Ginette Serrero

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.

Prostaglandin F2α inhibits the differentiation of adipocyte precursors in primary culture

Abstract Influence of arachidonate metabolite pathway on adipose differentiation was investigated using primary culture of adipocyte precursors in defined medium. Treatment of the cells with cyclooxygenase inhibitors stimulates adipose differentiation by at least 2-fold. Among the various arachidonate metabolites tested, only prostaglandin F2α (PGF2α) was found to inhibit the differentiation of adipocyte precursors in a dose dependent fashion. Other eicosanoids tested did not have any effect. A 50% inhibition of adipose differentiation was observed with a dose of PGF2α of 3×10 −9 M to 7×10 −9 M according to the strain of rats used. Maximal inhibition occurred at PGF2α concentrations equal or higher than 10 −8 M. PGF2α inhibited not only the expression of late markers of adipose differentiation such as G3PDH and triglycerides accumulation but also the mRNA expression of early markers of adipose differentiation such as clone 154, lipoprotein lipase and ap2 gene. These results indicate that PGF2α represents a physiological negative modulator of adipose differentiation.

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

SummaryNewborn 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.

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

SummaryThe 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.

Characterization of calcium-independent cytosolic phospholipase A2 activity in the submucosal regions of rat stomach and small intestine

This study was undertaken to compare the calcium-independent phospholipase A2 (PLA2) activities in the cytosols of twelve rat tissues and to determine whether their activities were distinct. 1-O-Alk-1′-enyl-2-[14C]-oleoyl-sn-glycero-3-phosphocho-line (PlsC) and 1-O-Alk-1′-enyl-2-[14C]oleoyl-sn-glycero-3-phosphethanolamine (PlsE) were synthesized and used as substrates, instead of phosphatidyl compounds, to exclude hydrolysis by cytosolic PLA1 activity that could be present in some of the cytosolic preparations. For each tissue, we examined substrate specificity, pH optimum, and effect of adenosine triphosphate (ATP) and ATP analogues. PLA2 activity was detected in eleven out of the twelve issues examined. Based on substrate specificity and pH optimum, cytosolic calcium-independent PLA2 were classified in three groups. The first group, which included PLA2 from small intestine, stomach and spleen, had the highest specific activity with PlsC as substrate (1253, 309 and 75 nmol/mg protein/hour, respectively) and an optimal pH at 6.5. Activity with PlsE as substrate was much lower (20–37%) than with PlsC. The second group of PLA2 activities included the cytosolic activities from thymus, lung, liver and pancreas that showed lower specific activities for both substrates (14–23 nmol/mg protein/hour with PlsC) and had a broader optimal pH range of 6.1 to 7.5. The cytosols from brain, kidney, heart and muscle comprised the third PLA2 group that was found to have a higher specific activity with PlsE (5–20 nmol/mg protein/hour) than PlsC and an optimal pH range from 7.4 to 7.9. Since the highest specific activity was found in the cytosol from small intestine, this PLA2 was examined further. PLA2 activity was found to be equally distributed in the cytosol of the submucosal portion of duodenum, jejunum and ileum with an optimal pH of 6.1 and a 5-fold higher activity with PlsC than PlsE as substrate. Moreover, this PLA2 activity was inhibited by treatment with detergents. These results indicate the presence in the submucosal portion of the intestine of a calcium-independent cytosolic PLA2 with a high specific activity toward PlsC and properties distinct from those described for the PLA2 found in the intestinal brush-border.

Fibroblast Growth Factor Inhibits Proliferation of a Highly Tumorigenic Insulin-Independent Teratoma-Derived Cell Line

The present paper examines the effect of basic fibroblast growth factor (bFGF) on the proliferation of teratoma-derived cell lines having increased tumorigenic properties isolated from the non-tumorigenic adipogenic cell line 1246. Although FGF is a mitogen for the non tumorigenic 1246 cells and for the moderately tumorigenic 1246-3A cells derived from the 1246 cells, bFGF inhibits the proliferation and DNA synthesis of the highly tumorigenic PC cells starting at concentration as low as 30 pg/ml. The inhibitory effect of FGF on PC cell growth is irreversible as demonstrated by the inability of the cells to resume proliferation once FGF is removed from the culture medium. Comparison of 125I-bFGF binding to the three cell lines was performed. Based on the Scatchard analysis of the binding data, PC cells display only low affinity class of FGF binding sites whereas 1246 and 1246-3A cells presented also high affinity binding sites. The inhibitory effect of FGF on PC cells did not go through activation of a PKC mediated pathway, which is also known to inhibit PC cell proliferation, since FGF inhibition of PC cell growth was still apparent after PKC down regulation. FGF was still able to transiently stimulate the expression of mRNA for early growth associated genes as demonstrated by c-myc and c-fos expression, although it inhibited cell proliferation on PC cells. Our data demonstrate that the highly tumorigenic teratoma cells acquire an inhibitory response for a factor which is growth stimulatory to non-tumorigenic and moderately tumorigenic cells from which they are derived.

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.

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.

Nucleotide sequence of the C-terminal region of the mouse epidermal growth factor receptor and expression in teratoma-derived cell lines with increased tumorigenic properties

The isolation of a cDNA corresponding to a portion (amino acid 943 to 1073) of the cytoplasmic domain of the mouse EGF receptor surrounding the auto phosphorylation sites was obtained by using the reverse transcriptase polymerase chain reaction (RT-PCR) approach. Deduced amino acid sequence of mouse EGF receptor (EGFr) shows a 92% and 76% homology to corresponding regions in the human and the chicken EGFr, respectively. This cDNA was used to develop a sensitive RNase protection assay to investigate EGF receptor mRNA expression in mouse C3H teratoma derived cell lines with increased tumorigenic properties which display a progressive decrease of EGF binding and response. The results show that increased tumorigenicity was not accompanied by a change in EGF receptor mRNA expression. Moreover, they indicate that the RNase protection assay developed using the probe described here is a sensitive approach to investigate EGF receptor expression in murine cells.

Assay for adipose differentiation using primary culture of adipocyte precursors

This paper describes a method to establish primary and secondary cultures of adipocyte precursors isolated from inguinal fat pads of 48-hr-old rats in defined medium. The culture medium consists of DME-F12 medium supplemented with fibronectin, insulin, transferrin, and fibroblast growth factor. Data presented indicate that 90% of the cells plated in the 4F medium differentiate in 7 d. These cultures provide appropriate differentiation assay systems to characterize regulators of differentiation acting on normal cells derived from the adipose tissue.