent peptide believed to mediate the growth-promoting actions of growth hormone, has been assessed using explants of fetal mouse tissues. Quantitation of this peptide in media of explants cultured for 3 days has been accomplished with a membrane receptor assay for somatomedin and a specific radioimmunoassay for somatomedin-C. Somatomedin-C is produced by the 11-day-gestation fetal mouse liver, increases exponentially in parallel with liver growth until the 16th day of gestation, and falls postnatally. Media somatomedin is believed to be derived by de novo synthesis since saline extracts of liver and most other fetal tissues contain only a small fraction of the activity in culture media. The immunoreactive material secreted into media appears to be closely related to human somatomedin-C since it produces dilution curves which are parallel to those of pure hormone, migrates on Sephacryl 200 at a size similar to that of one of the components of human serum somatomedin-C, dissociates into small molecular weight material with acid treatment, and isofocuses in a range comparable with that of somatomedin-C purified from human serum. Eleven-day limb bud mesenchymal micromass cultures and 17-day-gestation intestine, heart, brain, kidney, and lung also synthesize immunoreactive somatomedin-C in serum-free medium. For these tissues, the media activity was far in excess of the tissue extractable activity. Somatomedin activity in excess of the tissue extractable activity, however, was not found in media from 17-day-gestation placenta. The finding that multiple tissues synthesize somatomedin-C raises the possibility that the primary biological actions of this hormone are exerted locally at its sites of origin. Although a function of this type by a peptide has not been widely suspected, it seems plausible that the cells of fetal tissues are capable of producing local mitogens in much the same manner as the postulated inducers of tissue differentiation.     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Characterization of the binding of a rat somatomedin to receptors in human placental cell membranes.

The somatomedins presumably initiate their growth promoting effects by first binding to specific cell surface receptors in responsive tissues. The specific and high affinity binding of [¹²⁵I]-rat somatomedin to human placental membranes was saturable and reversible with a dissociation constant of 4.5 × 10^?9 M calculated from Scatchard analysis of competitive binding experiments. Competition for [¹²⁵I]-rat somatomedin binding to placental receptors by other somatomedins and growth factors suggest a close structural relationship between rat somatomedin and the human somatomedin, insulin-like growth factor I.     

Demonstration of the presence of an ultrafiltrable substance in human serum activating somatomedin A

Human serum contains an ultrafiltrable factor which stimulates the somatomedin activity measured by 35SO4 incorporation into pelvic cartilage of chick embryo, this ultrafiltrable factor activates native serum somatomedin or partially purified somatomedin. The molecular weight determined by fractionated ultrafiltration or chromatography on Biogel P2 is about 350-500 daltons.     

Regulation of the Balb/c-3T3 cell cycle-effects of growth factors

The platelet-derived growth factor (PDGF), which is found in serum but not in plasma, has been purified to homogeneity; it stimulates replication at a concentration of 10^?10M. Brief treatment with PDGF causes densityinhibited Balb/c-3T3 cells to become competent to synthesize DNA; pituitary fibroblast growth factor (FGF) or precipitates of calcium phosphate also induce competence. Continuous treatment with plasma allows competent, but not incompetent, cells to synthesize DNA. A critical component of plasma is somatomedin, a group of hormones with insulin-like activity; multiplication-stimulating activity (MSA) or insulin replace plasma somatomedin in promoting DNA synthesis. We have studied the molecular correlates of competence and the role of SV40 gene A products in regulating DNA synthesis. Treatment of quiescent cells with pure PDGF or FGF causes the preferential synthesis of five cytoplasmic proteins (approximate molecular weight 29,000, 35,000, 45,000, 60,000, and 72,000 detected by SDS-PAGE under reducing conditions). Two of these competence-associated proteins (29,000 and 35,000 daltons) are found within 40 min of PDGF addition; they are not induced by plasma, insulin, or epidermal growth factor (EGF), PDGF, FGF, or calcium phosphate induce an ultrastructure change within the centriole of 3T3 cells; this ultrastructural modification of the centriole is detectable by immunofluorescence within 2 h of PDGF treatment. Plasma, EGF, or MSA do not modify the centriole. SV40 induces replicative DNA synthesis in growth-arrested 3T3 cells but does not cause this alteration in centriole structure. Gene A variants of SV40, including a mutant with temperature-sensitive (ts) T-antigen (ts A209), a deletion in t-antigen (dl 884), and several ts A209 strains containing t-antigen deletions were used to induce DNA synthesis in Balb/c-3T3 cells. Like wild type SV40, all strains induced DNA synthesis equally well under permissive or nonpermissive conditions. Addition of PDGF or plasma had little effect on SV40-induced DNA synthesis. Thus, the viral function that induces replicative DNA synthesis in Balb/c-3T3 cells is not t and is not temperature sensitive. This SV40 gene function overrides the cellular requirement for hormonal growth factors. It does not induce transient centriole deciliation, a hormonally regulated event.     

Rete testis fluid contains a growth factor for cultured fibroblasts

A new polypeptide mitogen has been detected at high specific activity in the rete testis fluid of rams (oRTF). The factor, which stimulates DNA synthesis in quiescent Swiss 3T3 cells, has a molecular weight of 45,000 as assessed by gel filtration through Ultrogel AcA 34. The factor is heat stable but is inactivated by proteolytic enzymes and by β-mercaptoethanol. The growth-promoting activity in oRTF does not bind to concanavalin A.     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

No Decrease in Free IGF‐I with Increasing Insulin in Obesity‐Related Insulin Resistance

Objective: Different facts suggest that the insulin growth factor (IGF)/ insulin growth factor‐binding protein (IGFBP) system may be regulated by factors other than growth hormone. It has been proposed that, in healthy subjects, free IGF‐I plays a role in glucose metabolism. The role of free IGF‐I in glucose homeostasis in insulin resistance is poorly understood. This study was undertaken to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF‐I and IGFBP‐1 in obese and non‐obese subjects. Research Methods and Procedures: Nineteen lean and 24 obese subjects were investigated. A frequently sampled intravenous glucose tolerance test was performed. Free IGF‐I and IGFBP‐1 were determined at 0, 19, 22, 50, 100, and 180 minutes. Results: Basal free IGF‐I levels tended to be higher and IGFBP‐1 lower in obese than in lean subjects. IGFBP‐1 levels inversely correlated with basal insulin concentration. To determine the effects of insulin on the availability of free IGF‐I and IGFBP‐1, changes in their plasma concentrations were measured during a frequently sampled intravenous glucose tolerance test. After insulin administration, a significant suppression of free IGF‐I at 22% was observed in lean subjects. In contrast, plasma‐free IGF‐I levels remained essentially unchanged in the obese group. The differences between both groups were statistically significant at 100 minutes (p < 0.01) and 180 minutes (p < 0.05). Serum IGFBP‐1 was suppressed to a similar extent in both groups. Discussion: These data suggest that the concentrations of free IGF‐I and IGFBP‐1 are differentially regulated by obesity. Obesity‐related insulin resistance leads to unsuppressed free IGF‐I levels.     

Further purification of a fibroblast growth factor-like factor from chick embryo extract by heparin-affinity chromatography

Summary A mitogenic factor which promotes quail myoblast proliferation has been purified some 10⁵-fold from chick embryo extract by a combination of cation-exchange chromatography and heparin-affinity chromatography. The factor is eluted from heparin-Sepharose with 2M NaCl and is a single-chain polypeptide with an apparent molecular weight of 15000 to 17000. It is active at subnanogram level in triggering the proliferation and thereby delaying temporarily fusion of myoblasts. It also stimulates the proliferation of quail fibroblasts in a similar effective concentration range. For both myoblasts and fibroblasts the dose-response to the factor is quantitatively and qualitatively comparable with that of bovine pituitary fibroblast growth factor. These observations strongly suggest that the factor very probably corresponds to chicken fibroblast growth factor or to a closely related molecule(s) and that it is possibly involved in the regulation of myogenesis. This work was partly supported by a grant from the National Center of Neurology and Psychiatry (NCNP grant 86-01) of the Ministry of Health and Welfare, Japan.     

Platelet-derived growth factor stimulates the phosphorylation of ribosomal protein S6

The human platelet derived-growth factor (PDGF) is both a potent mitogen and a strong chemoattractant protein for cells involved in inflammation and repair. In seeking mechanisms by which PDGF might initiate specific activities in target cells, it was found that highly purified PDGF stimulates the phosphorylation of an Mr approximately 33000 protein in confluent Swiss mouse 3T3 cells [Biochem. Biophys. Res. Commun. (1981) 103, 355-361]. The Mr approximately 33000 protein has now been recovered in polysomes by differential centrifugation and identified as ribosomal protein S6 by two-dimensional polyacrylamide gel electrophoresis.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Phospholipase Cγ Activation, Phosphotidylinositol Hydrolysis, and Calcium Mobilization are Not Required for FGF Receptor-Mediated Chemotaxis

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoartractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transf ected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLOγ, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLCγ following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLCγ, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.     

Effects of lipids on cyclic-nucleotide phosphodiesterases

Several naturally-occurring lipids but not n-propanol, guanidine-HCl or a variety of synthetic detergents stimulate the 3′,5′-cyclic AMP-phosphodiesterase activities of a supernatant fraction of brain at 1.25 × 10^?7 M cAMP. The time courses of the reaction are linear in the presence and absence of lipid. On the other hand, lipid has different effects on various phosphodiesterase activities in fractions obtained after gel filtration of the crude extract. It stimulates the phosphodiesterase activities measured at 1.25 × 10^?7 M and 10^?4 M 3′,5′-cyclic-AMP and 1.25 × 10^?7 M 3′,5′-cyclic GMP in two of the fractions partially retained in the gel. However, lipid has little effect on the enzymatic hydrolysis of low concentrations of cAMP or cGMP and markedly inhibits the hydrolysis of high concentrations of cAMP by the fraction excluded from the gel.     

Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats

When rat serum was subjected to gel filtration on a Sephadex G-200 column, a factor, "hepatotropin", that promoted hepatocyte growth in primary culture was separated. Its Mr was about 150 KD and it was an anionic protein that was unstable on acid- and heat-treatments. Hepatotropin was purified 20-fold further by affinity chromatography on heparin-Sepharose CL-6B. The purified hepatotropin was effective at 20 micrograms/ml and maximally effective at 120 micrograms/ml, and its effect was additive with that of insulin plus epidermal growth factor. In rats after partial hepatectomy, the hepatotropin activity in the serum increased time-dependently reaching a maximum of about 5-fold the initial level 24 h after the operation. Various known growth factors, such as fibroblast growth factor, platelet derived growth factor, somatomedin, thrombin and transferrin, did not stimulate DNA synthesis in cultured hepatocytes. These results suggest that hepatotropin is a new growth factor.     

Production of an insulin-like growth factor by osteosarcoma

To test the possibility that osteosarcoma cells produce their own growth factors, we measured levels of insulin and somatomedin C (SMC), an insulin-like growth factor, in culture media of two cell lines derived from patients with that disease. SMC but not insulin levels increased three- to ten-fold over a period of 7 days paralleling the increases in cell number. Production of SMC was inhibited by cycloheximide.     

Adenylate cyclase enhancing factor from rat osteosarcoma cytosol.

A cytosol factor from a transplantable rat osteosarcoma stimulates the adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity of partially purified membranes 1.5 fold at pH 7.6 and over 5 fold at pH 6.5. This effect can also be seen at maximum Gpp(NH) stimulation of the enzyme. The cytosol factor is non-dialyzable, ultrafiltrable through a 100,000 dalton exclusion membrane, heat labile and trypsin digestible. The stimulation is immediate, is independent of Ca²⁺, exhibits sygmoidal concentration dependency and is enhanced by GTP. The factor did not bind GTP. The stimulatory activity was fully recovered in two Sephadex G-100 fractions of approximate molecular weights of 55,000 and 29,500. Unlike the starting material the fractions were not stable to freeze-thawing or lyophilization. A similar factor could not be found in embryonic bone, nor did the osteosarcoma factor affect bone adenylate cyclase.