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J Pairault

Publications and source records attributed to J Pairault.

At least 37 records · Page 2Linked to original sources
PubMed1990

Analysis of gene expression during adipogenesis in 3T3-F442A preadipocytes: insulin and dexamethasone control.

In the present study, we have investigated dexamethasone and insulin regulation of the expression of adipose-specific mRNA, namely, glycerophosphate dehydrogenase (G3PDH) and adipsin, at different stages of differentiation. During adipose conversion, insulin promotes an accumulation of G3PDH mRNA which is linked to cell differentiation; in fully differentiated cells, insulin is not required to maintain G3PDH gene expression. Differentiating cells in serum deprived medium already exhibit, at day 1, a maximal amount of mRNA encoding for adipsin, which is tenfold decreased by 10 nM of insulin; insulin also exerts a negative effect on the abundance of adipsin mRNA in mature cells. This result indicates that adipsin appears to be a very early marker of adipose conversion, the gene expression of which is down-regulated by the presence of insulin. Dexamethasone (DEX) decreases the G3PDH message at all stages of adipose conversion, while it promotes the accumulation of adipsin mRNA mainly in differentiating cells. In DEX-treated adipocytes, the transcription efficiency of the G3PDH gene is not altered, and reduction to 50% of the message is due essentially to an approximately twofold decrease in its half-life.

Adipose Tissue↗
PubMed1990

[Decrease of gene expression of glycerophosphate dehydrogenase by dexamethasone in differentiated 3T3-F442A cells: antagonism with insulin and antiglucocorticoid RU38486].

Preadipocyte subclones derived from mouse 3T3 cells differentiate into adipocytes; this differentiation is characterized by an increased activity of numerous enzymes required for triglyceride synthesis and/or mobilization. Among these enzymes, the role of glycerophosphate dehydrogenase in the differentiation process has been previously reported. In the present work, we studied the hormonal regulation of glycerophosphate dehydrogenase gene expression (G3PDH) in differentiated 3T3-F442A adipocytes. Dexamethasone (DEX) elicited a 50% decrease in both mRNA content and specific activity of G3PDH. This effect was due to a posttranscriptional event since DEX shortened the half life of the mRNA, whereas it did not modify the transcription rate of this gene. The DEX effect is specific to G3PDH, since the expression of another adipose-specific gene, namely adipsin, is not modified by DEX treatment. Insulin counteracts the inhibitory effect of DEX, mainly by stabilizing the mRNA encoding for G3PDH. The antiglucocorticoid RU38486 is able to reverse DEX inhibition. Latter phenomenon suggests that DEX action on G3PDH gene expression could be mediated by glucocorticoid receptors.

Animals↗
PubMed1989

Adipsin mRNA amounts are not decreased in the genetically obese Zucker rat.

Adipsin gene expression as assessed by mRNA amounts was examined in adipose tissue of genetically obese rats at the onset (16 days of age) or at later stages (30 and 60 days of age) of obesity. Amounts of mRNA were equivalent in obese and lean rats at 16 days of age. In adult rats, we observed a 2-fold decrease in adipsin mRNA in the obese rats compared with control lean rats, which was abolished by weaning the animals on a high-fat diet. Our data show that, in sharp contrast with genetically obese mice, adipsin mRNA is not suppressed in genetically obese Zucker rats.

Adipose Tissue↗
PubMed1989

The antiglucocorticoid RU38486 is a potent accelerator of adipose conversion of 3T3-F442A cells.

We examined the effects of RU38486, a potent glucocorticoid and progestin antagonist, upon several aspects of 3T3-F442A adipocyte differentiation. RU38486 accelerated the onset of differentiation, as monitored by cell morphological changes, accumulation of lipid droplets and widespread increases in the rate of expression of several enzyme adipose markers and specific mRNAs. RU38486, at a maximal concentration of 1 microM, dramatically hastened the emergence of both fatty-acid synthetase (FAS) and glycerol-3-phosphate dehydrogenase (G3PDH) enzyme activities (550% and 450% above control values 4 days after confluence, respectively). RU38486 induction of G3PDH-specific activity ran parallel to an increase in G3PDH mRNA content (2.4-fold the control content 4 days after confluence). Moreover, RU38486-treated cells exhibited enhancement of adenylate cyclase sensitivity to both isoproterenol and ACTH (160% and 350% above control activities 8 days after confluence, respectively). While the level of expression of lipogenic markers reached similar values at the mature stage, RU38486 enabled cells to acquire hypersensitivity in terms of ACTH-stimulated adenylate cyclase activity. Similarly, adipsin gene expression was highly potentiated by the drug at day 15 post-confluence (5-fold the control value). RU38486 responsiveness observed in differentiating 3T3-F442A cells is dependent upon their prior developmental activation; none of the studied markers could be induced by the drug in the undifferentiating 3T3-C2 cell subclone. Finally, this antiglucocorticoid appears to be a useful tool for studies on adipose conversion in vitro; it could permit a re-evaluation of the role of glucocorticoids in the understanding of adipocyte development.

Adenylyl Cyclases↗
PubMed1989

Decreased biosynthesis of actin and cellular fibronectin during adipose conversion of 3T3-F442A cells. Reorganization of the cytoarchitecture and extracellular matrix fibronectin.

Differentiation of 3T3-F442A cells was accompanied by changes in cell morphology, decreased synthesis and assembly of actin and fibronectin. The network of microfilament stress fibers detected with NBD-phallacidin was altered during adipose conversion of 3T3-F442A cells. Parallel to this, the disappearance of fibrillar bundles of extracellular matrix fibronectin was observed by immunofluorescence staining. The pericellular fibronectin content, detected by immunoblotting, strongly diminished during the differentiation process. An altered rate of biosynthesis of both proteins was also measured by [35S]-methionine pulse-labeling and immunoprecipitation. A 4-5-fold decrease in cellular fibronectin synthesis was observed in adipocytes compared to control preadipocytes. Conversely, non-differentiating 3T3-C2 control cells did not reorganize either the cytoskeletal architecture or the extracellular matrix fibronectin in the resting state. These results suggest that the decreased rate of biosynthesis of cell-associated fibronectin is correlated with that of actin. Moreover, both events can essentially be ascribed to differentiation.

Actin Cytoskeleton↗
PubMed1988

Differential effects of retinoic acid upon early and late events in adipose conversion of 3T3 preadipocytes.

When confluent 3T3-F442A cell cultures (Day 0) were grown for 3 days in fetal calf serum-supplemented medium containing isobutyl methyl xanthine and dexamethasone (induction phase) and then shifted to serum-free hormone-defined medium (expression phase), they spontaneously exhibited a sharp rise in lipoprotein lipase activity (LPL); at the peak (Day 7) the LPL activity was about 25 times higher than in control cultures and was further enhanced by insulin. Although this expression of LPL activity was spontaneous, the emergence of glycerophosphate dehydrogenase (G3PDH) activity was completely dependent upon insulin as well as upon the expression of the differentiated phenotype. In committed cells, insulin elicited sustained DNA synthesis associated with limited cell proliferation. The addition of retinoic acid during the phase of expression inhibited insulin-dependent terminal differentiation (i.e., the emergence of G3PDH activity and acquisition of the differentiated phenotype). In addition, retinoic acid counteracted the stimulating effect of insulin upon LPL activity, but affected neither the mitotic process nor the spontaneous emergence of LPL activity. When added during the phase of induction, it prevented the overall process of adipogenic differentiation. Thus, the use of retinoic acid can indicate independent control of the mitogenic and lipogenic effects of insulin following commitment to adipogenic differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Growth and differentiation of 3T3-F442A preadipocytes in three-dimensional gels of native collagen.

Three-dimensional gels of native type I collagen have been used as a substrate for growth and differentiation in 3T3 adipocyte precursors. Such hydrated lattices can support a sustained cell growth leading to several 10-fold increases in cell number within 2 weeks. During this period, the cells condense the hydrated collagen lattice to a tissue-like structure one-fourth of the area of the initial gel. From Days 10 to 12, the cells progressively exhibit morphological characteristics of adipocytes and accumulate lipid droplets as evidenced by Oil Red O staining. Lipoprotein lipase activity appears very early; between Days 8 and 22 it sharply increases 15-fold and then remains stable at a very high level (about 30 nmol/min/10(6) cells). The emergence of glycerophosphate dehydrogenase activity is delayed; it becomes detectable at Day 15 and progressively increases up to 700 nmol/min/10(6) cells at Days 35-40. Thus, this adipose tissue equivalent appears to be a potential model for studying adipocyte function.

Adipose Tissue↗
PubMed1987

Dexamethasone-dependent expression of beta 1-24 corticotropin stimulated adenylate cyclase during adipose conversion of 3T3-F442A cells.

When 3T3-F442A preadipocytes were grown in culture media supplemented with corticosteroid poor fetal calf serum and insulin they differentiated into adipocytes. Glycerophosphate dehydrogenase, a marker of terminal differentiation, developed a 600-fold increase of activity whereas the adenylate cyclase system remained unresponsive to the synthetic ACTH(1-24) analog. In contrast, 3T3-F442A adipocytes, differentiated in the presence of dexamethasone, exhibited an adenylate cyclase activity which was stimulated 4-fold by ACTH(1-24). The stimulation of the adenylate cyclase activity by GTP gamma S remained unchanged (about 20-25-fold) suggesting that the G regulatory coupling protein was not functionally modified by dexamethasone. Binding studies with 125I-ACTH revealed that specific cellular binding could be evidenced in dexamethasone-treated cells while control adipocytes did not exhibit any specific binding of 125I-ACTH. These findings lend support to the hypothesis that the setting off of this ACTH responsiveness in 3T3-F442A cells is regulated by dexamethasone after cells are committed to adipose differentiation.

Adenylyl Cyclases↗
PubMed1987

Control of the adipogenic differentiation of 3T3-F442A cells by retinoic acid, dexamethasone, and insulin: a topographic analysis.

Differentiation of 3T3-F442A adipocytes, monitored by accumulation of neutral lipid and by using the sensitive marker glycerophosphate dehydrogenase, is inhibited by incubation of confluent 3T3-F442A fibroblasts in medium containing retinoic acid or dexamethasone. When added together, dexamethasone (0.25 microM) potentiates about 50-fold the inhibitory effect of retinoic acid (10 microM). Insulin cannot counteract the retinoic acid blockade; however, it can overcome the inhibition of differentiation elicited by dexamethasone. These differential effects of insulin are used for characterizing the adipose conversion cycle. We describe cell culture conditions where terminal differentiation of 3T3-F442A preadipocytes is achieved by low, physiological levels of insulin. They include the switch from a high-serum medium containing isobutyl methyl xanthine and dexamethasone to a serum-free, hormone-supplemented medium. The data reported establish the existence of two successive states for commitment to adipogenic differentiation: a first commitment point (CA) to differentiation which requires serum adipogenic factors, and a second commitment point (CH) controlled by lipogenic hormones, namely insulin, after which terminal maturation can resume. We demonstrate that retinoic acid can prevent and interrupt differentiation by blocking the cells within the early differentiation phase.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Dihydrocytochalasin B promotes adipose conversion of 3T3 cells.

Differentiation of preadipose 3T3-F442A cells into adipose cells is accelerated by the addition of dihydrocytochalasin B. The effect of the drug on 3T3-C2 cells is more marked: these cells are practically unable to differentiate in the absence of H2CB but a long-term exposure to the drug enables the cells to accumulate lipid droplets in medium supplemented with fetal calf serum and insulin. During their differentiation under these conditions the 3T3-C2 cells develop markers typical of adipose cells: glycerophosphate dehydrogenase, ATP-citrate lyase, fatty acid synthetase and glycerophosphate acyltransferase.

Adipose Tissue↗
PubMed1979

A study of the adipose conversion of suspended 3T3 cells by using glycerophosphate dehydrogenase as differentiation marker.

The adipose conversion of 3T3 cells has been examined in stabilized suspension cultures. In 3T3-F442A cells, glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: NAD(+) 2-oxidoreductase, EC 1.1.1.8), a key enzyme in triglyceride synthesis, increases in specific activity by more than 5000-fold and can be used as a sensitive and precise measure of the conversion. The conversion depends on an adipogenic factor present in the serum, and this factor can be assayed by the cellular enzyme response. If the cells are growing at the time they receive the adipogenic factor, the enzyme response does not become detectable until after 3 days, during which the cells first enter a resting state. If the cells are resting at the time they receive the adipogenic factor, the enzyme activity begins to increase in 24 hr or less. Only resting cells seem susceptible to the reprogramming of their differentiated state necessary for the adipose conversion. Once the conversion begins, the increase in enzyme activity is exponential over at least 2 orders of magnitude. When cells in a resting state begin the adipose conversion, their biosynthetic processes are accelerated: the rate of protein synthesis increases, they accumulate cell protein, and they may replicate their DNA and divide. The cell multiplication is not essential for adipose conversion but is a form of clonal selection that increases the proportion of adipose cells relative to nonadipose cells.

Adipose Tissue↗
PubMed1976

[The coupling of beta1-24-corticotropin to the adenylate-cylase system in rat adipocytes. Evidence for hormone-nucleotides interaction (author's transl)].

The general aim was to define some of the most important parameters involved in the coupling step between the synthetic analog of adrenocoricotropin hormone (beta1-24-corticotropin tetracosa peptide) and the catalytic unit of the adenylate-cyclase system of fat cells. These studies were performed with a purified plasma membrane fraction from rat adipose tissue. In this regard, some effects of ions, pH, and nucleotides (ATP nad GTP) on this hormone sensitive system were studied A simple model based on a random association process of reactants yeilded a statisfactory approximation of the kinetic data. In contract to results obtained by two other groups, which were analyzed by De Haen, no evidence was found for a regulation of the adenylate-cyclase activity by the adenosine triphosphate which was not complexed to magnesium...

Adenosine Triphosphate↗