Decreased transforming growth factor-beta response and binding in insulin-independent teratoma-derived cell lines with increased tumorigenic properties.

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.     

Identification of insulin receptors on the insulin-independent variant 1246-3A cell line

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.     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.     

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.     

Characterization of transforming growth factors produced by the insulin-independent teratoma-derived cell line 1246-3A

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.     

Characterization of immortalized mouse granulosa cell lines

Summary Cell cultures of primary mouse granulosa cells were transfected with a v-myc-containing plasmid, and the resulting stable cell lines were tested for their steroidogenic properties and physiologic status. Granulosa cells were obtained from 22-day-old NMRI mice injected with 8 IU pregnant mare serum gonadotropin i.p. 2 days earlier. In Passage 1 the cells were transfected with pSVv-myc using calcium phosphate precipitation or lipofectin. The 3β- and 17β-hydroxy steroid dehydrogenase activity was visualized in control cultures. The three cell lines obtained have been in culture for over 1 yr and have been subcultured for more than 90 passages. The cell line GRM01, with a doubling time of 37±3 h and a diploid modal chromosome number, produced progesterone, estradiol, as well as inhibinlike and activinlike material under basal conditions. A combination of follicle-stimulating hormone and luteinizing hormone was able to increase the secretion of progesterone. GRM01L, a fast growing clone of the GRM01 line with a doubling time of 10±1 h, retained only the capacity to produce activinlike material and transforming growth factor-β, and it was the only one with a tumorigenic capacity. Epidermal growth factor, insulin, and interleukin-6 were able to induce the [³H]thymidine incorporation into DNA in these two cell lines. GRM02, with a doubling time of 36±2 h and a hypertriploid modal chromosome number, produced progesterone and activinlike and inhibinlike material. Follicle-stimulating hormone and luteinizing hormone were able to enhance the secretion of progesterone. For this cell line, only insulin was shown to induce [³H]thymidine incorporation into DNA.     

Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.

The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.     

Purification of an insulin-related factor secreted by a teratoma-derived mesodermal cell line

Insulin-related factor (IRF), a polypeptide secreted by the mouse teratoma-derived cell line (1246-3A), was purified 3210-fold to homogeneity from 1246-3A conditioned medium using a rapid three-step procedure including cation-exchange chromatography, immunoaffinity chromatography using a monoclonal antibody against porcine insulin coupled to an agarose gel support, and reverse phase high performance liquid chromatography. 10 micrograms of IRF was purified from 6 liters of 1246-3A conditioned medium. Pure IRF appeared as a single band with the same mobility as insulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRF stimulates cell proliferation of insulin-dependent cell line 1246 and competes with 125I-insulin for binding to 1246 cells; half-maximal growth stimulation and binding competition were achieved at an IRF concentration of 6.5 ng/ml (1.3 nM) and 25 ng/ml (4 nM), respectively, comparable with those for bovine insulin. The biochemical, biological, and immunological characteristics of IRF, as well as its amino acid composition, strongly suggest that it is closely related to pancreatic insulin in structure and function.     

Characterization of an insulin-related factor secreted by a teratoma cell line

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.     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described. This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant 4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006).     

An in vitro model to study adipose differentiation in serum-free medium

Adipose differentiation was studied in a teratoma-derived fibroadipogenic cell line (1246) cultured in serum-free medium. The addition of dexamethasone and 1-methyl-3-isobutylxanthine to the serum-free medium induced confluent 1246 cells to differentiate into adipocyte-like cells as evidenced by triglyceride accumulation and increased levels of lipolytic enzyme activities. Hormone-sensitive lipase activity measured 5 days after the addition of dexamethasone and 1-methyl-3-isobutylxanthine increased 17-fold and was activated by cAMP-dependent protein kinase. Neutral diglyceride lipase, monoglyceride lipase, and cholesterol ester hydrolase specific activities increased 23-, 75-, and 73-fold, respectively. Among these three activities, only cholesterol ester hydrolase was activated by cAMP-dependent protein kinase. Differentiated 1246 cells expressed receptors to lipolytic hormones as shown by the stimulation of glycerol release by epinephrine (8.6-fold), glucagon (2.2-fold), and adrenocorticotrophic hormone (5.5-fold). Heparin treatment of 1246 cells in serum-free medium resulted in the release of lipoprotein lipase activity into the culture medium. Thus, 1246 cells can serve as a model for the study of adipose differentiation under defined culture conditions since they are capable of growth and survival in the absence of serum while retaining their ability to differentiate into adipocytes.     

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

Receptor binding and mitogenic effects of insulin and insulinlike growth factors I and II for human myeloid leukemic cells

Insulin and insulinlike growth factors I and II (IGF-I and IGF-II) influence mesodermal cell proliferation and differentiation. As multiple growth factors are involved in hemopoietic cell proliferation and differentiation, we assessed the receptor binding and mitogenic effects of these peptides on a panel of mesodermally derived human myeloid leukemic cell lines. The promyelocytic cell line HL60 had the highest level of specific binding for these 125I-labeled ligands, with lower binding to the less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these cell lines (HL60blast, KG1a). Insulin binding affinity and receptor numbers were reduced significantly by chemically induced granulocytic differentiation of HL60 cells and was unchanged following induced monocytic differentiation. No substantial alteration in IGF-I or -II binding occurred with induced HL60 cell differentiation. Insulin and IGF-I demonstrated cross competition for receptor binding and down-regulated their homologous receptors without detectable cross modulation of the heterologous receptors on HL60 cells. IGF-I and insulin increased HL60 cell proliferation, as assessed by 3H-thymidine uptake, IGF-I greater than insulin. IGF-I binding and mitogenic effects were blocked by the monoclonal anti-IGF-I receptor antibody IR3, indicating that IGF-I-induced proliferative effects were mediated via its homologous receptor. In contrast, insulin binding and mitogenesis displayed blocking by both anti-IGI-I and anti-insulin receptor antibodies, indicating mediation of its activity through both receptors. These data demonstrate specific binding and mitogenic interactions between insulin, IGFs, and hemopoietic cells which are associated with their state of differentiation.     

Isolation of myoblastic, fibro-adipogenic, and fibroblastic clonal cell lines from a common precursor and study of their requirements for growth and differentiation

C17-S1-D-T984 (to be referred to as T984) is a myogenic clonal cell line isolated from a mouse teratocarcinoma. T984 exhibits phenotypic instability since it gives rise not only to myogenic but also to fibro-adipogenic and fibroblastic clones. A cell line of each clone type has been established and studied with respect to (1) phenotypic expression and stability; and (2) growth and differentiation in serum-free and serum-supplemented media. In both respects, marked differences between the three cell lines were observed. All three cell lines respond by increased growth in serum-free media to insulin, transferrin, fibroblast growth factor (FGF) and the serum-spreading factor of Holmes. The fibroblastic and the fibro-adipogenic cell lines can both be grown indefinitely in a serum-free medium which contains the above factors. The fibro-adipogenic cell line, which differentiates in serum-supplemented medium, exhibits very limited differentiation in the absence of serum; the serum factor(s) required for adipogenic differentiation is (are) probably proteins of molecular weight superior to 10 000. In direct contrast, the myogenic cell line exhibits limited growth in serum-free medium but readily differentiates under these conditions. Moreover, myogenic differentiation could be obtained in the defined medium at very low densities and was not influenced by the addition of medium conditioned by cells seeded at high densities. Thus, in this system, muscular differentiation is apparently independent of diffusible endogenous or exogenous factors and is probably triggered by the arrest of growth. While our results do not explain the reason why T984 exhibits phenotypic instability, they do indicate that this clonal cell line and its clonal derivatives could be used to identify the factors that influence the growth and the differentiation of cells of different mesenchymal phenotypes. The possible relationship of phenotypic instability to muscular dystrophies is also discussed.     

Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture.

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLC/PRF/5, and SK-Hep-1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3-L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100 degrees C, with protease, with 2-mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cell also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.     

Effects of growth and differentiation inducing factors on protein kinase-C of cultured intestinal crypt cells

To assess the role of protein kinase-C (PK-C) in the growth and differentiation of small intestinal enterocytes, IEC-6 cells (a cell line derived from the crypts of rat small intestine) were incubated with factors known to induce growth (insulin, epidermal growth factor, gastrin, somatostatin and transferrin) or differentiation (transforming growth factor-beta, retinoic acid and phorbol 12-myristate 13-acetate (PMA)). Cell proliferation (3H-thymidine incorporation) and PK-C activity (Ca++/phospholipid dependent) were measured. Among growth promoting factors only epidermal growth factor, insulin and transferrin were associated with increased 3H-thymidine incorporation, and none of these agents induced PK-C activation as measured by its translocation from cytosol to membrane fraction. Of the differentiation inducing factors, only PMA translocated PK-C from cytosol to membrane. PMA also inhibited 3H-thymidine incorporation in a dose dependent manner. These results suggest that growth and proliferation of enterocytes occur independent of PK-C signal transduction.     

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 chinese hamster cells

Summary A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed “growth inhibiting factor” (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity CIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.     

Phospholipase A2 is a differentiation-dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture

Phospholipase A2 enzymatic activity was measured in the teratoma-derived adipogenic cell line 1246 and in adipocyte precursors in primary cultures. It was shown that enzymatic activity was low while the cells were undifferentiated and increased by 20-24-fold after the cells had undergone adipocyte differentiation. The increase of phospholipase A2 activity follows the same time course as that observed for glycerol-3-phosphate dehydrogenase activity used as a marker of differentiation. In contrast, the differentiation-deficient, insulin-independent cell line 1246-3A always contained very low levels of phospholipase A2 activity. Phospholipase A2 activity measured in the 1246 cells was inhibited in a dose-dependent fashion by incubation with ONO-RS-082 and quinacrine which are inhibitors of phospholipase A2 activity. Measurements of arachidonate metabolites in 1246 cells showed that production of prostaglandin F2 alpha by the 1246 cells followed the same time course as the increase of phospholipase A2 activity during differentiation. Similar results were obtained with primary cultures of adipocyte precursors. These results indicate that phospholipase A2 is a differentiation-dependent enzymatic activity for the adipogenic cell line 1246 and for adipocyte precursors in primary culture. These data suggest that metabolic pathways controlled by phospholipase A2 activity could play an important physiological role in adipose tissue differentiation.     

The role of the insulin receptor in mediating the insulin-stimulated growth response in Reuber H-35 cells

Summary Insulin is able to stimulate a growth response in a variety of different cell types. However, the role of the insulin receptor in mediating this response is not clear. Indeed, it has been reported that the ability of insulin to stimulate a growth response is a result of its interaction with other growth factor receptors rather than the insulin receptor.We have previously reported that the H-35 hepatoma cell line responded to physiological concentrations of insulin as a growth factor and that the relative potency of proinsulin suggested that this response was mediated by the insulin receptor. In this report, two experimental approaches are used to demonstrate the involvement of the insulin receptor in mediating the growth response. Two different preparations of antibody to the insulin receptor are found to be capable of stimulating this response. In addition, the human insulin-like growth factors (IGF-I and II) show very low cross-reactivity with the insulin receptor and are significantly less potent than insulin in stimulating the growth response.Abbrevations IGF insulin-like growth factor - MSA multiplication stimulating activity - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid