Immunological cross-reactivity of multiplication-stimulating activity polypeptides

The immunoreactivity of the multiple species of multiplication-stimulating activity (MSA) purified from medium conditioned by a rat liver cell line (BRL-3A) has been examined. Antibodies were raised in rabbits following immunization with MSA II polypeptides. Subpopulations of antibodies were purified from one antiserum using DEAE-cellulose chromatography. One antibody subpopulation recognized common antigenic determinants on MSA I and MSA II polypeptides; whereas a second antibody subpopulation recognized common determinants on MSA I, II, and III polypeptides. In a radioimmunoassay utilizing 125I-MSA III-2 and a purified antibody subpopulation, the human somatomedins (somatomedin A, insulin-like growth factor I and II) showed weak, but significant cross-reactivity: insulin-like growth factor II was 10% as potent as MSA II. By contrast, somatomedin partially purified from rat serum, insulin, growth hormone, epidermal growth factor, nerve factor, and fibroblast growth factor, showed no reactivity in the radioimmunoassay.     

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.     

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.     

Purified multiplication-stimulating activity from rat liver cell conditioned medium: comparison of biological activities with calf serum, insulin, and somatomedin

Multiplication-stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum-free medium conditioned by the growth of a line of rat liver cells (CRL), The biological activities of purified CRL MSA for chicken embryo fibroblasts were compared with those of calf serum to determine which activities are important for the stimulation of DNA synthesis and mitosis. In a balanced salt solution, only glucose and amino acids were needed in addition to purified CRL MSA to stimulate DNA synthesis maximally. Purified CRL MSA stimulated the rates of uptake of glucose and α-aminoisobutyric acid. Only the stimulation of the rate of glucose uptake appeared to be a primary response to purified CRL MSA since the stimulation was not inhibited by actinomycin D or cycloheximide. The stimulation of the rate of uptake of α-aminoisobutyric acid was inhibited by actinomycin D. CRL MSA differed from calf serum in its inability to commit cells irreversibly to synthesize DNA after the removal of CRL MSA and in its lack of the ability to stimulate the migration or prolong the survival of chicken embryo fibroblasts. Comparative studies indicated that purified CRL MSA had functional similarities to insulin and somatomedin. CRL MSA may be representative of a family of small polypeptide hormones having insulin-like activity which are involved in the control of cell multiplication.     

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.     

Pygmy mouse fibroblasts in tissue culture: evaluation of multiplication stimulating activity (MSA) receptors and growth responses to MSA

The pygmy mouse has been proposed as a model for growth hormone resistance; it has normal serum somatomedin levels and does not respond to growth hormone treatment. In order to determine if the growth impairment is caused by a defect in somatomedin binding or in postreceptor action of somatomedin we compared fibroblasts derived from pygmy mice with those from normal appearing littermates. Using multiplication-stimulating activity (MSA) as a model somatomedin we found a normal Ka of binding to the cell surface MSA receptor but a significantly increased number of MSA receptors on the fibroblasts derived from pygmy mice. Studies of thymidine incorporation into DNA failed to demonstrate a difference between pygmy and normal fibroblasts in their responses to MSA alone, but there was a significantly greater thymidine incorporation into the DNA of normal fibroblasts when both competence factor (platelet-derived growth factor) and progression factors (somatomedins and growth hormone deficient platelet-poor plasma) were present in the test medium. On the other hand, cell proliferation studies did not demonstrate a consistent difference in the growth rate of normal versus pygmy fibroblasts. The data support the conclusion that the imparied growth of the pygmy mouse in vivo may be caused by factors which lie outside of the growth hormone-somatomedin pathway.     

Glutamine B16 insulin: Reduced insulin-like metabolic activity with moderately preserved mitogenic activity

An analogue of insulin in which the naturally occurring tyrosine residue in position B¹⁶ is replaced by a glutamine residue has been synthesized. Glutamine appears in the corresponding position in the B-domain of the insulin-like growth factors. This analogue displays 9% of the potency of insulin in binding to the insulin receptor from rat liver plasma membranes, 17% in stimulating the conversion of [3-³H] glucose into lipids in rat adipocytes, and 23% in insulin radioimmunoassay, but 40% of the potency of insulin in stimulating DNA synthesis in cultured chick fibroblasts. The analogue is a more potent mitogen than is a hybrid molecule which contains the A-chain of insulin and the entire B-domain sequence of IGF-I.     

胰岛素受体家族的结构与功能研究

胰岛素（insulin）与胰岛素样生长因子-1（IGF-1）分别是由胰岛β细胞和肝细胞分泌的 多肽类激素.它们通过结合并激活位于细胞膜上的受体酪氨酸激酶(RTKs),发挥重要的生理作用. 作为起始信号传导的第一步,胰岛素与IGF-1是如何与各自受体的膜外区域（ectodomain） 结合并进一步激活受体的细胞膜内酪氨酸激酶活性一直属于科学研究的关键基础问题.本文 概述了胰岛素受体家族（IR和IGF-1R）及其配体的结构与功能的特点和关系,并重点介绍 了近年来国内外在胰岛素受体家族复合体结构和功能上的研究手段和取得的突破性进展.     

The binding of rat liver cell multiplication-stimulating activity (MSA) to human placenta and serum proteins.

Multiplication-stimulation activity (MSA) from the medium of BRL-3A rat liver cells in culture binds to cell membrane and cytosol receptors from human placenta and to serum proteins. The binding of MSA to placental cell membranes is dependent on time, temperature, pH and divalent ion concentration. MSA bound to placental cytosol receptor and serum is not displaced by insulin, whereas that bound to placental cell membranes is displaced by insulin and insulin-like peptides. The affinity of the three receptors for MSA is similar [approximately 10(8) M(-1)]. An assay using 125I-MSA and placental membrane receptor detects somatomedin-like receoptor activity (SmLRA) in unextracted sera from man and animals. A binding protein in serum that competes for 125I-MSA with receptor could not be completely separated from SmLRA by heating, acidification, charcoal treatment and gel chromatography of the serum. The relative activities of SmLRA and serum binding protein remained constant in three disorders of human growth (acromegaly, growth hormone deficiency and Laron's dwarfism) in which values of SmLRA varied widely. However, the binding protein is only partly responsible for the apparent SmLRA of unextracted serum. It is concluded that MSA is a suitable radioligand for the investigation of somatomedin disorders in man either by receptor assays or by studies of tissue receptors.     

Serotonin receptors antagonistically modulate Caenorhabditis elegans longevity

The neurotransmitter serotonin has been implicated in affecting the variation of longevity in natural Drosophila populations and age-related diseases in mammals. Based on these observations, it has been predicted that serotonin signal, perhaps at levels of serotonin biosynthesis, may control lifespan. Here, we investigated a variety of mutations in serotonin-signal genes, including serotonin biosynthesis genes, a serotonin transporter gene, and serotonin receptor genes. Despite this prediction, mutations in the serotonin biosynthesis genes had little or modest effects on lifespan, while the mod-5 mutation with increased availability of serotonin caused a modest life-shortening effect. In contrast, a deletion mutation of the ser-1 serotonin receptor gene increased longevity by up to 46%, likely through the insulin/insulin-like growth factor 1 pathway. This result suggests an interaction between the serotonin pathway and the insulin/insulin-like growth factor 1 pathway. A deletion mutation of another serotonin receptor gene, ser-4 , shortened early to mid lifespan. The results suggest that serotonin signal antagonistically modulates longevity through different serotonin receptors. This study may indicate serotonin receptors as a potential target for antigeric interventions.     

Effects of the somatomedins and insulin on myoblast differentiation in vitro

The effects of insulin and the somatomedins on differentiation of rat myoblasts were investigated in experiments on cells cloned from Yaffe's L6 line. Incubation for 48 hr with either insulin or Temin's multiplication stimulating activity (MSA), a member of the somatomedin family, caused a dramatic increase in myoblast fusion. This stimulation of differentiation is not a simple consequence of the increased cell density resulting from the effects of these hormones on myoblast proliferation, and the increase in fusion is not an effect common to all mitogens (FGF inhibits the process). Other somatomedins (human somatomedin C and insulin-like growth factor I), were as effective as MSA in stimulating differentiation. The somatomedins were active at concentrations in the range of their levels in fetal blood, in contrast to insulin, which was inactive at concentrations below 10^?7, M. Growth hormone (GH) had no effect on muscle differentiation. In serum-free medium MM-1 (in which myoblasts maintain apparently normal morphology and metabolic activity), the very high levels of insulin required to stimulate differentiation could be replaced entirely by physiological levels (1.0 μg/ml) of MSA, further supporting our view that insulin at high concentrations serves primarily as an analogue of the somatomedins in stimulating the growth and development of muscle cells.     

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.     

Tyrosine hydroxylase and insulin-like growth factor II but not insulin are adjacent in the teleost species barramundi, Lates calcarifer

In humans, the genes encoding tyrosine hydroxylase (TH), insulin and insulin-like growth factor II (IGF-II) form an extremely tight linkage group on chromosome 11p15. Characterisation of the homologous genomic region of a teleost, the barramundi Lates calcarifer , revealed tight linkage of the TH and IGF-II genes, and the absence of the gene encoding insulin.     

A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts

A polypeptide fraction with multiplication-stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum-free medium conditioned by the growth of a line of rat liver cells. The specific multiplication-stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication-stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non-suppressible insulin-like activities, but did not have anti-trypsin activity. The rat liver cell multiplication-stimulating activity resembled both multiplication-stimulating activity from calf serum and somatomedin.     

Insulin but not IGF-I is required for the maintenance of the adipose phenotype in the adipogenic cell line 1246

Summary The mouse adipogenic cell line 1246 which possesses both insulin and insulin-like growth factor I (IGF-I) receptors was used to investigate the role of IGF-I and insulin on the proliferation of adipocyte precursors and their differentiation into mature adipocytes. Results indicate that both insulin and IGF-I stimulate the proliferation of the 1246 adipocyte precursors with IGF-I being slightly more potent than insulin. Dose-response studies indicated that both polypeptides acted at physiological concentrations corresponding to binding to their own receptors. In contrast, comparison of insulin and IGF-I capacity to stimulate terminal adipose differentiation indicated that only insulin was active when added at physiological concentrations. IGF-I could not stimulate adipocyte differentiation except at supraphysiological concentrations (100 ng/ml and above) permitting its binding to the insulin receptors on 1246 cells. Time course study of expression of early and late markers of adipose differentiation indicated that the induction of markers such as adipose differentiation-related protein (ADRP), lipoprotein lipase (LPL) and fatty acid binding protein (FAB) took place even in the absence of insulin. However, the level of early and late differentiation markers decreased to a level below the one found in undifferentiated cells when cells had been maintained in the absence of insulin after differentiation had been initiated. These data indicate that although insulin is not necessary for the early onset of the adipose differentiation program, it is stringently required for the maintenance of the adipocyte phenotype and cannot be substituted by IGF-I.     

Regulation of hepatic growth hormone receptors by insulin.

Induction of diabetes in the rat with streptozotocin caused a decrease in the specific binding of human growth hormone to liver receptors. The decrease was due to a loss of binding sites, with no change in the affinity constant for growth hormone (5.6 × 10⁹M^?1). A highly significant correlation was seen between serum insulin levels and hepatic growth hormone binding. Specific insulin binding to hepatic receptors was increased in diabetes, with a highly significant negative correlation between serum insulin levels and insulin binding. The loss of growth hormone receptors was reversed by treating diabetic rats with insulin. Since hormones which bind to “lactogenic” binding sites in the liver are reported to regulate somatomedin levels, the insulin dependence of human growth hormone receptors might account for the decrease in serum somatomedin in diabetes.     

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.     

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.     

Insulin-like growth factors, IGF-1, IGF-2 and somatomedin C trigger cell proliferation in mammalian epithelial cells cultured in a serum-free medium

A single insulin-like growth factor which constitutes part of a defined serum-free medium is sufficient to stimulate DNA synthesis and mitosis in mammalian lens epithelial cells. Rabbit lenses were cultured in KEI-4, a medium which mimics rabbit aqueous humor, or in KEI-4 containing insulin growth factor I (IGF I), insulin growth factor II (IGF II) or somatomedin C. The magnitude of DNA synthesis and mitosis was evaluated on whole mount preparations of the epithelium at various times of culture. IGF I and II, the most highly purified of the insulin-like growth factors, and somatomedin C were equipotent lens mitogens, were active at the ng level, were more mitogenic toward lens epithelial cells than insulin, and initiated cell proliferation throughout the normally amitotic central region of the lens epithelium. The time course of the mitotic response elicited by the insulin-like growth factors was identical to that noted in lenses cultured in medium supplemented with serum or insulin. The present results, coupled with those of other investigators, suggest that insulin-like factors may regulate cell division in the mammalian lens in vivo.