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Biomedical subjects

G Serrero

Publications and source records attributed to G Serrero.

At least 55 records · Page 3Linked to original sources
PubMed1991

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.

Adipose Tissue↗
PubMed1991

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.

Animals↗
PubMed1991

Decrease in transforming growth factor beta 1 binding during differentiation of rat adipocyte precursors in primary culture.

Transforming growth factor beta 1 (TGF-beta 1) binding and action were investigated during differentiation of adipocyte precursors freshly isolated from rat inguinal fat-pad cultivated in defined medium. The data presented in this paper indicate that TGF-beta 1 inhibits differentiation of adipocyte precursors with a 50% effective dose of 9 pM. Time course experiments demonstrate that TGF-beta 1 is active only when it is added to the cells while they are still undifferentiated. If added after the cells have started to differentiate, TGF-beta 1 is less active or becomes inactive. 125I-TGF-beta 1 binding studies on adipocyte precursors before and after differentiation indicate a 10-fold decrease in the number of TGF-beta 1 binding sites after the cells have differentiated. Blocking of the differentiation process by treating the cells with fetal bovine serum or with prostaglandin F2 alpha prevented the decrease in the number of TGF-beta 1 receptors, thereby demonstrating that this change in binding was specifically linked to the differentiation process. Experiments cross-linking 125I-TGF-beta 1 to adipocyte precursors showed that 125I-TGF-beta 1 is specifically cross-linked to two bands with molecular weights of 92,000 and 70,000. After differentiation, a decrease in the intensity of the cross-linked bands was observed. These results demonstrate that loss of cell surface TGF-beta 1 binding sites follows differentiation of adipocyte precursors.

Adipose Tissue↗
PubMed1990

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.

Adipose Tissue↗
PubMed1990

Pedersen fetuin contains three adipogenic factors with distinct biochemical characteristics.

Crude Pedersen fetuin, derived from fetal bovine serum, contains adipogenic activity. Biochemical characterization was undertaken by following the differentiation of the 1246 adipogenic cell line. The present paper provides evidence that crude fetuin contains distinct proteins with adipogenic activity. By molecular sieve fractionation using Sephacryl S-300, the majority of adipogenic activity eluted in two distinct peaks, FI (molecular weight greater than 669 kDa) and FII (molecular weight ranging from 445 and 232 kDa). In addition a minor activity was found in a third peak, FIII (molecular weight around 69 kDa). Partial purification and biochemical characterization indicate that FI and FII are two distinct factors. FI has a PI higher than 9.4, is destroyed by alkaline treatment, and is stable when treated with acid. FII has a PI lower than 4.0, is alkali stable, but is destroyed completely by treatment with acid. Moreover, our data show that adipogenic factors are distinct from another protein alpha 2 macroglobulin known to be found in crude Pedersen fetuin. These results suggest that serum contains two large molecular weight proteins bearing adipogenic activity which could play an important role in the control of the adipose differentiation process.

Adipose Tissue↗
PubMed1989

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Autocrine growth induced by the insulin-related factor in the insulin-independent teratoma cell line 1246-3A.

An insulin-independent teratoma-derived cell line, called 1246-3A, has been isolated from the adipogenic cell line 1246, which stringently requires insulin for proliferation. The 1246-3A cell line, which can proliferate in the absence of exogenous insulin, produces in its conditioned medium a growth factor similar to pancreatic insulin by its biological and immunological properties. This factor, called "insulin-related factor" (IRF), was purified and iodinated to study its binding to cell surface receptors. 125I-labeled IRF binding to intact 1246-3A cells is lower than to 1246 cells. Cell surface binding can be restored by culturing the 1246-3A cells in the presence of an anti-porcine insulin monoclonal antibody or by acid prewash of the cells prior to performing the binding. Scatchard analysis of binding indicates that IRF secreted by the 1246-3A cells partially occupies high-affinity binding sites on the producer cells. Moreover, insulin monoclonal antibody inhibits the proliferation of the IRF-producing 1246-3A cells, suggesting that these cells are dependent on the secreted IRF for growth in culture. We conclude that the insulin-related factor secreted by the insulin-independent 1246-3A cells stimulates their proliferation in an autocrine fashion.

Animals↗
PubMed1987

EGF receptors on TEA3A1 endocrine thymic epithelial cells.

Thymic endocrine epithelial cell line TEA3A1 can be maintained and passaged in a serum-free WAJC404A medium supplemented with insulin, transferrin, dexamethasone and EGF. EGF not only promotes the growth of these cells but also regulates the activation of phospholipase A2 enzyme activity. The binding of [125I]EGF to the TEA3A1 cells is temperature and time dependent, saturable and can be blocked by excess unlabelled EGF. Two classes of EGF receptors are found on these cells. One with Kd of 5 X 10(-11)M (approximately 3000 sites/cell) and the other with Kd of 5 X 10(-9)M (approximately 30,000 sites/cell). The resynthesis of EGF receptor in TEA3A1 cells after down-regulation requires about 24 hrs and can be blocked by both actinomycin D and cycloheximide.

Animals↗
PubMed1987

EGF inhibits the differentiation of adipocyte precursors in primary cultures.

Adipocyte precursors, isolated from inguinal fat pads of 2-day old NBR rats, proliferate and differentiate in defined medium. EGF stimulates the proliferation of adipocyte precursors with an ED50 of 2 ng/ml and inhibits their differentiation with an ED50 of 0.45 ng/ml. EGF has no effect on differentiated adipocytes. EGF binding studies indicate that adipocyte precursors have two classes of receptors (Kd 4 X 10(-11) M, 4800 receptors/cell and Kd 1.4 X 10(-9) M, 22,000 receptors/cell). TGF-alpha has the same effect as EGF on adipocyte precursors. In contrast, basic and acidic FGF stimulate adipocyte precursor proliferation without inhibiting their differentiation.

Adipose Tissue↗
PubMed1987

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.

Animals↗
PubMed1987

Differentiation of newborn rat adipocyte precursors in defined serum-free medium.

Newborn rat adipocyte precursors, isolated from inguinal fat pads of 2 day-old NBR rats proliferate and undergo adipose differentiation in defined medium in the absence of serum when cultivated on polylysine coated dishes in DME-F12 medium supplemented with fibronectin, insulin, transferrin and FGF. After 7 days in culture in these conditions, 90% of the cells have undergone differentiation as measured by the increase of G3PDH specific activity and by the accumulation of triglycerides in their cytoplasm. In contrast, the cells cultivated in the presence of 10% fetal bovine serum, have a limited ability to differentiate. These results indicate that newborn rat adipocyte precursors from inguinal fat pads do not require the presence of an undefined adipogenic factor in order to differentiate in culture. In contrast, proliferation and differentiation are dependent on the presence of insulin in the culture medium. Moreover, the data presented in this paper show that the rat adipocyte precursor culture represents a rapid and reproducible system for investigating the processes of adipose tissue development and for studying the negative and positive regulators of the adipose differentiation in a controlled environment.

Adipose Tissue↗
PubMed1986

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.

Animals↗
PubMed1985

Tumorigenicity associated with loss of differentiation and of response to insulin in the adipogenic cell line 1246.

The adipogenic cell line 1246, which grows and differentiates in defined medium, stringently requires insulin for both processes. From this cell line, insulin-independent variants were isolated and characterized. Unlike 1246 cells, the variant cell lines proliferate without insulin, have lost their differentiation ability, produce factor(s) able to replace insulin to stimulate 1246 cell growth but not differentiation and are tumorigenic. Because of these properties, this system is appropriate to examine the correlation (if any) between the loss of response to an extra-cellular factor and of ability to differentiate, and between the production of endogenous growth factor and the acquisition of tumorigenic properties.

Adipose Tissue↗
PubMed1980

Effects of a serum spreading factor on growth and morphology of cells in serum-free medium.

A heat-sensitive, trypsin-sensitive factor that promoted growth and spreading of cells in serum-free, hormone-supplemented medium was partially purified from human serum. The major portion of the proteins in these preparations migrated upon SDS-polyacrylamide gel electrophoresis with a mobility consistent with molecular weights between 60,000 and 90,000. The spreading activity, which we have termed serum spreading factor, stimulated growth and spreading of a wide variety of cell types. The serum spreading factor was similar to fibronectin in that it showed an affinity for the plastic cell culture substrate but was shown to be distinct from fibronectin by several criteria. This factor may prove useful in studies of cell attachment and spreading and in studies of the relationship of cell shape and cell proliferation.

Animals↗