Danielle Gaillard

Expression of Peroxisome Proliferator-activated Receptor PPARδ Promotes Induction of PPARγ and Adipocyte Differentiation in 3T3C2 Fibroblasts

Nutritional long chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. The molecular mechanisms implicated in this action of fatty acids remain poorly understood. It has been well established that peroxisome proliferator-activated receptor (PPAR) γ, activated by specific prostanoids, plays a central role in the control of adipocyte gene expression and terminal differentiation. Thus far, the role of PPARδ in the control of adipose tissue mass has remained unclear. Herein, we report the effects of ectopically expressed PPARδ on the control of adipose-related gene expression and adipogenesis of 3T3C2 fibroblasts. Treatment of PPARδ-expressing fibroblasts with fatty acids alone did not stimulate adipogenesis, whereas exposure of cells to a combination of fatty acids and PPARγ activators promoted lipid accumulation and expression of a typical adipocyte program. At the molecular level, activation of PPARδ by fatty acids induced transcription of the genes encoding fatty acid transporter, adipocyte lipid-binding protein, and PPARγ. Subsequent activation of PPARγ by specific agonists appeared to be required to promote terminal differentiation. These data demonstrate that PPARγ gene expression is under the control of PPARδ activated by fatty acids and could explain, at least partially, the adipogenic action of nutritional fatty acids.

Structural and functional characterization of the mouse fatty acid translocase promoter : activation during adipose differentiation

Fatty acid translocase (FAT/CD36) is a cell-surface glycoprotein that functions as a receptor/transporter for long-chain fatty acids (LCFAs), and interacts with other protein and lipid ligands. FAT/CD36 is expressed by various cell types, including platelets, monocytes/macrophages and endothelial cells, and tissues with an active LCFA metabolism, such as adipose, small intestine and heart. FAT/CD36 expression is induced during adipose cell differentiation and is transcriptionally up-regulated by LCFAs and thiazolidinediones in pre-adipocytes via a peroxisome-proliferator-activated receptor (PPAR)-mediated process. We isolated and analysed the murine FAT/CD36 promoter employing C(2)C(12)N cells directed to differentiate to either adipose or muscle. Transient transfection studies revealed that the 309 bp upstream from the start of exon 1 confer adipose specific activity. Sequence analysis of this DNA fragment revealed the presence of two imperfect direct repeat-1 elements. Electrophoretic mobility-shift assay demonstrated that these elements were peroxisome-proliferator-responsive elements (PPREs). Mutagenesis and transfection experiments indicated that both PPREs co-operate to drive strong promoter activity in adipose cells. We conclude that murine FAT/CD36 expression in adipose tissue is dependent upon transcriptional activation via PPARs through binding to two PPREs located at -245 to -233 bp and -120 to -108 bp from the transcription start site.

Trans-differentiation of myoblasts to adipoblasts: triggering effects of fatty acids and thiazolidinediones

Long-chain fatty acids (LCFA) and thiazolidinediones are potent activators of differentiation of preadipose cells. These adipogenic effects are, at least in part, mediated by nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) subfamily. This report describes the effects of these agents on the differentiation pathway of myoblasts. Exposure of C2C12 myoblasts to LCFA or thiazolidinediones prevents the formation of multinucleated myotubes and the expression of specific muscle markers, leading in parallel to the expression of a typical adipose differentiation program. Similar transdifferentiation also occurs in mouse muscle satellite cells maintained in primary cell culture. These observations indicate that PPAR activators, such as LCFA or thiazolidinediones, convert the differentiation pathway of myoblasts into that of adipoblasts. This phenomenon could explain the appearance of adipocytes into muscle which occurs in some pathological states characterized by an increase of fatty acid disposal, such as obesity or mitochondrial myopathy.

Autocrine control of adipose cell differentiation by prostacyclin and PGF2α

The mitogenic-adipogenic effect exerted by arachidonic acid, which leads to terminal differentiation of Ob1771 mouse preadipocytes, has been shown to be (i) blocked by cyclooxygenase inhibitors, (ii) mimicked by a stable analogue of prostacyclin (carbaprostacyclin) and (iii) potentiated by PGF2 alpha. Since these prostanoids are known to be synthesized and secreted by preadipocytes, we have proposed that both prostacyclin as the key mediator and PGF2 alpha as a modulator control the expression of terminal events of adipose conversion by means of an autocrine mechanism (Gaillard, D. et al. and Negrel, R. et al. Biochem. J. (1989) 257, 389-397 and 399-405). In order to test this hypothesis, the release of prostacyclin, characterized under the form of its stable degradation product 6-keto-PGF1 alpha, and that of PGF2 alpha have been studied in the culture medium of Ob1771 cells. A striking increase in the release of 6-keto-PGF1 alpha and to a minor degree of PGF2 alpha was observed when cells were exposed to arachidonic acid as shown by using [3H]arachidonic acid prelabelled cells or by radio-immunoassays. Since antagonists of PGF2 alpha and PGI2 receptors were not available, specific antibodies directed against PGF2 alpha and 6 beta-PGI1, another stable analogue of prostacyclin, were added as neutralizing agents in the culture medium. These antibodies were able to counteract the mitogenic-adipogenic effect of arachidonic acid. Prostacyclin and PGF2 alpha thus appear as autocrine mediators in the process of adipose conversion.

Terminal differentiation of mouse preadipocyte cells : adipogenic and antimitogenic role of triiodothyronine

The role of triiodothyronine (T3) in the differentiation process of Ob1771 mouse preadipocyte cells has been studied under serum-free and hormone supplemented culture conditions which were previously shown to lead to terminal differentiation. In the absence of T3, a dramatic decrease in the adipogenic activity of the culture medium (EC50 = 0.1 nM) could be observed, as indicated 12 days after confluence by the low levels of late markers of differentiation such as adipsin, lipid-binding protein aP2 and glycerol-3-phosphate dehydrogenase as well as the sharp reduction of the number of triacyglycerol-containing cells. This decrease in adipogenic activity was accompanied by a parallel increase of the mitogenic potency of the culture medium. Therefore, T3 appears to be a hormone capable of modulating both proliferation and differentiation of preadipocytes. T3 ceased to be necessary provided the culture medium was supplemented with high concentrations of inducers of differentiation, such as 8-bromo-cAMP or carbaprostacyclin.

GROWTH OF PREADIPOCYTE CELL LINES AND CELL STRAINS FROM RODENTS IN SERUM-FREE HORMONE-SUPPLEMENTED MEDIUM

SummaryOb17 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.

Terminal Differentiation of Mouse Preadipocyte Cells: The Mitogenic-Adipogenic Role of Growth Hormone is Mediated by the Protein Kinase C Signalling Pathway

The role of growth hormone (GH) in the differentiation process of Ob1771 mouse preadipocyte cells has been studied under culture conditions that were serum-free and hormone-supplemented and which were previously shown to lead to terminal differentiation. In the absence of GH, a dramatic decrease in the adipogenic activity of the culture medium could be observed, as indicated 12 days after confluence by the low levels of glycerol-3-phosphate dehydrogenase activity and the sharp reduction of the number of triacylglycerol-containing cells. This decrease in adipogenic activity was accompanied by a parallel loss of the mitogenic potency of the culture medium. Determination of the half-maximal and maximal concentrations of GH required for the restoration of growth and differentiation were identical, 0.5 and 2 nM, respectively. Despite the presence of insulin-like growth factor-I (IGF-I) to substitute for supraphysiological concentrations of insulin and to saturate IGF-I receptor, GH was still required to induce terminal differentiation of a maximal number of cells. However, protein kinase C activators such as prostaglandin F2 alpha, phorbol esters and diacylglycerol were able to mimic GH in promoting a maximal mitogenic-adipogenic response, indicating that the ability of GH to induce diacylglycerol production (Doglio et al., 1989; Catalioto et al., 1990) plays a prominent role in this process. Furthermore, in agreement with the fact that the mitoses which precede terminal differentiation of Ob1771 preadipocytes are strictly controlled by cAMP and only modulated by protein kinase C, terminal differentiation of Ob1771 preadipocytes occurred in the absence of GH upon supplementation with high concentrations of carbaprostacyclin, added as a cAMP-elevating agent or with 8-Br-cAMP, added as a cAMP analogue. It is concluded that the control exerted by GH on terminal differentiation of mouse preadipocytes corresponds to a modulating mitogenic effect mediated through protein kinase C activation and leading to a potentiation of the cAMP and IGF-I mitogenic signalling pathways.

Long chain fatty acids as modulators of gene transcription in preadipose cells

During the last years, it has been clearly established that long-chain fatty acids act as modulators of gene expression in various tissues, such as adipose tissue, intestine and liver. This transcriptional action of fatty acids explains in part adaptation mechanisms of tissues to nutritional changes and especially to high-fat diets by increasing expression of proteins involved in lipid catabolism in liver and fatty acid uptake and utilization in other tissues. It is now clearly demonstrated that some of these transcriptional effects of fatty acids are mediated by activation of specific nuclear hormone receptors, called peroxisome proliferator-activated receptors (PPARs). These findings will be discussed with a special reference to control of gene expression in preadipocytes and adipose tissue development.

Changes in adenosine A1- and A2-receptor expression during adipose cell differentiation.

Two adenosine receptors A1 and A2 are associated with either stimulation (A2) or inhibition (A1) of adenylate cyclase. Using the clonal cell line Ob1771, we have studied the expression of the two receptors during the process of adipose conversion accelerated by exposure to dexamethasone and 3-isobutyl-l-methylxanthine (IBMX) during the first 3 days post-confluence. The effects mediated by the two receptors on preadipocyte differentiation and adipocyte metabolism were also investigated. The two adenosine agonists NECA and PIA were used as preferential agonists of the A2- and A1-receptor, respectively. In preadipose cells (just confluent), both of the mouse clonal line and human primary culture, NECA dose-dependently stimulated cAMP production with a significant higher potency (P < 0.01) than did PIA. In adipose cells (16-day post-confluent) NECA was found to exert a biphasic effect on forskolin-stimulated cAMP production: i.e., NECA was clearly inhibitory in the femto- to picomolar concentration range whereas this effect gradually diminished at higher concentrations. The effect of PIA in 16-day post-confluent adipose cells however, was purely inhibitory on both cAMP production (IC50: 33.52 +/- 0.44 fM) and lipolysis (64% +/- 7%; P < 0.01). These findings were corroborated by Northern blot analysis which revealed A1-receptor mRNA to be exclusively expressed in the mature adipocytes, whereas A2-receptor mRNA gradually declined during the differentiation process except in 16-day post-confluent cells. In addition, NECA significantly enhanced the effect of corticosterone-induced differentiation by 46.8% (P < 0.05) but failed to have any adipogenic potency acting either alone or in concert with carbaprostacyclin (cPGI2). Thus, endogenous adenosine may have a bimodal action on adipose tissue metabolism mediated through stimulatory A2- and inhibitory A1-receptors, respectively, as a function of adipose conversion.

Characterization of ouabain-resistant mutants of the preadipocyte Ob17 clonal line. Adipose conversion in vitro and in vivo.

After exposure to a mutagenic drug (ethylmethanesulfonate), mutant clones of Ob17 preadipose cells resistant to the cytotoxic action of ouabain have been isolated. Their ability to grow in serum-supplemented medium containing greater than or equal to 3 mM ouabain is consistent with the decreased ouabain sensitivity of K+ transport observed in one of these clones (Ob17-OR11). Ouabain-resistant mutants retain their ability to convert into adipose-like cells in vitro with a frequency similar to that of original Ob17 cells. Long-term maintenance of Ob17-OR11 cells in ouabain-enriched medium leads to a higher frequency of adipose conversion, which is linked to a rather specific and limited growth-promoting effect of the drug. Undifferentiated Ob17-OR11 cells, when injected subcutaneously into athymic mouse, give rise in vivo to a fat pad containing ouabain-resistant mature adipocytes. Therefore, Ob17-OR11 clone should be a useful tool to distinguish in vivo between intrinsic and extrinsic factors involved in the differentiation of adipose precursor cells.