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Biomedical subjects

M M Rechler

Publications and source records attributed to M M Rechler.

At least 109 records · Page 6Linked to original sources
PubMed1984

Potential mechanism of the stimulatory action of insulin on insulin-like growth factor II binding to the isolated rat adipose cell. Apparent redistribution of receptors cycling between a large intracellular pool and the plasma membrane.

Previous studies have proposed that insulin increases the binding of insulin-like growth factor II (IGF-II) in isolated rat adipose cells at 24 degrees C by increasing receptor affinity (Ka). This study re-examines these observations under conditions in which receptor-ligand internalization is blocked by 1 mM KCN. In the absence of KCN, adipose cells bind 0.71 amol of IGF-II/cell with low apparent affinity (0.030 nM-1), of which greater than 75% is not accessible to trypsin. In contrast, in the presence of KCN, IGF-II binding is decreased by 95% and its apparent affinity increased to 0.21 nM-1. Moreover, greater than 60% of the bound IGF-II now is sensitive to trypsin. In either the absence or presence of KCN, approximately 20% of the cell's total IGF-II receptors are present in the plasma membranes and approximately 80% in the low density microsomes. Insulin induces a 5-fold increase in cell surface IGF-II receptors without a change in affinity when IGF-II binding is measured in the presence of KCN. Similarly, insulin increases IGF-II receptor concentration in the plasma membranes and concomitantly decreases that in the low density microsomes. Receptor affinity in these two subcellular membrane fractions is not affected by incubation of intact cells with either insulin or KCN and is similar to that observed in intact cells in the presence of KCN. Addition of KCN prior to insulin abolishes all of these effects of insulin. These data suggest that (a) the effects of KCN reflect a selective blockade of endocytosis; (b) in the absence of KCN, IGF-II binds to receptors of constant affinity that cycle between the plasma membrane and an intracellular pool resulting in an accumulation of intracellular IGF-II; (c) insulin induces an increase in IGF-II binding by causing a steady state redistribution of receptors from this intracellular pool to the plasma membrane; and (d) this redistribution in the intact cell can only be detected using Scatchard analysis when recycling of the receptors is prevented by KCN.

Adipose Tissue↗
PubMed1984

Insulin-like growth factor-I (IGF-I) stimulates tyrosine kinase activity in purified receptors from a rat liver cell line.

Solubilized, lectin-purified receptor preparations from BRL 3A2 rat liver cells are rich in Type I and Type II IGF receptors, but possess few insulin receptors. High concentrations of IGF-I or insulin stimulate phosphorylation of a Mr congruent to 98K membrane protein in these preparations. Phosphorylation of a synthetic polymer of tyrosine and glutamic acid was stimulated by IGF-I greater than IGF-II congruent to insulin. These relative potencies, together with the results of immunodepletion experiments using an autoantibody to the insulin receptor, suggest that the effects of each of these hormones is mediated by the Type I IGF receptor. Our results are consistent with the Type I IGF receptor having intrinsic tyrosine kinase activity capable of phosphorylating the receptor itself and other substrates.

Animals↗
PubMed1984

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

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

Animals↗
PubMed1984

Somatomedin/insulin-like growth factor tissue receptors.

There are two types of Sm/IGF receptors based on results of competitive binding experiments and investigations of receptor structure. The type I receptor preferentially interacts with IGF I rather than IGF II and interacts weakly with insulin. This receptor has a binding subunit of Mr = 130 000 which is disulphide bonded to form larger structures of Mr greater than 300 000. The type II receptor prefers IGF II to IGF I and does not interact with insulin. Its binding subunit is not linked by disulphide bonds to other membrane components (Mr = 260 000 with reduction, 220 000 without reduction). Subunit organization of the type I receptor appears to be similar to that of the insulin receptor but it is unlikely that the insulin and Sm/IGF binding sites are on a common alpha subunit. The type I receptor is down-regulated by IGFs and insulin. A rapid increase in ligand binding to the type II receptor by insulin has been described in intact rat adipocytes. The original idea that an IGF receptor mediates the growth-promoting action of both IGFs and insulin while acute metabolic effects of insulin and IGFs are mediated by the insulin receptor is an oversimplification . There now are clear examples of insulin stimulating growth by acting through the insulin receptor and, conversely, instances of IGF stimulating glucose transport by acting through an IGF receptor. Radioreceptor assays which measure IGF I in preference to IGF II (human placental membrane) and which measure IGF II in preference to IGF I (rat liver and rat placental membranes) have been utilized for clinical measurements of Sm/IGF levels, but are less specific than the respective radioimmunoassays. With the demonstration of Sm/IGF receptors on circulating human mononuclear cells and human skin fibroblasts, it is expected that these systems will be useful for investigations of patients with possible end-organ resistance to Sm/IGF.

Animals↗
PubMed1984

Interaction of insulin-like growth factors with a nonfusing mouse muscle cell line: binding, action, and receptor down-regulation.

Insulin and the insulin-like growth factors (IGFs) are chemically related polypeptides that interact with distinct receptors and elicit the same biological responses. We have sought a readily propagated cell line from a potential target tissue in which to probe the multiple and complex interrelationships among receptor and effector pathways for these polypeptides. We now report that the mouse muscle cell line BC3H-1 represents such a model system. BC3H-1 cells differentiate spontaneously at high density to form cells with muscle-specific properties, but do not fuse. Standaert et al. reported that differentiated BC3H-1 myocytes possess insulin receptors that mediate glucose and amino acid uptake and are down-regulated by prolonged incubation with insulin. The present report demonstrates that BC3H-1 myocytes also possess functional and regulated IGF receptors. Two subtypes of IGF receptors, types I and II, differing in structure and peptide specificity, were demonstrated by competitive binding and affinity cross-linking experiments. Low concentrations of IGFs stimulated glucose incorporation and alpha-aminoisobutyric acid uptake by BC3H-1 myocytes, suggesting that these effects were mediated primarily by IGF receptors rather than insulin receptors. Preincubation with IGFs (or high concentrations of insulin) selectively down-regulated type I IGF receptors without affecting type II IGF receptors. Since [125I]IGF-I binds to both type I and type II receptors in BC3H-1 cells, and since type I receptors have a higher affinity for IGF-I, the selective down-regulation of type I IGF receptors results in an apparent decrease in affinity for IGF-I. This difference in the regulation of type I and type II receptors in BC3H-1 myocytes is consistent with observations in other systems in which only one IGF receptor was present or examined. In their ability to be down-regulated by IGFs and insulin, type I IGF receptors are more similar to the structurally homologous insulin receptors than to the structurally dissimilar type II IGF receptors. These findings indicate that the BC3H-1 cell line provides an excellent model system in which to study the structure-function relationships of the receptor and effector pathways for insulin and the IGFs.

Amino Acids↗
PubMed1983

Purification of an insulin-like growth factor II receptor from rat chondrosarcoma cells.

An insulin-like growth factor II (IGF-II) receptor was purified from rat chondrosarcoma cells by Triton X-100 solubilization of a 100,000 X g membrane preparation and affinity chromatography on a multiplication-stimulating activity (MSA)-Sepharose column. Analysis of the purified receptor by sodium dodecyl sulfate-gel electrophoresis and silver staining showed a major band of Mr = 210,000 (Mr = 250,000 after reduction with dithiothreitol). When 125I-MSA was chemically cross-linked to the purified receptor and analyzed by sodium dodecyl sulfate-gel electrophoresis (with and without dithiothreitol) and autoradiography, the radioactive bands coincided with the Mr = 210,000 and 250,000 bands identified by silver staining. The purified receptor also appeared to contain an Mr = less than 68,000 species identified by silver staining in addition to the Mr = 250,000 binding component. IGF-I, IGF-II, and MSA-II inhibited binding of 125I-MSA to the purified receptor with the same relative potency as for binding to the intact chondrosarcoma cell (IGF-II greater than MSA-II greater than IGF-I), and insulin did not inhibit binding. The association constant (K alpha) for MSA-II binding to the purified receptor was 2 X 10(9) M-1. The purified receptor bound to concanavalin A-Sepharose and wheat germ lectin-Sepharose columns and was eluted with alpha-methyl-D-mannoside and N-acetyl-D-glucosamine, respectively, showing that the receptor is a glycoprotein.

Animals↗
PubMed1983

Antireceptor antibodies as probes of insulinlike growth factor receptor structure.

Insulin receptors and Type I insulinlike growth factor (IGF) receptors have a similar structure with a major binding subunit of Mr approximately 130,000 linked by disulfide bonds to other membrane proteins to form a Mr greater than 300,000 complex. Both insulin and Type I IGF receptors also interact with both insulin and IGF, although with different binding affinities. We used a panel of human and rabbit sera containing antibodies to insulin receptors to determine whether these sera also interact with Type I IGF receptors. Immunoglobulins from five of five human sera inhibited binding of 125I-insulin and 125I-IGF-I to insulin receptors and Type I IGF receptors in human placenta and human lymphocytes. The rank order of reactivity with both receptors was the same; two sera, however, appeared to be selectively less reactive with the Type I IGF receptor, especially in placenta. Sera from five of seven patients and from a rabbit immunized with purified insulin receptor effectively immunoprecipitated both placental insulin receptors and Type I IGF receptors. Of the remaining sera, one had only a low titer against the insulin receptor and did not immunoprecipitate the IGF receptor, whereas the second serum effectively immunoprecipitated cross-linked and surface-iodinated insulin receptors, but had negligible reactivity against the Type I IGF receptor. These results suggest that most antisera to the insulin receptor also contain antibodies to Type I IGF receptors. Whether both specificities are inherent in the same or different antibody molecules remains to be determined. These data support the hypothesis that the insulin and IGF-I receptors are separate but related molecules, although there remains a small possibility that both receptors are domains on the same protein.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

Synthesis of multiplication-stimulating activity (rat insulin-like growth factor II) by rat embryo fibroblasts.

Multiplication-stimulating activity, a family of insulin-like growth factors previously identified in medium conditioned by the BRL-3A rat liver cell line, is also synthesized by third passage cultures of rat embryo fibroblasts (REFs) maintained in serum-free medium. Conditioned serum-free medium from REFs was chromatographed on Sephadex G-75 in 1 m acetic acid, and fractions in the size range of BRL-MSA contained MSA immunoreactivity. A dose-response curve of pooled G-75 fractions was parallel to BRL-MSA standard, and levels in the REF-conditioned medium were 0.5-1 microgram/ml. REF-MSA from the Sephadex G-75 pool was equipotent to BRL-MSA in stimulating [3H]thymidine incorporation into DNA in chick embryo fibroblasts and in stimulating DNA synthesis in REFs, as measured by autoradiography. In receptor binding assays using REFs, chick embryo fibroblasts, Swarm rat chondrosarcoma cells, and rat liver membranes, REF MSA was equal to BRL MSA in competition for binding of [125I]iodo-MSA. REF MSA also behaved identically to BRL MSA in a competitive binding protein assay using binding proteins in rat serum. Further characterization of REF MSA using Bio-Gel P-10 chromatography, followed by high pressure liquid chromatography or polyacrylamide gel electrophoresis, indicated that immunoreactive polypeptides in the fibroblast medium corresponded to BRL MSA II (mol wt, 8700) and BRL MSA III (mol wt, 7100). The amount of REF MSA released into the medium increased linearly over time. Cycloheximide decreased the amount of MSA in the medium, and during a recovery period, the amount of MSA returned nearly to control levels. In summary, rat embryo fibroblasts synthesize MSA which is biologically, immunologically, and chemically identical to MSA produced by the BRL-3A rat liver cell line.

Animals↗
PubMed1983

Growth hormone stimulates islet B-cell replication in neonatal rat pancreatic monolayer cultures.

A possible role for growth hormone (GH) in stimulating islet B-cell replication was examined in neonatal rat pancreatic monolayer cultures. Addition of ovine GH (1000 ng/ml) to serum-free medium for 2 days resulted in a significant increase (+114%) in [3H]thymidine labeling of B-cells in aldehyde-thionine-stained autoradiographs, and a similar increase in the B-cell mitotic index. The stimulatory effects of GH on islet B-cell replication were greatest in serum-free medium, unaffected by glucose concentrations (2.8--16.7 mM), and not accompanied by any stimulation of insulin release. The threshold concentration of GH for a significant stimulatory effect on B-cell replication was 30 ng/ml. The insulin-like growth factor, multiplication stimulating activity (MSA), also effectively stimulated B-cell replication. Although some effects of GH are mediated by the insulin-like growth factors (IGFs), the effects of GH on B-cell replication did not appear to be mediated by IGFs since (1) the maximal effect of GH (+156%) was significantly greater than that of MSA (+91%); (2) the combination of maximal stimulatory concentrations of GH and MSA produced an additive effect; and (3) a significant effect of GH on B-cell replication was observed as early (8 h) as that produced by MSA. These results suggest that GH can stimulate islet B-cell replication directly, and that this effect may not depend on production of either insulin-like growth factors.

Animals↗
PubMed1982

Demonstration of receptors for insulin and insulin-like growth factors on Swarm rat chondrosarcoma chondrocytes. Evidence that insulin stimulates proteoglycan synthesis through the insulin receptor.

The insulin-like growth factor, multiplication stimulating activity (MSA), and insulin were recently shown to stimulate proteoglycan synthesis in monolayer cultures of chondrocytes derived from the Swarm rat chondrosarcoma. Insulin produced significant stimulation at a concentration of less than 1 ng/ml, suggesting that insulin was acting through the insulin receptor rather than through a somatomedin receptor. In this paper we have shown that the insulin-like growth factors IGF-I and IGF-II also are potent stimulators of [35S]sulfate incorporation into macromolecules recovered from the medium and cell layer matrix of the chondrosarcoma chondrocytes. Proinsulin was 3% as potent as insulin in stimulating [35S]sulfate incorporation. We identified receptors for insulin and the insulin-like growth factors, MSA, IGF-I, and IGF-II. Insulin, at concentrations 1000 times the concentration required to produce the biologic response, did not compete for binding of 125I-MSA-II-1 or of 125I-IGF-II and only partially competed for 125I-IGF-I binding. Anti-insulin receptor IgG stimulated proteoglycan synthesis and competed for 125I-insulin binding. Fab fragment prepared from anti-insulin receptor IgG completely blocked the stimulation of [35S]sulfate incorporation into macromolecules by insulin while only partially inhibiting the biologic response to insulin-like growth factors, MSA, IGF-I, and IGF-II. Similarly, the anti-insulin receptor IgG only partially inhibited the binding of 125I-IGF-I and 125I-IGF-II while completely blocking the binding of 125I-insulin. We conclude that insulin stimulates proteoglycan synthesis in the chondrosarcoma chondrocytes by acting through the insulin receptor whereas the insulin-like growth factors probably act through their own receptors.

Animals↗
PubMed1982

Structure of the insulin-like growth factor receptor in chicken embryo fibroblasts.

The insulin-like growth factors (IGFs) and insulin stimulate DNA synthesis and cell multiplication in chicken embryo fibroblasts in culture. This response appears to be mediated by interaction with a single type of IGF receptor. The present study examines the subunit structure of this receptor by covalently crosslinking two 125I-labeled IGFs, IGF-I and multiplication-stimulating activity (MSA), to chicken embryo fibroblasts by using disuccinimidyl suberate. After solubilization, NaDodSO4/polyacrylamide gel electrophoresis, and autoradiography, IGF receptor complexes of appropriate specificity were identified; they had Mr approximately 130,000 (major band) and approximately 260,000 (minor band) under reducing conditions and Mr greater than 300,000 without disulfide reduction. The proportion of the Mr 260,000 component increased with increasing concentration of crosslinking agent, suggesting that it was formed from smaller proteins during the crosslinking procedure. The IGF receptor in chicken embryo fibroblasts resembles the insulin receptor in size and structure but can be distinguished by a higher affinity for IGF-I and MSA than for insulin. Although IGF receptors with different structure and specificity have been recognized in other tissues, the function of these binding sites is unknown. The present study demonstrates that the IGF receptor of chicken embryo fibroblasts that appears to mediate the growth-promoting effects of the IGFs contains a Mr approximately 130,000 binding subunit and exists as a native receptor complex of Mr greater than 300,000.

Animals↗
PubMed1982

Developmental pattern of a serum binding protein for multiplication stimulating activity in the rat.

The concentration of multiplication stimulating activity (MSA), an insulinlike growth factor (IGF), is high in fetal rat serum. We now report that MSA is exclusively associated wth an albumin-size binding protein in fetal rat serum; the growth hormone-dependent, gamma globulin-size binding protein, which predominates in the older animal, is absent from fetal rat serum. When (125)I-MSA was incubated with fetal rat serum and then gel filtered on Sephadex G-200, specific radioactivity eluted in the void volume (peak I) and the albumin region (peak III); by contrast, specific radioactivity eluted mainly in the gamma globulin region (peak II) in adult rat serum. Pools of the Sephadex G-200 fractions were chromatographed on Sephadex G-50, in 1 M acetic acid, to separate the binding protein from IGF activity. Analysis of IGF activity by chick embryo fibroblast bioassay, competitive protein binding assay, and MSA by radioimmunoassay revealed that all the IGF activity and MSA in fetal rat serum resided in peak III. Measurement of MSA binding capacity of the stripped binding protein by Scatchard analysis demonstrated that the majority of binding capacity also was found in peak III in fetal rat serum; most of MSA binding capacity was in peak II in adult rat serum. In fetal rat sera, in addition to the peak III binding protein, which is the major carrier of endogenous MSA, there is a component in peak I capable of specifically binding (125)I-MSA. This component elutes as a single species from a Sepharose-6B column. As MSA associated with peak III gradually declined in early neonatal life, peak II-associated IGF activity measured by chick embryo fibroblast bioassay showed a rise of activity with a peak at 5 d of neonatal life, a nadir at 20 d, with an increase again to attain adult levels. These studies demonstrate that the MSA binding protein in the fetus is different from the growth hormone-dependent binding protein in adult life.

Aging↗
PubMed1982

Interactions of multiplication-stimulating activity with bovine endothelium: comparative studies in primary, passaged, and cloned cultures from the pulmonary and systemic circulations.

Endothelial cells were prepared from adult bovine pulmonary and systemic vessels and maintained as primary cultures, passaged cultures, and cloned cell strains. All cultures were shown to contain endothelial cells on the basis of several endothelial-specific and endothelial-associated traits. Both primary and passaged cells demonstrated specific receptors for the insulin-like growth factor, multiplication-stimulating activity (MSA). [125I]Iodo-MSA binding was greatest in cells derived from aortas, least in pulmonary venous cells, and intermediate in pulmonary arterial cells. When compared to MSA receptors on primary endothelial cell cultures derived from human umbilical veins or arteries, the bovine cells bound 4-10 times more MSA per cell. In all bovine cells, insulin competed weakly or not at all with [125I]iodo-MSA binding. The presence of MSA receptors on primary and passaged cells from five different vascular sources (three bovine and two human) suggests that receptors for the insulin-like growth factors may be an intrinsic component of the vascular endothelium.

Animals↗
PubMed1982

Multiplication-stimulating activity stimulates the multiplication of F9 embryonal carcinoma cells.

The present study was designed to assess the effectiveness of multiplication-stimulating activity (MSA), an insulin-like growth factor, in supporting the growth of F9 cells (an embryonal carcinoma line with properties similar to embryonic stem cells). Under serum-free growth conditions in a medium supplemented with transferrin and fibronectin, MSA is an effective mitogen. At 100 ng/ml, MSA completely replaces the growth requirement for fetal calf serum. Biologically active MSA polypeptides II and III act as potent growth promoters of F9 cells, whereas MSA I appears to be somewhat less effective. Binding studies carried out with [125I]iodo-MSA indicate that F9 cells possess specific receptors for MSA. Scatchard analysis indicates a single class of MSA receptors with a Ka of 8.2 x 10(9) M-1; about 55,000 MSA molecules can bind per F9 cell. Insulin-like growth factor I and II both complete for MSA binding, whereas insulin does not compete. Since insulin is an effective promoter of F9 cell growth, these results indicate that the mitogenic action of insulin toward F9 cells is not mediated through MSA receptors. It is apparent from these results that MSA is capable of serving as an early embryonic growth factor.

Animals↗