Progesterone Stimulates Adipocyte Determination and Differentiation 1/Sterol Regulatory Element-binding Protein 1c Gene Expression

POTENTIAL MECHANISM FOR THE LIPOGENIC EFFECT OF PROGESTERONE IN ADIPOSE TISSUE*

Danièle LacasaDagger , Xavier Le Liepvre, Pascal Ferre, and Isabelle Dugail§

From the Dagger  Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Médecine Paris Ouest, Université René Descartes, 75270 Paris, France and INSERM U 465, Nutrition, Métabolisme, Obésité, 15 Rue de l'École de Médecine, 75006 Paris, France

Received for publication, September 19, 2000, and in revised form, December 14, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fatty acid synthase (FAS), a nutritionally regulated lipogenic enzyme, is transcriptionally controlled by ADD1/SREBP1c (adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c), through insulin-mediated stimulation of ADD1/SREBP1c expression. Progesterone exerts lipogenic effects on adipocytes, and FAS is highly induced in breast tumor cell lines upon progesterone treatment. We show here that progesterone up-regulates ADD1/SREBP1c expression in the MCF7 breast cancer cell line and the primary cultured preadipocyte from rat parametrial adipose tissue. In MCF7, progesterone induced ADD1/SREBP1c and Metallothionein II (a well known progesterone-regulated gene) mRNAs, with comparable potency. In preadipocytes, progesterone increased ADD1/SREBP1c mRNA dose-dependently, but not SREBP1a or SREBP2. Run-on experiments demonstrated that progesterone action on ADD1/SREBP1c was primarily at the transcriptional level. The membrane-bound and mature nuclear forms of ADD1/SREBP1 protein accumulated in preadipocytes cultured with progesterone, and FAS induction could be abolished by adenovirus-mediated overexpression of a dominant negative form of ADD1/SREBP1 in these cells. Finally, in the presence of insulin, progesterone was unable to up-regulate ADD1/SREBP1c mRNA in preadipocytes, whereas its effect was restored after 24 h of insulin deprivation. Together these results demonstrate that ADD1/SREBP1c is controlled by progesterone, which, like insulin, acts by increasing ADD1/SREBP1c gene transcription. This provides a potential mechanism for the lipogenic actions of progesterone on adipose tissue.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fatty acid synthase (FAS)1 is a multifunctional enzyme that catalyzes all the steps in the synthesis of long chain fatty acids from malonyl CoA. As a key lipogenic enzyme, FAS is expressed mainly in liver and adipose tissue, where it turns dietary carbohydrates to fat. In these tissues, the transcription of the FAS gene is under nutritional control, leading to commensurately regulated activity of the enzyme. Briefly, feeding a high carbohydrate diet induces, whereas fasting or consuming a high fat diet decreases, FAS gene expression. Insulin (1), glucose (2), fatty acids (3-5), and cAMP (6) are direct effectors of the nutritional regulation of FAS, exerting coordinated effects on FAS gene transcription at the promoter level.

High levels of FAS expression are also found in some tumor cells of breast cancer (7-9) and derived cell lines, where it is associated with a worsened prognosis (10). cDNA for FAS has been cloned initially as a progestin-responsive mRNA by differential screening of the MCF7 breast cancer cell line (11), and further studies have established that FAS expression was induced by progestins in the normal mammary gland also (12). The mechanism of FAS induction by progestins relies primarily on transcriptional activation, as shown by run-on studies (13). However, the direct implication of the progesterone receptor in the FAS gene-stimulated transcription has not been clearly established.

Recently, significant progress has been made in the elucidation of the mechanisms of FAS gene regulation. Particularly, the role of a key transcription factor, ADD1/SREBP1c (adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c), has been uncovered. ADD1/SREBP1c, a member of the basic helix loop helix family of transcription factors (14, 15), has been identified as a potent activator of the FAS promoter in cultured cells (16) and in transgenic mice (17). The ability of ADD1/SREBP1c to transactivate the FAS gene seems to be physiologically relevant, because ADD1/SREBP1c is induced by insulin in primary hepatocytes (18) and adipose cell lines (19), down-regulated by cAMP and glucagon in hepatocytes (18), and nutritionally regulated in the liver of mice (20). For these reasons, it has been proposed that ADD1/SREBP1c is the mediator of insulin action on FAS gene expression (21).

In the light of these new insights, the present study was designed to investigate the mechanisms of FAS gene regulation by progesterone. We found that progesterone is able to stimulate ADD1/SREBP1c expression, making it likely that progesterone-induced stimulation of FAS expression is exerted through activation of the same transcription factor as insulin, ADD1/SREBP1c.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Fetal bovine serum was obtained from Life Technologies, Inc. Phenol red-free DMEM containing 4.5 g/liter glucose and DMEM, Ham's F12 (50:50 mix) were obtained from Sigma.

Animals-- Procedures with experimental animals were authorized and followed the guidelines of the Ministry of Agriculture (France) (authorization 006614). Female Harlan Sprague-Dawley rats (125-150 g) were killed by decapitation, and parametrial fat pads were removed aseptically.

Cell Culture and Adenovirus-mediated Gene Transfer-- Cell preparation and culture were performed as described in Ref. 22. Briefly, preadipocytes obtained from the stroma-vascular fraction of adipose tissue by collagenase digestion were plated at a density of 1-2 × 104 cells/cm2 in 8% fetal bovine serum/DMEM. After 12 h, cultures were washed and fed with 8% fetal bovine serum/DMEM. Medium was changed every other day. At confluence (3 days post-plating), cells were allowed to differentiate in DMEM/Ham's F12 containing 5 µg/ml insulin, 10 µg/ml transferrin, and 200 pM T3 (ITT medium) in the absence of serum, as described in Ref. 22. Early differentiating preadipocytes (day 2 post-confluence) were treated with progesterone in serum-free medium for 24 h unless otherwise stated. Progesterone treatment was provided to cell dishes as an ethanol solution. An equivalent volume of ethanol alone (never exceeding 0.1% v/v) was added in untreated controls.

In some experiments, cells in serum-free medium were infected (100 plaque-forming units/cell) with an adenovirus encoding a dominant negative form of ADD1 under the control of the cytomegalovirus promoter (ad-DN) or with a control empty virus (ad-null) as described (23). 16 h post-infection, the medium was changed, and progesterone was added or not for the next 24 h. MCF7 cells were obtained from ATCC (Manassas, Va) and cultured as recommended by the supplier. Progesterone treatment was performed in serum-free medium 1 day after confluence.

RNA Isolation and Northern Blot Analysis-- Total RNA was isolated from 3-5 culture dishes (90 mm) by the guanidium thiocyanate method, as described in Ref. 24. RNAs were then separated on formaldehyde-agarose gels and transferred onto nylon membranes (Hybond N+, Amersham Pharmacia Biotech). Hybridization was as described previously (23), and blots were washed in 0.1% SSC, 0.1% SDS at 60 °C. Hybridization probes were as follows. ADD1/SREBP1c probe was a rat ADD1 cDNA fragment encompassing the first 403 amino acids of the ADD1 protein cloned in pSVSPORT1 (provided by B. Spiegelman, Boston, MA). The SREBP1a probe was a polymerase chain reaction fragment described previously (18). The plasmid encoding full-length MTII is described in Ref. 25 and was a kind gift of Dr. P. Hainaut (International Agency for Research on Cancer, Lyon, France). An RNA probe for 18 S was used for normalization of the results.

Run-on Experiments-- The effect of progesterone on gene transcription was assessed by run-on experiments as described in Ref. 26.

Western Blot Analysis-- Nuclear extracts and crude membranes were prepared from cultured preadipocytes as described previously (27), separated on 8% polyacrylamide-SDS gels, and electrotransferred to nylon membranes (Amersham Pharmacia Biotech). SREBP1 was probed using a 5 µg/ml dilution of the polyclonal antibody IgG-2A4 (ATCC). A C/EBPbeta antibody (SC130, Santa Cruz Biotechnology) was also used as a control for the specificity of progesterone effect. The blots were revealed using the ECL system (Pierce), as described by the manufacturer.

Statistical Analysis of the Results-- The effect of progesterone was evaluated by Dunnet's Post test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The stimulatory effect of progesterone on FAS gene expression was originally described in MCF7, a breast carcinoma cell line. As a first step, the ability of progesterone to induce FAS mRNA in cultured preadipocytes was compared with the MCF7 cell system. A model of cultured preadipocytes isolated from female parametrial adipose tissue was chosen because of the presence of well characterized progesterone receptors on these cells (28) and the induction of FAS activity upon differentiation. MCF7 cells or early differentiating preadipocytes (day 2 post-confluence) were incubated for 24 h in serum-free medium in the presence of increasing concentrations of progesterone, and FAS mRNA levels were assessed by Northern blot analysis. Fig. 1 shows that progesterone dose-dependently increases FAS mRNA in both MCF7 cells and preadipocytes. We have also observed that induction of FAS mRNA by progesterone in preadipocytes was accompanied by a significant increase in lipogenic activity, as assessed by the conversion of [U-14C]glucose into lipids (data not shown). This establishes that the system of primary cultured preadipocytes behaves as the MCF7 cell line and is suitable to study progesterone regulation of FAS activity.



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Fig. 1.   Northern blot analysis of FAS mRNA in MCF7 cells (left) or in primary cultured preadipocytes (right) treated for 24 h in the presence of increasing concentrations of progesterone, as described under "Experimental Procedures." For MCF7 cells, a typical dose-response curve obtained from two experiments is shown. For preadipocytes, each bar represents the mean value ± S.E. from three independent cultures. The effect of progesterone is statistically significant at the p < 0.05 level by Dunnet's Post test. C, control.

In the light of the importance of ADD1/SREBP1c in insulin-mediated regulation of FAS gene expression, the effect of progesterone on SREBPs was next investigated. Fig. 2 shows that using an ADD1/SREBP1 cDNA probe, a dose-dependent induction of progesterone on ADD1/SREBP1 mRNA levels could be detected in preadipocytes. The EC50 for the progesterone effect was 90 nM, in good relation to that observed for the induction of FAS gene expression (220 nM) by the hormone (see Fig. 1). The ADD1/SREBP1 gene can be transcribed from two alternate promoters, generating two different (ADD1/SREBP1c and SREBP1a) transcripts of approximately the same size. Because the ADD1/SREBP1 probe used did not distinguish between the 1c and the 1a transcript, we also used a probe specific for SREBP1a to examine the effect of progesterone. We show in Fig. 2 that this 1a probe generated very weak hybridization signals that required a long time exposure and did not reveal any effect of progesterone. This suggests that in preadipocytes, the expression of the 1a isoform is low and unaffected by hormone treatment. Thus we concluded that the progesterone-induced ADD1/SREBP1 mRNA represented mainly the ADD1/SREBP1c transcript. For these reasons, the signals generated by the ADD1/SREBP1 probe were identified as ADD1/SREBP1c mRNA in quantitative analysis in Fig. 2B. Because it has been demonstrated that progesterone was able to modify cholesterol trafficking at the plasma membrane, by virtue of its amphiphile properties, we also probed the blots for the mRNA encoding SREBP2, the cholesterol-sensitive isoform of SREBP that is derived from an independent gene. No hybridization signal for SREBP2 could be detected by Northern blot analysis, and no induction of SREBP2 mRNA could be seen upon progesterone treatment (data not shown). These data indicate that progesterone selectively stimulates the expression of the ADD1/SREBP1c mRNA, but not SREBP1a or SREBP2. The time course of the induction of ADD1/SREBP1c by progesterone was also examined (Fig. 2C). ADD1/SREBP1c mRNA levels increased between 0 and 24 h in control cells treated with ethanol only, reflecting the differentiation-dependent expression of ADD1/SREBP1c during adipose conversion. In progesterone-treated cells, ADD1/SREBP1c induction was more marked than in controls, indicating an effect of progesterone beginning after 6 h of treatment, sustained for at least a 24-h period.



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Fig. 2.   Effect of progesterone on ADD1/SREBP1 mRNA levels in primary cultured preadipocytes. A shows Northern blots from primary cultured preadipocytes treated for 24 h with progesterone. B presents quantification of the dose-response curves for ADD1/SREBP1c mRNA. Values were obtained from at least four independent cultures. The effect of progesterone is statistically significant at the p < 0.05 level by Dunnet's Post test. C, control. C shows time course experiments of the effect of progesterone on ADD1/SREBP1c mRNA. Progesterone (1 µM) was added as an ethanol solution, and ethanol only was added in control cells. A typical experiment of two is shown.

Among well characterized progesterone target genes is the Metallothionein IIA (MT-IIA) (29). Thus, we next compared the effects of progesterone on MT-IIA and ADD1/SREBP1c mRNA levels in the MCF7 cell line. Only background levels of hybridization could be detected in MCF7 cells with the SREBP1a-specific probe (data not shown), suggesting that, as in preadipocytes, the signals obtained with the ADD1/SREBP1 probe were generated by ADD1/SREBP1c. Fig. 3 shows that ADD1/SREBP1c mRNA is expressed, albeit at low levels, in MCF7 cells in the absence of progesterone. This basal level of ADD1/SREBP1c mRNA can be dose-dependently induced by progesterone, in a very similar manner to the induction observed for the mRNA of MT-IIA, a well known progesterone-responsive gene. EC5O values were 110 and 70 nM for ADD1/SREBP1c and MT-IIA mRNAs, respectively. The time course of progesterone induction of ADD1/SREBP1c mRNA was also very similar to that reported in preadipocytes, with a stimulatory effect detectable after 6 h (data not shown). Thus these results demonstrate that ADD1/SREBP1c, like MT-IIA, is a progesterone-inducible gene. To further investigate the mechanism by which ADD1/SREBP1c mRNA concentrations are increased by progesterone, we measured gene transcription rates in run-on experiments. Fig. 4 shows that the ADD1/SREBP1 transcription rate is increased by progesterone treatment in both MCF7 cells and preadipocytes. Moreover, the amplitude of the progesterone effect on transcription (6.5-fold in MCF7 cells and 3.2-fold in preadipocytes) closely parallels that observed for steady state mRNA levels (9.7-fold in MCF7 cells and 3.1-fold in preadipocytes). Thus progesterone acts primarily at the transcriptional level in the regulation of ADD1/SREBP1c.



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Fig. 3.   Effect of progesterone on ADD1/SREBP1c and MT-IIA mRNA levels in MCF7 cells. MCF7 cells were treated with increasing concentrations of progesterone for 24 h as described under "Experimental Procedures." Representative blots are shown on the left, and quantification of the signals (normalized to 18 S) is shown on the right. C, control.



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Fig. 4.   Effect of progesterone on ADD1/SREBP1c transcription rate. MCF7 cells (upper panel) and preadipocytes (lower panel) were treated for 24 h with 10 µM progesterone (Pg) as described under "Experimental Procedures," and nuclei were prepared. Labeled RNAs were hybridized to 10 µg of dot-blotted plasmids. The control plasmid was the pSVsport1 vector in which ADD1/SREBP1 is cloned. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plasmid (full-length rat glyceraldehyde-3-phosphate dehydrogenase cDNA in pUC9) was used as a negative control. Autoradiograms show representative results obtained with two independent preparations of nuclei. Quantification of the blots is shown on the right.

To establish the link between the induction of ADD1/SREBP1c mRNA expression by progesterone and the induction of the FAS gene, an ADD1/SREBP1c target, we first investigated whether progesterone was able to increase the amount of the ADD1/SREBP1 protein in the cells. Crude membranes and nuclear extracts of preadipocytes were prepared and probed with a monoclonal anti-SREBP1 antibody in Western blots. As shown in Fig. 5A, cells treated with progesterone for 24 h showed increased levels of the precursor form in membrane fractions and higher contents of the active nuclear (cleaved) form of the ADD1/SREBP1 protein. As a control for the specificity of the progesterone effect, nuclear extracts were also probed with a C/EBPbeta antibody, which did not reveal any change in C/EBPbeta protein content. Collectively, these data demonstrate that progesterone, by increasing transcription rates of the gene, raises the levels of ADD1/SREBP1 mRNA in cells, which in turn leads to the accumulation of the membrane-bound precursor and mature nuclear forms of the ADD1/SREBP1 protein.



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Fig. 5.   The effect of progesterone on the FAS gene is mediated through ADD1/SREBP1. A, Western blot analysis of nuclear extracts and crude membrane fractions from preadipocytes treated or not with progesterone. Blots were probed with a monoclonal SREBP1 antibody. A C/EBPbeta antibody was used as a control for the specificity of the progesterone effect in nuclear extracts. This antibody recognized several C/EBPbeta isoforms, which were not resolved on a 8% polyacrylamide-SDS gel. Similar results were obtained in three independent preparations of nuclear extracts and membranes. kD, kilodaltons. B, effect of adenovirus-mediated overexpression of a dominant negative form of ADD1/SREBP1c on progesterone (Prog)-induced FAS gene expression. Preadipocytes from parametrial adipose tissue from females (1 day after confluence) were infected with 100 plaque-forming units/cell of an adenovirus encoding or not (ad-null) a dominant negative form of ADD1 (ad-DN). 16 h post-infection, the serum-free medium was supplemented or not with 10-6 M progesterone. Total RNA was extracted from five pooled dishes 24 h later. A representative blot of two independent experiments is shown.

To definitely establish the link between the induction of the nuclear mature form of ADD1/SREBP1 and FAS gene expression, we examined whether overexpression of a dominant negative form of ADD1/SREBP1c in progesterone-treated cells was able to block FAS gene induction. Preadipocytes were thus infected with an adenovirus overexpressing a dominant negative mutant for ADD1, described in Ref. 23, or a control null vector and then stimulated by progesterone. The dominant negative form of ADD1 has been demonstrated to sequester endogenous ADD1/SREBP1c in cells, by dimerizing with the wild type protein and preventing DNA binding (30). Results in Fig. 5B show that the 4-fold induction of FAS expression by progesterone that occurred in cells infected by the null vector was abolished when cells were infected by the adenovirus encoding the dominant negative form of ADD1. This demonstrates that ADD1/SREBP1 is required for progesterone-induced FAS gene expression.

Finally, we investigated the relationship between insulin and progesterone in the control of ADD1/SREBP1c expression. Preadipocytes, which are known to acquire insulin sensitivity upon differentiation, were induced to fully differentiate in the presence of insulin (day 7 post-confluence), and the ability of progesterone to induce ADD1/SREBP1c expression was tested. Fig. 6 shows that when cells differentiated in the presence of insulin, the ability of progesterone to induce ADD1/SREBP1c expression was abolished. The lack of progesterone effect was not due to a general desensitizing effect of insulin to progesterone, because MT-IIA gene expression still responded normally in insulin-differentiated cells. In agreement, Fig. 6 also shows that progesterone response was restored when cells were allowed to fully differentiate in the presence of insulin and then deprived of the hormone 24 h before progesterone treatment. Thus this experiment shows that insulin and progesterone exert nonadditive effects on ADD1/SREBP1c gene expression.



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Fig. 6.   Interaction between insulin and progesterone on ADD1/SREBP1c, MT-IIA, and FAS mRNA levels in differentiated preadipocytes. Preadipocytes from parametrial adipose tissue from female rats were differentiated for 7 days post-confluence in the presence of insulin. At day 7 post-confluence, insulin was withdrawn for 24 h or not, and progesterone treatment (1 µM) was then performed as above. A representative of two experiments is shown.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we provide evidence that the expression of ADD1/SREBP1c mRNA is stimulated by progesterone. We show that high levels of ADD1/SREBP1c mRNA can be induced upon progesterone stimulation in both the MCF7 cell line and primary cultured preadipocytes. We show that the effect of progesterone selectively involves the ADD1/SREBP1c isoform, which can be functionally distinguished from other cholesterol-regulated SREBP isoforms (for review, see Ref. 31) and which preferentially targets the expression of lipogenic genes in transgenic mice (17). We also demonstrate that the progesterone effect on ADD1/SREBP1c is exerted through increased gene transcription and leads to increased levels of the protein product, especially the membrane-bound precursor and the mature nuclear active form of ADD1/SREBP1c. Changes in the levels of active SREBPs in the nucleus implicate complex post-translational control, such as regulated proteolytic cleavage of the membrane-bound precursor (31), and also activation through phosphorylation (32). Our results showing the accumulation of both the high molecular weight precursor in membranes and the mature cleaved forms of ADD1/SREBP1 in nuclei upon progesterone treatment might suggest that proteolytic cleavage is not rate-limiting in the control of ADD1/SREBP1c. However, we cannot exclude the possibility that progesterone might also activate (directly or indirectly) ADD1/SREBP1c transcriptional activity. Our present data agree with a previous report (33) showing that the induction of several lipogenic genes was paralleled by increased levels of the mRNAs encoding SREBP1 and 2 and showing the accumulation of SREBP1 in the nucleus of human prostate cancer cells upon androgen treatment. These observations suggest that the SREBP factors might be a common control point through which sex hormones might signal for metabolic effects. Here we provide direct evidence that nuclear ADD1/SREBP1c protein is the factor through which the stimulatory effect of progesterone on FAS is exerted. This is supported mainly by the fact that the progesterone effect on FAS mRNA can be abolished by adenovirus-mediated overexpression of a dominant negative form of ADD1 within the cells, demonstrating that transcriptionally active ADD1/SREBP1c is required for the progesterone action on the FAS gene. This suggests that the effect of the hormone is not mediated through progesterone receptor target DNA sequences, which have not been found in the 5' regulatory region of the FAS gene. Moreover, in the context of the regulation of ADD1/SREBP1c expression by insulin, the present study points out that the expression of ADD1/SREBP1c might be a key control point to which several hormone signaling pathways might converge for the regulation of lipogenesis. This is supported by our data showing that progesterone can act as insulin, to stimulate ADD1/SREBP1c expression. The effect of these two hormones appear to be mutually exclusive, at least in the cultured preadipocyte system, which exhibits both insulin and progesterone sensitivity. This is in agreement with the fact that both hormones act with similar kinetics on ADD1/SREBP1c mRNA, through the same mechanism, i.e. stimulation of ADD1/SREBP1 gene transcription (present data and 19). The finding of the present study that progesterone might be able in some situations to replace insulin for the control of ADD1/SREBP1c expression might have some physiological significance. During late pregnancy, an insulin-resistant state (34, 35) develops, and maternal glucose utilization is reduced, hence sparing carbohydrates for the rapidly growing fetus. However, lipogenesis in the parametrial adipose tissue remains active (36). In this context, the induction of ADD1/SREBP1c by progesterone might serve to maintain lipogenesis in maternal adipose tissue, to preserve energy fat stores required for lactation, a highly energy-consuming process. On the other hand, it has been observed that progesterone treatment of diabetic rats was able to induce lipogenesis in fat (37), further suggesting that in the absence of insulin, progesterone can serve as an alternative stimulating factor of adipose tissue lipogenesis. Thus, our present observation that ADD1/SREBP1c is a progesterone-regulated transcription factor might provide a mechanism for the understanding of the physiological regulation of lipogenesis by progesterone.


    FOOTNOTES

* This work was supported by grants from the European community program, FAIR 97/3011 and ARC 5858.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 33 1 42 34 69 22; Fax: 33 1 40 51 85 86; E-mail: idugail @bhdc.jussieu.fr.

Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M008556200


    ABBREVIATIONS

The abbreviations used are: FAS, fatty acid synthase; ADD1, adipocyte determination and differentiation 1; SREBP, sterol regulatory element-binding protein; DMEM, Dulbecco's modified Eagle's medium; MT-IIA, Metallothionein IIA; C/EBP, CAAT enhancer-binding protein.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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