©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence of Increased Glyceraldehyde-3-phosphate Dehydrogenase and Fatty Acid Synthetase Promoter Activities in Transiently Transfected Adipocytes from Genetically Obese Rats (*)

(Received for publication, June 6, 1994; and in revised form, October 13, 1994)

Violaine Rolland (§) Isabelle Dugail Xavier Le Liepvre Marcelle Lavau

From the From INSERM U177, Unité de Recherches sur la Physiopathologie de la Nutrition, Institut Biomédical des Cordeliers, 15, Rue de l'École de Médecine, 75006 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5` deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cisacting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.


INTRODUCTION

The obesity of the Zucker rat first described by Zucker and Zucker (1) is inherited as an autosomal recessive disorder. It is due to a single gene defect, recently mapped to chromosome 5(2) , that remains totally unknown. The disease evolves from early phenotypic alterations expressed only in adipose tissue to a complex metabolic syndrome including hyperlipidemia and insulin resistance, in close resemblance to human obesity for which the Zucker rat provides a valuable model. As an approach to the nature of the mutation we and others (3) have attempted to delineate the primary functional effect of the genetic lesion. One very earliest trait to develop in the mutant pup, prior to hyperphagia and to hyperinsulinemia, is an increase in fat cell size concomitant with an adipose tissue-specific overexpression of a subset of lipid storage genes such as lipoprotein lipase, malic enzyme, fatty acid synthetase, Glut 4, and glyceraldehyde-3-phosphate dehydrogenase (4, 5, 6, 7) . Transcription rate studies and Southern analysis led to the conclusion that these genes were the target rather than the site of the mutation(5, 7) , suggesting that the coordinate over transcription of lipogenic genes in adipocytes from mutant pups was triggered by (a) fa genotype-dependent trans-acting factor(s). To test this hypothesis, we developed the transfection of adipocytes with plasmids containing GAPDH (^1)and FAS proximal promoter regions fused to CAT, providing the demonstration that transiently transfected rat adipocytes are a well suited system to study the regulation of metabolic gene promoter activity. We show that the fatty mutation induces a large increase in the adipocyte capacity to trans-activate both GAPDH and FAS genes. We have delineated a functional region of 159 bp in the GAPDH promoter responsible for the fa responsiveness of this gene.


MATERIALS AND METHODS

Plasmids and Constructs

The plasmid p(-488+21)GAPDH-CAT containing the -488+21 region of the human GAPDH promoter fused to the CAT gene and the plasmid p(-2187+65)FAS-CAT containing 2 kilobases of rat FAS promoter in front of CAT have been described previously(8, 9) . pPL1 plasmid including the region -2100+69 of the PEPCK promoter coupled to the CAT gene was provided by D. K. Granner (Vanderbilt University, Nashville, TN). pTZbeta-actin-nlslacZA1 containing the beta-actin promoter driving the expression of beta-galactosidase was provided by Claire Bonnerot (Institut Pasteur, Paris). RSV-beta-gal or RSV-CAT vectors containing the Rous sarcoma virus long terminal repeat were used in cotransfections as references for transfection efficiency. 5` deletions of the GAPDH promoter were made by digestion with restriction endonuclease of the -488+21 plasmid, fill in of ends with Klenow fragment, and recircularization. The location of the 5` end points of the deletions were -329 and -269 after digestion with Ppum1 and Dpn1, respectively. The sequence and orientation of each construct were controlled using T7 sequencing kit (Pharmacia Biotech Inc.) in the dideoxy chain termination method. The promoterless plasmid pCAT-basic (Promega) was used as a control.

Transfection of Isolated Rat Adipocytes by Electroporation

Lean (Fa/fa) and obese (fa/fa) Zucker rats, bred in our laboratory, were used at 30 days of age. Isolated adipose cells were prepared by collagenase digestion of inguinal fat pads as described previously(10) . Cells were washed three times and resuspended in DMEM. 0.2 ml of cell suspension (about 4 times 10^5 cells) was distributed in 0.4-cm gap electroporation cuvettes (Eurogentec, Seraing, Belgium) containing plasmid DNA as specified in 20 µl of DMEM, pH 7.4. After preliminary experiments to determine optimal transfection conditions, electroporation was performed at a voltage of 200 V and a capacitance of 960 µF using a gene pulser apparatus (Bio-Rad). Cells were then transferred to 2-ml Eppendorf tubes containing 1.5 ml of DMEM supplemented with 10% fetal calf serum (Life Technologies, Inc.), 20 mM glucose, antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin), 0 or 150 nM insulin, and incubated at 37 °C in 7.5% CO(2), 92.5% O(2) humidified air atmosphere for 40 h. Cells were then washed with phosphate-buffered saline, infranatant was removed, and 100 µl of lysis buffer (0.25 M Tris-HCl, pH 8, 5 mM dithiothreitol) were added. After sonication and centrifugation at 12,000 times g for 15 min, a clear cell lysate was obtained, and aliquots were used for CAT and beta-galactosidase activity determinations according to previously described methods(11, 12) . CAT activity was expressed as the percentage of conversion of chloramphenicol to its acylated products per h and 1% conversion corresponded to 35.7 pmol of chloramphenicol converted. 1 unit of beta-galactosidase represents 1 µmol of o-nitrophenyl beta-D-galactoside hydrolyzed per min at 37 °C. Gene reporter activities driven by eukaryotic promoters were normalized to viral promoter activities. No intrinsic beta-galactosidase-like activities could be measured in adipocytes. Results were obtained from three to seven independent transfection experiments in which different plasmid preparations were used. Within each experiment, individual data were calculated from triplicate electroporation cuvettes, the standard deviation never exceeding 20%.

Primer Extension Analysis

A synthetic oligonucleotide 5`-TAGCTTCCTTAGCTCCTGAAAATCTCGCCA-3` complementary to CAT cDNA (+21+51 from the AccI site of GAPDH-CAT plasmid, and +22+52 from the SalI site of FAS-CAT plasmid) was end-labeled with [-P]ATP (5000 Ci/mmol) and T4 polynucleotide kinase. Total RNA (20-50 µg) from adipocytes transfected or not with GAPDH-CAT or FAS-CAT constructs and P-labeled primer (100-500 fmol) were incubated at 60 °C in 20 µl of total volume of annealing buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1 mM EDTA) for 1 h 30 min. The extension reaction (37 °C for 30 min) was started by adding 130 µl of 50 mM Tris-HCl, 75 mM NaCl, 15 mM MgCl(2), 10 mM dithiothreitol, 0.5 mM dNTP, 77 µg/ml actinomycin D, and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). Then 100 µl of a DNase-free RNase/calf thymus DNA mixture (20 µg/ml and 100 µg/ml, respectively) were added. DNA was isolated by phenol extraction and ethanol precipitation and analyzed on a gel containing 8% polyacrylamide and 7 M urea. Sequencing ladders, obtained with the same primer on the GAPDH-CAT or FAS-CAT constructs to determine the size of the extended products, were run in parallel.

Preparation of Nuclear Extracts

Nuclear extracts from isolated fat cell nuclei were prepared essentially as described by Dignam et al.(13) . Briefly, isolated mature fat cells were broken in a Dounce homogenizer (10 strokes) in 10 mM Hepes, pH 7.9, 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM dithiothreitol, 1% Triton X-100, and protease inhibitor mixture containing 20 µM leupeptin, 20 µM pepstatin, 2 µM aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 2 mM sodium vanadate. Nuclei were pelleted by 10-min centrifugation at 600 times g, at 4 °C, and washed once in the same buffer without Triton X-100. The nuclear pellet was then resuspended in 10 µl of hypertonic buffer (25% glycerol, 20 mM Hepes, pH 7.9, 1.5 mM MgCl(2), 0.2 mM EDTA, 420 mM NaCl, and protease inhibitors). Nuclei were allowed to swell for 30 min at 4 °C and a clear nuclear extract was obtained by centrifugation 45,000 times g, 30 min, 4 °C. We checked that nuclear protein contents were identical in extracts from lean and obese rat adipocytes by similar binding to the probe Oct-1 consensus oligonucleotide (data not shown).

Gel Retardation

Nuclear proteins were incubated with the P- end-labeled -488-329 DNA fragment of GAPDH promoter (10,000 cpm/ng) in a 20-µl reaction containing 10 mM Tris-HCl, pH 7.5, 10% glycerol, 0.05% Nonidet P-40, 100 mM NaCl, 5 mM dithiothreitol, 50 ng/µl poly(dI-dC), and competitor or noncompetitor DNA, for 20 min at room temperature. The binding reaction was loaded on a 5% polyacrylamide gel containing glycerol 2.5% in 50 mM Tris, pH 8.2, 40 mM glycine, 2 mM EDTA. After migration (40 mA, 10 °C), gels were dried and subjected to autoradiography.

DNase I Footprinting Analysis

The antisense strand of -488+21 GAPDH fragment was end-labeled at the HindIII site using Klenow DNA polymerase with [alpha-P]dATP and [alpha-P]dCTP (3000 Ci/mmol). Following restriction with Ppum1, the -488-329 fragment was isolated by polyacrylamide gel electrophoresis. Binding assays were performed using 50 pg of labeled DNA (5000 dpm) and adipocyte nuclear proteins as described above except that the total volume reaction was increased to 40 µl. 4 µl of 25 mM CaCl(2) were added, followed by addition of 1.5-3 units of DNase I (Promega). The reaction was stopped after a 1-min incubation at room temperature by addition of a DNase stop solution (100 mM EDTA, 1% SDS). The mixture was incubated for 30 min at 42 °C in the presence of 10 µg of tRNA and 30 µg of proteinase K. The samples were phenol/chloroform-extracted, ethanol-precipitated, and resolved on a 8% polyacrylamide, 7 M urea sequencing gel. Sequence analysis was determined following the Maxam and Gilbert ``G+A'' reaction sequence procedure (14) to give molecular size markers.


RESULTS

The prerequisite to our work was the development of a system of transfected rat adipocytes suitable to investigate the regulation of metabolic gene promoter activity. This question, which has been investigated in several cell types, has not been previously addressed directly to adipocytes but only to a model for adipocytes, the adipose cell lines. In these cells, transfection of metabolic promoter-gene reporter plasmids was achieved by using the calcium-phosphate co-precipitation technique(15, 16, 17) . However, this method was not applicable to the floating rat adipocytes. Therefore, we turned to the electroporation procedure, and preliminary assays with RSV-CAT (Fig. 1) enabled us to define the optimal conditions of voltage and capacitance for transfection efficiency: one shock at 200 V and 960 µF, parameters which substantially differ from those reported very lately by others(18) .


Figure 1: Effect of voltage on electroporation efficiency in transfected rat adipocytes. Different voltages were applied with constant capacitance setting at 960 µF. 10 µg of RSV-beta-gal plasmid were added in each electroporation cuvette in a total volume of 200 µl. Points represent mean values of triplicate determinations. An independent experiment representative of three is shown.



The results of the transfection of metabolic gene promoters fused to CAT into primary cultured rat adipocytes are illustrated in Fig. 2. Co-transfection with RSV-beta-galactosidase gene reporter plasmids was routinely carried out in order to correct for transfection efficiency or unspecific effects, and CAT activities were normalized to beta-galactosidase activities. Fig. 2shows that normalized CAT activity in adipocytes transfected with the promoterless-CAT plasmid was extremely low. This activity was increased 40- or 130-fold when 500-bp GAPDH or 2100-bp FAS promoter fragments, respectively, were inserted upstream from the CAT gene. The specific CAT activity was very weak in adipocytes transfected with the promoterless-CAT plasmid: 2.8 ± 1.1 (n = 4) pmol of acylated chloramphenicol/h/mg of protein rose to 260 ± 92 (n = 10) and 296 ± 27 (n = 6) in adipocytes transfected with pGAPDH- and pFAS-CAT plasmids, respectively. Concomitantly, the specific beta-galactosidase activity, generated by the co-transfected RSV-beta-gal plasmid, fluctuated from 26 ± 13 (n = 4) to 58 ± 18 (n = 10) and 21 ± 9 (n = 6) milliunits/mg of protein in cells transfected with basic, GAPDH-, or FAS-CAT constructs, respectively. It is noteworthy that the level of specific CAT activities observed here in rat adipocytes transfected with p(-2187+65)FAS-CAT plasmid was substantial as compared to those reported in chick embryo hepatocytes transfected with 1.6EHCAT containing a 1.6-kilobase 5`-flanking sequence of goose FAS gene(19) . Those data clearly demonstrate the capacity of primary cultured electroporated rat adipocytes to transactivate metabolic gene promoters.


Figure 2: Expression of CAT driven by different promoters in transfected adipocytes from lean rats. 20 µg of each CAT construct and 5 µg of RSV-beta-gal plasmid were cotransfected. Reporter gene activities were measured after 48 h in the presence or not of 150 nM insulin. CAT activities (percentage of conversion of [^14C]chloramphenicol to its acylated forms per h) were normalized to beta-galactosidase activity (milliunits). Means ± S.E. obtained from at least four independent experiments are shown.



To examine whether transcription from the pGAPDH- and pFAS-CAT chimeric genes initiated at the normal GAPDH or FAS start sites in transfected rat adipocytes, we carried out primer extension analysis using a 30-mer synthetic oligonucleotide complementary to cDNA CAT gene (+21+51 from AccI site of GAPDH-CAT plasmid, and +22+52 from SalI site of FAS-CAT plasmid). Fig. 3shows that the size of the resulting products was 72 and 117 nucleotides in p(-488+21)GAPDH- and p(-2187+65)FAS-CAT-transfected adipocytes, respectively, indicating that initiation of mRNA synthesis from these promoters occurred in the expected manner.


Figure 3: Determination of the transcription initiation sites from p(-488+21)GAPDH-CAT or p(-2187+65)FAS-CAT plasmids in transfected adipocytes. Primer extension analysis was performed with a synthetic 30-mer oligonucleotide complementary to CAT cDNA (+21+51 from the AccI site of GAPDH-CAT plasmid and +22+52 from the SalI site of FAS-CAT plasmid) and total RNA from adipocytes transfected (lane 1) or not transfected (lane 2) with GAPDH- or FAS-CAT plasmids. The size of the fragments was determined by A, T, G, and C dideoxynucleotide sequencing ladders of the GAPDH- and FAS-CAT plasmids primed with the same CAT complementary oligonucleotide. Arrows show the length of fragments in nucleotides and the +1 denotes the transcription start sites.



We next addressed the question of the ability of the system to be modulated by physiological regulators. We used insulin since functional insulin-responsive elements have been demonstrated within these two promoters in adipose cell lines(17, 20) . Insulin treatment of adipocytes had a significant but minor effect (+40 ± 16%, n = 16) on viral promoter-driven gene reporter activity. In contrast both GAPDH and FAS promoter-driven CAT activities were substantially stimulated by insulin, resulting in a 3-fold increase in normalized CAT activities (Fig. 2), an effect of the same magnitude as that previously reported in transfected 3T3L1 adipose cells(17, 20) . These data provide the first evidence that insulin is capable of inducing transcription of GAPDH and FAS genes in rat adipocytes through promoter activation. Taken together, these results demonstrate that transfected rat adipocytes are a well suited system to study the regulation of metabolic gene promoters providing a new physiologically relevant tool to delineate the mechanisms implicated in the regulation of gene transcription.

We next investigated the genotype effect on the adipocyte capacity to transactivate GAPDH and FAS gene promoters. The results summarized in Table 1show that there was no significant difference in CAT activity between the two genotypes when the promoterless construct (p-basic-CAT) was used. In contrast, when the plasmids contained FAS or GAPDH promoters in front of CAT, a large genotype effect was observed with an increase in transcription ranging 3-fold with GAPDH and 8-fold with FAS promoters in obese as compared to lean rat adipocytes. However, there was no difference between the two groups when the reporter gene activity was directed by either PEPCK or beta-actin promoter, pointing out the promoter specificity of the genotype effect. It is noteworthy that the genotype effect observed here on the activity of GAPDH and FAS promoters closely parallels the genotype effect previously observed on the expression of these genes in adipose tissue and confirms the conclusions drawn from nuclear run-on analysis (5, 6) that transcription is the altered step in this genetic disorder. The present functional assays of GAPDH and FAS promoters, providing evidence that obese rat fat cells have an increased capacity to transcribe these two genes as compared to lean rat fat cells, indicate the presence of (a) fa genotype-dependent transactivating factor(s) in adipocytes from obese rats. They also establish that -488+21 region of GAPDH promoter and the -2187+65 region of FAS promoter harbor fa-responsive regions.



To further delineate the fa-responsive element(s) within these promoters we have examined here, as a first step, the activity of a series of 5`-deleted GAPDH promoter fragments. Two constructs having deletion end points at -329 and -269 were linked to the CAT gene and transfected into lean and obese rat adipocytes. As shown in Fig. 4the removal of the -488 to -329 promoter fragment decreased CAT activity in obese rat adipocytes to the lean rat values. Likewise the -269-bp construct showed comparable levels of CAT activity in the two groups of cells. These results indicate that the 159-bp sequence spanning -488 to -329 contains (a) cis-acting element(s) which is essential for the responsiveness of GAPDH to fa.


Figure 4: 5` deletion analysis of GAPDH promoter activity in transfected adipocytes. 20 µg of three series of promoter constructs were cotransfected with 5 µg of RSV-beta-gal plasmid. Within each construct, results are expressed as the ratio of normalized CAT activity measured in adipocytes of obese versus lean rats. Means ± S.E. obtained from four independent experiments are shown.



To determine whether this region binds to specific proteins we used the 159-bp fragment as a probe and investigated the protein-DNA interactions by band shift assay using nuclear extracts from obese or lean rat adipocytes. Fig. 5illustrates the complexes formed on the probe. The binding pattern was similar in the two genotypes with two main retarded bands that were competed away specifically by a 30-fold molar excess of the cold probe. No displacement occurred with a 100-fold molar excess of an heterologous DNA, the 63-bp fragment containing binding site for Oct-1 protein (data not shown). Fig. 5also shows that DNA binding was barely detectable with lower than 4 µg of nuclear proteins whatever the genotype. In addition the dose response curves of gel-retained bands to nuclear protein amounts were identical in lean and obese rats. These data indicated that the functional difference of the -488-329 GAPDH promoter region between lean and obese rat adipocytes could not be accounted for by a differential abundance of DNA binding factors.


Figure 5: Gel retardation assay obtained after binding of increasing amounts of nuclear extracts from lean and obese adipocytes to -488-329 region of GAPDH promoter. Competitor DNA (-488-329) was added in a 30-fold (+) or 70-fold (++) molar excess relative to labeled DNA (1 ng/lane, 10,000 cpm). The autoradiogram is representative of four independent experiments using different nuclear protein preparations.



To define the sites of protein-DNA interactions the same probe was subjected to DNase I footprinting analysis in the presence of increasing amounts of nuclear proteins. Fig. 6shows that with large amounts of nuclear extracts one footprint spanning -442-422 was clearly apparent in both groups. In addition, the binding of adipocyte nuclear proteins produced distinct hypersensitive sites, one at position -409 and two others downstream. There was no detectable footprint at low protein concentration in either lean or obese rat adipocytes. No additional footprint could be revealed when large amounts of nuclear proteins were used whatever the genotype.


Figure 6: DNase I footprinting analysis of the GAPDH promoter in the presence of adipocyte nuclear extracts from lean or obese rats. The promoter fragment from GAPDH sequence between -488 and -329 was subjected to DNase I footprinting as described under ``Materials and Methods.'' Increasing amounts of adipocyte nuclear extracts (NE), as indicated, were incubated with the [P]DNA fragment (5000 cpm) labeled on the antisense strand as described above. The asterisks indicate hypersensitive sites and the white rectangle the footprinted region.




DISCUSSION

The ability of viral promoters to function in electroporated rat adipocytes has been recently reported(18) . We provide here the first evidence that transiently transfected rat adipocytes in primary culture are able to transactivate metabolic gene promoters. FAS or GAPDH promoters fused to CAT were able to drive a high level of CAT activity in these cells. Importantly, primer extension analysis demonstrated that transcription from the transfected GAPDH-CAT and FAS-CAT was correctly initiated representing appropriate transcriptional activation. Moreover we show here that insulin acted directly on rat adipocytes to stimulate FAS and GAPDH gene transcription through their promoter regions. Therefore transfected rat or human adipocytes represent a physiologically relevant model of great potential interest for the investigation of cis and trans factors involved in hormonal and metabolic transcriptional regulations of adipose tissue specific genes.

The main finding of this study is that the fa mutation induces an increase in the capacity of adipocytes to activate the promoters of metabolic genes. Obese rat adipocytes electroporated with constructs containing the 5`-flanking region of either GAPDH or FAS gene fused to CAT reporter gene exhibited a severalfold increase in CAT expression as compared to lean rat adipocytes, mimicking the overtranscription of these genes previously observed in obese rat adipocyte nuclei(5, 6) . We next attempted to identify the cis-acting elements and trans-acting factors involved in the up-regulation of GAPDH promoter activity in fatty rat adipocytes. The functional assays of 5`deleted promoter CAT constructs revealed that the GAPDH promoter region between -488 and -329 was critical to the genotype-mediated transcriptional increase in GAPDH gene, suggesting that this region harbored (a) fa responsive element(s). However, as assessed here by band shift assay and DNase I footprinting analysis, DNA-protein interactions with the -488-329 GAPDH fragment did not show any differences that could account for the functional data. The nuclear binding factors to this region were found to be present in similar amounts in lean and obese rat adipocytes and to footprint the same 20-bp(-442-422) nucleotide sequence. These results tend to suggest that the factor responsible for the observed footprint/gel retained band represents a constitutively expressed molecule that is present at the same level in both cell types, and that increased promoter activity in obese rat adipocytes does not arise simply as a consequence of differential abundance of factors binding to this region. A possibility is that the transcriptional activity of this basic constitutive complex equally abundant in both cell types is modulated by (a) fa dependent protein(s) along the mechanism recently described for Sp1 and protein p74(21) . However, we cannot exclude that the real regulator of the differential activity may have escaped detection as a consequence of a low abundance or stability. Or, if the fa gene encodes a mutated transcription factor, the lesion would concern the activation domain of the molecule and not the distinct DNA-binding domain. Such a phenomenon has been described for c-Jun in which a single amino acid change can modify the trans-acting function without altering DNA binding(22) . Alternatively, our present data might indicate that the fatty genotype activation of GAPDH promoter does not rely on a trans-acting factor unique to obese rat adipocytes but on an increased activity of transfactors common to both genotypes. The fa gene would interfere with one of the putative mechanisms (phosphorylation, dimerization, glycosylation) controlling the activity of transcription factors.

In summary, this work, with the demonstration that the fa gene affects adipocyte transcription factors, has allowed to narrow down the primary functional effect of the fa mutation. Through their activation of GAPDH and FAS promoters these fa gene-induced transcription factors are to play a crucial pathogenic role in the disruption of the caloric homeostasis of adipocytes. As they are likely to act also on an array of other metabolic genes, the fa gene-induced transcription factors have the potential to account for the pleiotropic effects that characterize this mutation. The identification of these trans-activators is a challenging task that will contribute to the understanding of the fa lesion.


FOOTNOTES

*
Part of this paper has been presented at Keystone Symposia on Molecular and Cellular Biology, January 1994 (Rolland, V., Dugail, I., Le Liepvre, X., and Lavau, M.(1994) J. Cell. Biochem., Suppl. 18A, 177). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-43-29-29-23; Fax: 33-1-40-51-85-86.

(^1)
The abbreviations used are: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FAS, fatty acid synthetase; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; PEPCK, phosphoenolpyruvate carboxykinase; RSV, Rous sarcoma virus; bp, base pair(s).


ACKNOWLEDGEMENTS

We are very grateful to Dr. Maria Alexander (Howard Hughes Medical Institute, Boston, MA) for providing the GAPDH promoter construct. We also thank Dr. Donald B. Jump (Michigan State University) for kindly providing the FAS promoter, Daryl. K. Granner (Vanderbilt University, Nashville, TN) for the pPL1 construct, and J. Antras-Ferry (CEREMOD, Meudon, France) for the CAT oligonucleotide.


REFERENCES

  1. Zucker, L. M., and Zucker, T. F. (1961) J. Hered. 62, 275-278
  2. Truett, G., Bahary, N., Friedman, J. M., and Liebel, R. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7806-7809 [Abstract]
  3. Krief, S., and Bazin, R. (1991) Proc. Exp. Biol. Med. Soc. 528-538
  4. Dugail, I., Quignard-Boulangé, A., Bazin, R., Le Liepvre, X., and Lavau, M. (1988) Biochem. J. 254, 483-487 [Medline] [Order article via Infotrieve]
  5. Dugail, I., Quignard-Boulangé, A., Le Liepvre, X., Ardouin, B., and Lavau, M. (1992) Biochem. J. 281, 607-611 [Medline] [Order article via Infotrieve]
  6. Guichard, C., Dugail, I., Le Liepvre, X., and Lavau, M. (1992) J. Lipid Res. 33, 679-687 [Abstract]
  7. Hainault, I., Guerre-Millo, M., Guichard, C., and Lavau, M. (1991) J. Clin. Invest. 87, 1127-1131 [Medline] [Order article via Infotrieve]
  8. Alexander, M. C., Lomanto, M., Nasrin, N., and Ramaika, C. (1988) Proc. Natl. Acad. Sci U. S. A. 85, 5092-5096 [Abstract]
  9. Amy, C. M., Williams-Ahlf, B., Naggert, J., and Smith, S. (1990) Biochem. J. 271, 675-679 [Medline] [Order article via Infotrieve]
  10. Rodbell, M. (1964) J. Biol. Chem. 239, 375-380 [Free Full Text]
  11. Seed, B., and Sheen, J. Y. (1988) Gene 67, 271-277 [CrossRef][Medline] [Order article via Infotrieve]
  12. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., pp. 16.63-16.67, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  13. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489 [Abstract]
  14. Maxam, A., and Gilbert, W. (1980) Methods Enzymol. 152, 721-735
  15. Kaestner, K. H., Christy, R. J., and Lane, M. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 251-255 [Abstract]
  16. Graves, R. A., Tontonoz, P., Ross, S. R., and Spiegelman, B. M. (1991) Genes & Dev. 5, 428-437
  17. Moustaid, N., Beyer, S., and Sul, H. S. (1994) J. Biol. Chem. 269, 5629-5634 [Abstract/Free Full Text]
  18. Quon, M. J., Zarnowski, M. J., Guerre-Millo, M., de la Luz Sierra, M., Taylor, S. I., and Cushman, S. W. (1993) Biochem. Biophys. Res. Commun. 194, 338-346 [CrossRef][Medline] [Order article via Infotrieve]
  19. Kameda, K., and Goodridge, A. G. (1991) J. Biol. Chem. 266, 419-126 [Abstract/Free Full Text]
  20. Nasrin, N., Ercolani, L., Denaro, M., Kong, X. F., Kang, I., and Alexander, M. (1990) Proc. Natl. Acad. Sci U. S. A. 87, 5273-5277 [Abstract]
  21. Murata, Y., Kim, H. G., Rogers, K. T., Udvadia, A. J., and Horowitz, J. M. (1994) J. Biol. Chem. 269, 20674-20681 [Abstract/Free Full Text]
  22. Boyle, W. J., Smeal, T., Defize, L. H. K., Angel, P., Woodgett, J. R., Karin, M., and Hunter, T. (1991) Cell 64, 573-584 [Medline] [Order article via Infotrieve]

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