From the Department of Biochemistry, School
of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274, Japan, the ¶ Laboratoíre de Biologie Moléculaire et
Cellulaire, Universite de Bourgogne, BP138, 21004 Dijon, France,
and the
Laboratory of Metabolism, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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Regulation of gene expression of three putative
long-chain fatty acid transport proteins, fatty acid translocase (FAT),
mitochondrial aspartate aminotransferase (mAspAT), and fatty acid
transport protein (FATP), by drugs that activate peroxisome
proliferator-activated receptor (PPAR) and
were studied using
normal and obese mice and rat hepatoma cells. FAT mRNA was induced
in liver and intestine of normal mice and in hepatoma cells to various
extents only by PPAR
-activating drugs. FATP mRNA was similarly
induced in liver, but to a lesser extent in intestine. The induction
time course in the liver was slower for FAT and FATP mRNA than that
of an mRNA encoding a peroxisomal enzyme. An obligatory role of
PPAR
in hepatic FAT and FATP induction was demonstrated, since an
increase in these mRNAs was not observed in PPAR
-null mice.
Levels of mAspAT mRNA were higher in liver and intestine of mice
treated with peroxisome proliferators, while levels in hepatoma cells were similar regardless of treatment. In white adipose tissue of KKAy
obese mice, thiazolidinedione PPAR
activators (pioglitazone and
troglitazone) induced FAT and FATP more efficiently than the PPAR
activator, clofibrate. This effect was absent in brown adipose tissue.
Under the same conditions, levels of mAspAT mRNA did not change
significantly in these tissues. In conclusion, tissue-specific expression of FAT and FATP genes involves both PPAR
and -
. Our data suggest that among the three putative long-chain fatty acid transporters, FAT and FATP appear to have physiological roles. Thus,
peroxisome proliferators not only influence the metabolism of
intracellular fatty acids but also cellular uptake, which is likely to
be an important regulatory step in lipid homeostasis.
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INTRODUCTION |
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Long-chain fatty acids (LCFAs)1 are an efficient energy source for many cells. Cells metabolize and/or store intracellular LCFAs, depending on cell type and energy requirement. In addition to their ability to biosynthesize LCFAs, cells can utilize plasma LCFAs released by lipoprotein lipase-catalyzed hydrolysis of triglycerides from circulating chyromicrons and very low density lipoproteins (1). Thus, uptake of LCFAs into cells can be an important step in energy metabolism. However, the mechanism and regulation of uptake of extracellular LCFAs into mammalian cells is not well understood.
Because of their hydrophobic properties, it has been suggested that LCFAs are transported across the plasma membrane by simple diffusion (2, 3). However, other studies have suggested that this process is mediated by proteins. Three independent transport proteins have been identified that may contribute to this process, including fatty acid translocase (FAT)(4), mitochondrial aspartate aminotransferase (mAspAT) (5), and fatty acid transport protein (FATP) (6). The precise role of these proteins in mediating LCFA uptake is not known and the mechanisms of LCFA uptake into various mammalian cells may not be the same. Nevertheless, there is evidence that all three may, at least in part, contribute to LCFA cellular uptake.
Peroxisome proliferator-activated receptors (PPARs) have unique roles
in lipid homeostasis. PPARs are part of the nuclear hormone receptor
superfamily, and there are three subtypes that have been described,
,
(
), and
. Each is encoded by a separate gene and have
unique tissue distribution patterns. Furthermore, their roles in
mediating changes in gene expression appear to be cell- and
tissue-specific. For example, PPAR
mediates fibrate and dietary
polyunsaturated fatty acid induction of hepatic peroxisomal lipid-metabolizing enzymes, including acyl-CoA oxidase a key enzyme in
regulating peroxisomal lipid catabolism (7, 8). Furthermore, PPAR
modulates hepatic gene expression of apolipoprotein A-I and C-III in
response to the peroxisome proliferator Wy 14,643 (9). It is of
interest to note that recently, polyunsaturated fatty acids Wy 14,643 and leukotriene B4 have been shown to bind to PPAR
(10-12). Upon
binding, PPAR
-dependent gene transcription is activated,
and these effects are most pronounced in the liver, a tissue with a
high capacity for
-oxidation of fatty acids. Compounds that bind to
another subtype, PPAR
, have also been identified. The antidiabetic
drugs, thiazolidinediones and prostaglandin J2 derivatives, have been
shown to preferentially bind to and activate PPAR
, and these effects
occur predominantly in adipose cells (11-15). In summary, roles for
PPAR
include those involved in fatty acid catabolism, while those
for PPAR
include those involved in adipogenesis.
Since PPARs are known to be key transcription factors of different genes participating in lipid homeostasis (16), we examined the effects of specific activators of PPARs on mRNA levels of the three putative fatty acid transporters.
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EXPERIMENTAL PROCEDURES |
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Materials-- 2-(p-Chlorophenoxy)isobutyric acid ethyl ester (clofibrate), 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-thio)acetic acid (Wy 14,643), di(2-ethyhexyl)adipate (DEHA), and di(2-ethylhexyl)phthalate (DEHP) were purchased from Tokyo-Kasei (Tokyo, Japan). Troglitazone was a generous gift from Sankyo Co. Ltd. (Tokyo, Japan).
Animals and Treatment--
Normal male NZB mice (5-6 weeks of
age) were kept on a 12-h light-dark cycle and provided with food and
water ad libitum. Mice were fed either a control diet (CE7,
Clea Japan) or one containing 0.05% Wy 14,643, 0.5% clofibrate, 2%
DEHA or 2% DEHP for 1-5 days. Animals were euthanized at 1330 h
to minimize the effect of diurnal rhythms. Male PPAR wild-type (+/+)
or homozygote-null (
/
) mice (F3 generation, 10-12
weeks of age) (7) were fed either a control diet (Bioserv, Frenchtown,
NJ) or one containing 0.1% Wy 14,643 for 14 days as described
previously (17). Male KKAy obese mice (11 weeks of age) were obtained
from Clea Japan (18) and fed either a control diet (MF, Oriental Kobo,
Japan) or one containing 0.5% clofibrate, 0.03% pioglitazone, or
0.1% or 0.3% troglitazone for 8 days.
Cell Culture-- The Fao cell line, a subclone of rat hepatoma HII4E, were cultured under conditions as reported previously (19). When a PPAR activator was added, a concentrated solution in Me2SO was added to medium before adding serum and sonicated to dissolve completely prior to adding to the cells. Treatment was initiated by changing the medium to a prewarmed drug-containing medium.
RNA Preparation and Northern Blot Analysis--
Total RNA was
prepared from the liver, intestine, adipose tissue, or the cultured
hepatoma cells by the acid guanidium thiocyanate-phenol-chloroform extraction method (20). Northern blot analysis was carried out essentially as described previously (17). Among the cDNAs used for
probes included peroxisomal HD, 2u-globulin, apolipoprotein E
(apoE), and acyl-CoA oxidase, which have been described previously (17). The remaining cDNAs used for probes were obtained by cloning of PCR products of cDNA synthesized from poly(A) RNA isolated from
the liver of Wy 14,643-fed mice. Their identities were confirmed by
sequencing from both ends to 300-400 bases inside after cloning into
the SmaI site of pUC18. The synthetic oligonucleotides
used to amplify respective cDNA sequences were
5'-TCTGACATTTGCAGGTCCATCTATGCTG and 5'-ATCTCAACCAGGCCCAGGAGCTTTATTT for
FAT (corresponding to nucleotide number from 873 to 1410 of the
published rat sequence (4) (GeneBankTM accession number
L19658); 5'-CTTACGTGCTCCCCAGTGTCTGGAAG and 5'-GGAGGAGGACACTCTGCTCTGGGATT for mAspAT (corresponding to nucleotide number from 134 to 538 of the published mouse sequence (5) (GeneBankTM accession number U82470));
5'-GCCATTGTGGTGCACAGCAGGTACTA and 5'-TCGTGTCCTCATTGACCTTGACCAGA for
FATP (corresponding to nucleotide number from 775 to 1285 of the
published mouse sequence (6)(U15976)); 5'-GGGAGTTTGGCTCCAGAGTTTGACCG
and 5'-GGAACACTTTGTAGGGCATCTGAGAGCG for lipoprotein lipase
(corresponding to nucleotide number from 8 to 1000 of the published
mouse sequence (21) (GeneBankTM accession numbers J03302
and J02740); 5'-AGGCTTTTCTGAAAGGGTGAGGCATTTT and
5'-ACCATAGGAGTGGATGCTAATGTGCCCT for leptin (corresponding to nucleotide
number from 1422 to 2280 of the published sequence (22)
(GeneBankTM accession number U18812); and
5'-CCTACAGATGTGGTAAAGGTCCGCTTCC and 5'-GAGTCATCAGTACAGAGGCACAGGGAGG for
uncoupling protein 2 (corresponding to nucleotide number from 701 to
1368 of the reported mouse sequence (GeneBankTM accession
number U69135). The cDNA clones for fatty acid-binding proteins
were obtained by reverse transcription-PCR after adding oligo(dC) tails
to the cDNA synthesized as above by terminal deoxynucleotidyl tansferase and dCTP. The PCR primers were oligo(dG) and three specific
primers: 5'-GTACCAAGTGCAGAGCCAAGAGAACTTT for rat liver FAB
corresponding to nucleotide number from 353 to 380 of the published
sequence (23) (GeneBankTM accession number J00732),
5'-CCAAGCTTCCTCTTCATCACATTAATGCCCATTTT for mouse intestinal FAB
corresponding to nucleotide number from 96 to 130 of the published
sequence (24) (GeneBankTM accession number M65034), and
5'-TCATGTAATCATCGAAGTTTTCACTGGAGACAAGC for mouse adipocyte FAB
corresponding to nucleotide number from 604 to 638 of the published
genomic sequence (25) (GeneBankTM accession number M13385).
After hybridization, membranes were washed and autoradiographed with an
intensifying screen at
80 °C. For quantitative analysis, a BAS2000
image analyzer (Fujix, Japan) was used. [
-32P]dCTP
(3000 Ci/mmol) and a random prime labeling kit were purchased from
Muromachi Kagaku (Tokyo, Japan) and Takara (Osaka, Japan).
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RESULTS AND DISCUSSION |
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All Three Fatty Acid Transporters, FAT, FATP, and mAspAT, Are Induced by the Peroxisome Proliferator Wy 14,643-- FATP mRNA is highly expressed in skeletal muscle, heart, and fat, but only found at low levels in liver of normal mice (6). We observed that FATP mRNA is expressed significantly higher in the liver of Wy 14,643-treated mice as compared with controls during efforts to isolate the cDNA by reverse transcription-PCR. To examine the possibility that fatty acid transporters mRNAs are induced by the peroxisome proliferator Wy 14,643, we measured the time course effect of the drug on the levels of FAT, FATP, and mAspAT mRNAs in the liver and intestine (Fig. 1). Feeding a diet containing Wy 14,643 resulted in a large induction of peroxisomal HD mRNA in liver, reaching maximal level within a day as reported previously (26). In the intestine HD mRNA had not yet reached a plateau by 5 days. Intestinal fatty acid-binding protein mRNA was also gradually induced by Wy 14,643 and expressed only in the intestine. During these periods, the levels of apolipoprotein AI mRNA did not change significantly both in the liver and intestine. mRNAs for FAT, FATP, and mAspAT all responded to Wy 14,643 administration, but with different patterns. FAT, FATP, and mAspAT mRNAs were all gradually induced in the liver with a different time course than that of HD mRNA. FAT and mAspAT mRNA were also detected in higher levels in the intestine. Longer exposure of autoradiographic film revealed that FATP was also induced in the intestine with a similar time course as in the liver, but this increase by day 5 was less than 10% of the liver level (not shown). It should be pointed out that for FAT, we did observe two different sizes of mRNA as reported previously (4). Northern blot analysis showed that all three putative LCFA transport proteins FAT, FATP, and mAspAT were induced in a tissue-specific manner by the peroxisome proliferator Wy 14,643.
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FAT and FATP mRNAs Are Induced in Mouse Liver by Various
PPAR Activators--
To examine whether PPAR
activators other
than Wy 14,643 induce FAT and FATP in the liver, the effects of three
other peroxisome proliferators were compared. The mRNA levels in
liver from mice treated with these compounds for 5 days were determined
by Northern blot analysis as shown in Fig.
2. All four PPAR
activators induced liver fatty acid binding protein compared with controls. In addition, peroxisomal HD mRNA was markedly increased in response to all 4 PPAR
activators consistent with previous work (not shown; see Ref.
17). Similarly, FAT mRNA was markedly induced by all four PPAR
activators compared with controls. Interestingly, mRNA of FATP and
lipoprotein lipase were higher, and
2u-globulin was lower, as a
result of exposure to Wy 14,643, clofibrate, or DEHA, while these
effects were less pronounced or absent in mice treated with DEHP. This
could be due to the fact that DEHP is less effective at activating
PPAR
-dependent processes, including increasing reporter
gene activity in transient co-transfections (27), peroxisome proliferation (28), increasing replicative DNA synthesis (29), and
formation of hepatic tumors (29). In contrast, mAspAT mRNA levels
were not significantly different in response to any of the PPAR
activators, although Wy 14,643 did cause a small increase in this
mRNA compared with controls. These results suggest that expression
of FAT and FATP, but not mAspAT, mRNA in the liver is under the
control of PPAR
.
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FAT and FATP mRNAs Are Induced by Various PPAR Activators
but Not by a PPAR
Activator in Rat Hepatoma Cells--
To further
examine the effects of a wide variety of PPAR activators on the
induction of FAT and FATP mRNAs, we used the rat hepatoma Fao cells
line that is responsive to peroxisome proliferators (19). Fao cells
were treated with various PPAR activators, including Wy 14,643, ciprofibrate, troglitazone, carbacyclin, indomethacin, ibuprofen, and
perfluorooleate for 6 or 24 h, and the levels of FAT, FATP, and
mAspAT mRNAs, together with control mRNAs, were measured by
Northern blots as shown in Fig. 3. Fao
cells responded to most PPAR
activators examined (Wy 14,643, ciprofibrate, indomethacin, ibuprofen, and perfluorooleate) as assessed
by the increased levels of peroxisomal acyl-CoA oxidase and HD
mRNAs. Time courses of induction was different among the
activators, and indomethacin was the least effective. FAT and FATP
mRNAs were increased by PPAR
activators similarly as the
peroxisomal mRNAs were while the effect of indomethacin on these
mRNAs was negligible. Thus the relative abilities of various
PPAR
activators to induce FAT and FATP mRNAs were of similar
magnitude to those observed with peroxisomal enzyme mRNAs. Levels
of mAspAT and apoE mRNA were not affected by any treatment. The
results obtained using cultured hepatoma cells are consistent with
those observed in the liver of mice treated with PPAR
activators. In
contrast to the PPAR
activators, a typical PPAR
activator
Troglitazone did not cause any change in the levels of all the
mRNAs examined. Combined, these results suggest that both the FAT
and FATP genes are PPAR
target genes.
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Induction of FAT and FATP in the Liver Is Mediated by
PPAR--
To directly examine the role of PPAR
in the induction
of FAT and FATP mRNAs, we utilized the PPAR
-null mouse (7).
Northern blot analyses of liver RNA from (+/+) and (
/
) mice fed
either a control diet or one containing 0.1% Wy 14,643 are shown in
Fig. 4. FAT and FATP mRNAs as well as
HD mRNA were higher in the wild-type mice (+/+) fed Wy 14,643, and
this effect was not observed in the (
/
) mice fed Wy 14,643. In
addition,
2u-globulin mRNA was significantly lower in (+/+) mice
fed Wy 14,643 but unaffected by Wy 14,643 feeding in (
/
) mice as
reported previously (17). These results indicate that PPAR
has an
obligatory role in Wy 14,643 induction of FAT and FATP mRNAs in the
mouse liver. This could be due to direct interaction of PPAR
with
peroxisome proliferator responsive elements, although peroxisome
proliferator responsive elements have not been indentified in these
genes to date. Alternatively, the increase in gene transcription of
FATP as a result of PPAR
activators (30) could be the result of
other PPAR
-dependent events that indirectly result in
altering gene expression of fatty acid transport proteins to compensate
for the increase in fatty acid catabolism.
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FAT and FATP Are Induced by PPAR Activators in White Adipose
Tissue--
Although FAT and FATP mRNAs were induced by PPAR
activators in hepatoma cells and in a PPAR
-dependent
manner in the liver, the possible involvement of PPAR
in other
tissues where the
-type receptor is predominant over the
-type
cannot be excluded (31). Therefore, we examined the effect of PPAR
activators in adipose tissue of mice. We used KKAy obese mice (18) for
this purpose to facilitate adipose tissue preparation. We utilized two
white adipose tissue stores, interintestinal and subcutaneous, and
brown adipose tissue. Northern blots from the three adipose tissues are
shown in Fig. 5. Changes in the levels of
several mRNAs were different among the three adipose stores. Brown
adipose did not respond to the PPAR
or PPAR
activators compared
with controls. Basal levels of FAT, FATP, A-FAB (adipose-type fatty
acid-binding protein), lipoprotein lipase, and leptin mRNAs were
lower in interintestinal white adipose than those in subcutaneous white
adipose tissue. The greatest effect induced by PPAR activators was
observed in interintestinal adipose. In both white adipose tissues, FAT
and FATP were induced most efficiently by pioglitazone followed by troglitazone and then clofibrate, just as other characterized PPAR
target genes such as A-FAB, lipoprotein lipase, and leptin. Thus, both
FAT and FATP were induced by PPAR
activators in white adipose
tissue.
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Comparison of FAT and FATP-- The responses of both putative LCFA transporter mRNAs to PPAR activators were similar, but not exactly coordinated. The differences were: 1) FATP was not induced as much in the intestine as FAT (see Fig. 1); 2) most peroxisome proliferators induced both mRNAs in the liver, but DEHP only induced FAT (see Fig. 2); and 3) FATP responded by rapid or transient induction, but FAT did not (see the lanes of RNA samples from the cells treated for 6 h with Wy 14,643, carbacyclin, and perfluorooleate in Fig. 3). These results suggest a complexity in the PPAR-mediated transcriptional activation (32) and/or differences in their physiological roles.
During the completion of this manuscript, another paper concerning FATP and PPAR activators was published (30). Their conclusion on regulation of the expression of FATP is essentially the same as ours. We extend these observations by suggesting that FAT, in addition to FATP, may have an important role in fatty acid uptake and lipid homeostasis. Furthermore, we demonstrate that in liver, PPAR ![]() |
FOOTNOTES |
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* 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. Fax: 81-474-76-6195. E-mail: motojima{at}phar.toho-u.ac.jp.
1 The abbreviations used are: LCFA, long chain fatty acid; FAT, fatty acid translocase; mAspAT, mitochondrial aspartate aminotransferase; FATP, fatty acid transport protein; PPAR, peroxisome proliferator-activated receptor; Wy 14,643, 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-thio)acetic acid; DEHA, di(2-ethyhexyl)adipate; DEHP, di(2-ethylhexyl)phthalate; HD, hydratase-dehydrogenase; PCR, polymerase chain reaction; FAB, fatty acid-binding protein.
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REFERENCES |
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