(Received for publication, September 5, 1994; and in revised form, October 31, 1994)
From the
Exposure of preadipocytes to long chain fatty acids induces expression of several gene markers of adipocyte differentiation. This report describes the cloning, from a preadipocyte library, of a cDNA encoding a fatty acid-activated receptor, FAAR. The cDNA had the characteristics and ligand-binding domains of nuclear hormone receptors and encoded a 440 amino acid protein related to peroxisome proliferator-activated receptors, PPAR. The deduced protein sequence was 88% homologous to that of hNUC I, isolated from human osteosarcoma cells. FAAR mRNA was abundant in adipose tissue, intestine, brain, heart, and skeletal muscles and less abundant in kidney, liver, testis, and spleen. The mRNA was undetectable in growing Ob1771 and 3T3-F442A preadipocytes, was strongly induced early during differentiation, and was increased by fatty acid. Transcription assays using hybrid receptor showed strong stimulation by fatty acid and weaker induction by fibrates. Transfection of 3T3-C2 fibroblasts, with a FAAR expression vector, conferred fatty acid inducibility of the adipocyte lipid-binding protein and the fatty acid transporter. Transcriptional induction of these genes exhibited inducer specificity identical to that described in preadipocytes. In summary, the data indicate that FAAR is likely a mediator of fatty acid transcriptional effects in preadipocytes.
Fatty acid (FA) ()treatment of preadipocytes induces
expression of several genes encoding proteins implicated in FA
metabolism. These include the adipocyte lipid-binding protein,
ALBP(1, 2, 3) , the acyl-CoA synthase (1, 2) and a recently cloned (4) membrane
protein implicated in FA binding and transport, FAT. (
)Some
features of the transcriptional effects of FA in preadipocytes have
been described. Both saturated and unsaturated long chain FA were
effective in preadipocytes(1, 2) , unlike findings
with mammary cells(5) . Induction did not require FA metabolism
since it was observed with 2-bromopalmitate, which is not metabolized
by preadipocytes(6) , but it required protein synthesis and was
fully reversed upon FA removal(1, 2) . These effects
of FA have potentially important nutritional and clinical significance.
It is likely that FA, in vivo, as in vitro(7) promote adipose conversion of preadipocytes. In
addition, transcriptional regulation by FA applies to cell types other
than adipocytes and to multiple lipid-related
genes(8, 9, 10) .
The molecular mechanism
mediating the effect of FA on gene expression in preadipocytes remains
unknown. Recent evidence has suggested that FA may act through nuclear
receptors of the steroid-thyroid superfamily, the peroxisome
proliferator-activated receptors (PPARs). Activation of these receptors
by FA has been reported(8, 9, 10) , and an
arachidonic acid analogue (ETYA) was shown to be even more potent than
fibrates in activating one of these receptors, xPPAR(11) .
In this report we have investigated whether a member of the PPAR family
mediates the transcriptional effects of FA in preadipocytes. Using a
DNA fragment containing conserved PPAR sequence, we have isolated a
mouse PPAR-like protein that is activated by FA and related molecules
but weakly by fibrates. Furthermore, expression of this protein in
fibroblasts is shown to confer FA-responsive gene expression.
The deduced amino acid sequence for FAAR was different
from those previously reported (9, 17, 18) for mouse PPARs (PPAR and
). However, sequences with 86 and 84% identities to putative
DNA-binding domains of mPPAR and mPPAR
, respectively, were
identified in FAAR. The putative ligand-binding domain and the D
domains of mouse PPARs exhibited 70 and 50% identities, respectively,
with corresponding domains of FAAR. A search of data base nucleotide
sequences revealed near complete homology (99%) of FAAR sequence to
that of a 503-base pair cDNA recently isolated from a mouse brain cDNA
library (17) and postulated to represent the A/B and
DNA-binding domains of hNUC I, a member of the PPAR family isolated
from a human osteosarcoma cDNA library(19) . FAAR and hNUC I
exhibited strong similarities of their DNA- binding (97%),
ligand-binding (95%), and A/B (80%) and D domains (93%). The deduced
protein sequences for FAAR and hNUC I were 88% similar. The above
showed that FAAR was the mouse homologue of human NUC I.
Figure 1:
Tissue
distribution of FAAR mRNA in adult mouse. RNA (30 µg/lane) from
tissues of 8-week-old mice were analyzed and quantitated as described
under ``Materials and Methods.'' Actin mRNA ( and/or
) is shown as internal standard. Li, liver; W,
epididymal adipose tissue; I, intestine; M, skeletal
muscle; K, kidney; S, spleen; Lu, lung; H, heart; T, testis; B,
brain.
Figure 2:
Induction of FAAR mRNA with adipose
differentiation and by fatty acids. A, RNA (20 µg/lane)
was analyzed by Northern blotting as described under ``Materials
and Methods.'' 1, RNA from Ob1771 subconfluent cells; 2, RNA from 11-day post-confluent differentiated Ob1771; 3, RNA from subconfluent 3T3-442A cells; 4, RNA
from 11-day post-confluent differentiated 3T3-442A cells; 5, RNA from 11-day post-confluent 3T3-C2 cells. B,
Ob1771 cells were maintained in differentiation medium, and RNA was
prepared at the indicated times and analyzed by Northern blotting. FAAR
mRNA (), PPAR
(
), and ALBP mRNA (
) signals
were quantitated by densitometry and standardized to GAPDH mRNA
signals. C, 1-day post-confluent Ob1771 cells were maintained
for 24 h in standard medium (lane 1) in the presence of 250
µM palmitate (lane 2) or 100 µM 2-bromopalmitate (lane 3). RNA was analyzed as in A. The results shown in A-C are representative of
three separate experiments.
Fig. 2C shows that exposure of Ob1771
preadipocytes (day 1 post-confluence) to palmitate or 2-bromopalmitate
increased level of FAAR mRNA and induced ALBP expression.
Interestingly, PPAR mRNA, which is undetectable at day 1, remained
not expressed in cells exposed for 24 h to FA.
Figure 3:
FAAR and RXR or RXR
bind
cooperatively to the acyl-CoA oxidase PPRE. In vitro translated proteins were incubated according to the indicated
combinations in the presence of labeled oligonucleotide. The
protein
DNA complexes were resolved by polyacrylamide gel
electrophoresis as described under ``Materials and
Methods.''
Figure 4: Activation of hGR/FAAR hybrid receptor by FA. COS-1 cells were transfected with MMTV-CAT vector and either the hGR/FAAR or hGR (insert) expression vectors. The transfected cells were treated for 40 h in the following conditions: a, 0.1 µM dexamethasone; b-e, 1, 3, 10, and 30 µM 2-bromopalmitate; f, 100 µM palmitate; g, 10 µM ETYA; h, 100 µM bromooctanoate; i, 500 µM clofibric acid; and j, 100 µM Wy 14,643. CAT enzyme activity was determined as described under ``Materials and Methods.'' Results are presented by taking as 1 the value obtained in cells maintained in control medium and are presented as the mean ± S.D. of triplicate transfections.
Figure 5:
Stable transfectants 3T3-C2 expressing
FAAR are responsive to fatty acids. A, control transfected (lanes 1 and 2), FAAR-45 (lanes 3 and 4), -47 (lanes 5 and 6), and -27 (lanes
7 and 8) cells were maintained from confluence to day 2
post-confluence in standard medium (lanes 1, 3, 5, and 7) or exposed for the last day to 100
µM 2-bromopalmitate (lanes 2, 4, 6, and 8). RNA was analyzed by Northern blot as
described under ``Materials and Methods.'' Autoradiographic
times were: 8 h for GAPDH mRNA, 15 h for ALBP and FAT mRNAs, and 6 h
for FAAR mRNA. The data are representative of three independent
experiments. B, 1-day post-confluent FAAR-27 (), -45
(
), -47 (
), and control cells (
) maintained in
standard medium were exposed for 24 h to increasing concentrations of
2-bromopalmitate. FAT mRNA signals were quantified as in Fig. 2B.
The effects of FA treatment of FAAR and control cells (100 µM 2-bromopalmitate for 24 h) on ALBP and FAT gene expression are shown in Fig. 5A. As expected, ALBP and FAT mRNAs remained undetectable in all (six were tested) control-transfected cells (lane 2versuslane 1). In contrast, exposure of FAAR-expressing cells to 2-bromopalmitate induced expression of ALBP and FAT mRNAs (lanes 4, 6, and 8).
The response to FA was characterized with respect to inducer concentration on FAT gene expression in confluent transfected cells (Fig. 5B). For the three FAAR expressing clones, response to 2-bromopalmitate was detectable beginning at 3 µM with a half-maximal effect observed at about 20 µM. In contrast, FAT mRNA remained undetectable in control 3T3-C2 cells subjected to the same treatments. Similar results were obtained for the ALBP gene expression (data not shown).
The specificity of FAAR-mediated FAT gene induction was next investigated in FAAR-45 cells exposed for 24 h to increasing concentrations of various inducers. Fig. 6reports the maximal response obtained for each compound. FA analogues, i.e. 2-bromopalmitate and ETYA (columns b and e) as well as natural FA, i.e. palmitate and linolenate (columns c and d), were strong inducers of FAT mRNA expression. In contrast, middle chain FA derivative, 2-bromooctanoate (column f), clofibric acid (column g), and Wy 14,643 (column h) were found to be ineffective or weak inducers of FAT mRNA. Nuclear run-on experiments (Fig. 6, inset) were carried out using nuclei from FAAR-45 cells maintained for 24 h in standard medium in the absence (lane 1) or presence (lane 2) of 100 µM 2-bromopalmitate. FAT and ALBP transcription rates, which were very low in cells maintained in standard medium, were strongly increased by FA treatment indicating that induction of FAT and ALBP mRNA expression in response to FA was primarily due to transcriptional activation of the corresponding genes.
Figure 6: Activation of FAT mRNA expression by various inducers in FAAR-45 cells. 1-day post-confluent FAAR-45 cells were maintained for 24 h in the following conditions: a, standard medium; b, 100 µM 2-bromopalmitate; c, 200 µM palmitate; d, 200 µM linolenate; e, 100 µM ETYA; f, 100 µM 2-bromooctanoate; g, 500 µM clofibric acid; and h, 100 µM Wy 14,643. RNA was analyzed as in Fig. 2. The data are representative of three independent experiments. Inset, run-on assays from nuclei of 1-day post-confluent FAAR-45 cells exposed (2) or not (1) for 24 h to 100 µM 2-bromopalmitate.
The present work, which dealt with transcriptional effects of FA in preadipocytes, reported the following: 1) a cDNA, termed FAAR, encoding a member of the PPAR superfamily, was isolated from a library constructed from FA-treated preadipocytes; 2) FAAR was activated by FA; 3) FAAR was shown to be one of the earliest markers of preadipocyte differentiation; 4) the ability of FAAR, when expressed in 3T3-C2 fibroblasts unresponsive to FA, to confer FA-specific induction of two gene markers of adipose differentiation, ALBP and FAT, was documented; 5) FA induction in preadipocytes and in FAAR-expressing 3T3-C2 cells was shown to exhibit similar features consistent with FAAR mediation of the FA effects. The findings constitute the first direct demonstration of a receptor's role in mediating FA regulation of differentiation-linked genes in preadipocytes.
The preadipocyte FAAR is homologous to hNUC I, previously isolated from a human osteosarcoma cDNA library(19) . The deduced protein sequences for FAAR and hNUC I were 88% similar. Like hNUC I, FAAR is related to the mouse (9, 17, 18) and rat (10) PPARs as evidenced by the high identity of the respective DNA- and ligand-binding domains. In addition, FAAR binds to PPREs and the binding is enhanced by heterodimerization with RXRs (Fig. 3). However, weak similarity of other domains of FAAR with those of mouse and rat PPARs suggests that FAAR, and possibly hNUC I, form a distinct subtype of the PPAR family. This is further supported by two other lines of evidence. First, the observed tissue distribution of FAAR is different from that previously reported for mPPAR. FAAR is highly expressed in adipose tissue, muscle, and intestine and weakly expressed in liver and kidney (Fig. 1) while mPPAR is preferentially expressed in liver, kidney, and heart(9) . Second, the inducer responsiveness of FAAR and PPAR are different. FAAR appears equally sensitive to saturated and unsaturated FA and is less sensitive to fibrates ( Fig. 4and Fig. 6). In contrast, PPAR are activated by fibrates and unsaturated FA better than by saturated FA(10, 11) . The data would suggest that FAAR might be typical of a subgroup of PPAR preferentially responsive to long chain FA.
Multiple lines of evidence support the interpretation that FAAR mediates the transcriptional effects of FA in preadipose cells. FAAR is not expressed in 3T3-C2 and preconfluent preadipose cells which are unresponsive to FA. Its appearance at confluence in Ob1771 preadipocytes (Fig. 2) coincides with acquisition of the response to FA (2, 6) . Expression of FAAR in FA-unresponsive 3T3-C2 fibroblasts conferred FA-responsive expression of ALBP and FAT genes. Furthermore, the response observed exhibited the same specificity previously demonstrated in Ob1771 preadipocytes. In both cases, 2-bromopalmitate was more effective than native FA ( Fig. 6and (6) ). Both saturated and unsaturated FA were effective. Fibrates were weaker inducers, and middle chain FA were ineffective ( Fig. 6and (2) and (6) ).
The
mechanism of FAAR action to mediate induction of ALBP and FAT remains
to be determined. FAAR, in combination with RXRs, may act by binding to
the same DNA motif as PPARs(20, 21) . PPAR is
thought to bind to a direct repeat-like element in the ALBP promoter (26) which has been also involved in the tissue-specific
expression of ALBP(27) . A direct repeat-like element was found
in the promoter of human CD36, a homologue of FAT(28) . Thus,
it is tempting to speculate that such direct repeat-like elements might
constitute DNA-binding motifs for FAAR. Clearly, FAAR mRNA emerged
earlier than PPAR
mRNA during adipose conversion process of Ob1771
cells (Fig. 2B) as well as in 3T3-L1
cells(29, 30) . It is clear also that early after
confluence, the time at which the FA responsiveness appears, preadipose
cells express FAAR but not PPAR
mRNA (Fig. 2B).
PPAR
was not expressed in FAAR-transfected 3T3-C2 cells treated
with FA where induction of FAT and ALBP mRNA expression was observed
(data not shown). Taken together, these observations indicate that FAAR
is likely involved in FA activation of lipid metabolism-related genes
in preadipose cells (2, 6) and in the adipogenic
action of FA which occurs during the first days of the confluent
phase(7) . PPAR
, which is expressed at a later stage,
might mediate, possibly in combination with FAAR, regulatory effects of
FA occurring in partially to fully differentiated cells(1) .
Since FA promote terminal differentiation of preadipocytes in culture (7) , it will be important to determine whether 3T3-C2 fibroblasts, expressing high levels of FAAR, can be induced to differentiate in the presence of FA. Activation of FAAR could, in vivo, constitute an important part of the molecular mechanism behind the adipogenic effects of overfeeding. The data documenting FA induction of FAT expression adds one more differentiation-linked gene to those known to be sensitive to FA in preadipocytes. Furthermore, FA induction of FAT expression would amplify the effects of FA on preadipocyte differentiation since the FAT protein is implicated in the membrane binding and transport of FA into the cell(4) . While ALBP is adipose tissue specific, FAAR and FAT are more ubiquitous in expression and might respond to FA regulation in other cells, for example in developing muscle and intestinal cells. Determination of whether FAAR mediates transcriptional effects of FA in cells other than adipocytes will be crucial to understanding differential regulation of lipid-related genes in various tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L28116[GenBank].