(Received for publication, June 6, 1994; and in revised form, October 13, 1994)
From the
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 -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.
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 ()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.
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--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--galactosidase gene reporter plasmids was
routinely carried out in order to correct for transfection efficiency
or unspecific effects, and CAT activities were normalized to
-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
-galactosidase activity,
generated by the co-transfected RSV-
-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--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 [
C]chloramphenicol to its acylated
forms per h) were normalized to
-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 -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--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.
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.