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J. Biol. Chem., Vol. 278, Issue 28, 25468-25480, July 11, 2003
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The Human Apolipoprotein AV Gene Is Regulated by Peroxisome Proliferator-activated Receptor-
and Contains a Novel Farnesoid X-activated Receptor Response Element*
From the GlaxoSmithKline, 25 Avenue du Québec, 91951 Les Ulis cedex, France
Received for publication, February 6, 2003 , and in revised form, March 21, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The newly identified apolipoprotein AV (apoAV) gene is a key player in determining plasma triglyceride concentrations. Because hypertriglyceridemia is a major independent risk factor in coronary artery disease, the understanding of the regulation of the expression of this gene is of considerable importance. We presently characterize the structure, the transcription start site, and the promoter of the human apoAV gene. Since the peroxisome proliferator-activated receptor-
(PPAR) and the
farnesoid X-activated receptor (FXR) have been shown to modulate the
expression of genes involved in triglyceride metabolism, we evaluated the
potential role of these nuclear receptors in the regulation of apoAV
transcription. Bile acids and FXR induced the apoAV gene promoter activity.
5'-Deletion, mutagenesis, and gel shift analysis identified a heretofore
unknown element at positions –103/–84 consisting of an inverted
repeat of two consensus receptor-binding hexads separated by 8 nucleotides
(IR8), which was required for the response to bile acid-activated FXR. The
isolated IR8 element conferred FXR responsiveness on a heterologous promoter.
On the other hand, in apoAV-expressing human hepatic Hep3B cells, transfection
of PPAR specifically enhanced apoAV promoter activity. By deletion,
site-directed mutagenesis, and binding analysis, a PPAR response
element located 271 bp upstream of the transcription start site was
identified. Finally, treatment with a specific PPAR activator led to a
significant induction of apoAV mRNA expression in hepatocytes. The
identification of apoAV as a PPAR target gene has major implications
with respect to mechanisms whereby pharmacological PPAR agonists may
exert their beneficial hypotriglyceridemic actions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Recent epidemiological studies have shown that hypertriglyceridemia is a major independent risk factor for coronary heart disease, which remains as a major cause of mortality in the Western world (1–6). Understanding the factors that control plasma triglyceride levels and the genes that primarily reflect circulating concentrations of triglyceride-rich lipoproteins is thus of major importance and may provide new opportunities for therapeutic intervention in atherogenic dyslipidemia.
Pharmacological activation of peroxisome proliferator-activated
receptor-
(PPAR1; NR1C1)
lowers plasma levels of triglyceride-rich lipoproteins markedly
(7,
8). PPAR is a fatty
acid-activated nuclear transcription factor that regulates the expression of
genes involved in lipid and energy metabolism
(9,
10). PPAR
heterodimerizes with the retinoid X receptor (RXR; NR2B1) and
binds to specific DNA sequence elements, designated PPREs, which consist of a
direct repeat of the hexanucleotide core motif PuGGTCA separated by 1 or 2
nucleotides (DR1 or DR2). PPAR mediates the hypotriglyceridemic effect
of fibrates by regulating the transcription of key genes associated with
different intra- and extracellular metabolic pathways. Fibrates increase
cellular fatty acid transport and uptake, conversion to acyl-CoA derivatives,
and catabolism by 
-oxidation, which results in decreased substrate
availability for triglyceride synthesis and very low density lipoprotein
production by the liver. In addition, activated PPAR promotes lipolysis
and clearance of triglyceride-rich lipoproteins, thus decreasing the plasma
levels of triglycerides (11).
These extracellular effects have been attributed to an increase in LPL
activity via induction of its expression in the liver and reduction of hepatic
expression (and subsequently serum levels) of apolipoprotein CIII (apoCIII),
an established inhibitor of LPL activity and remnant particle catabolism
(7,
12–15).
The farnesoid X-activated receptor (FXR, NR1H4) is another member of the
nuclear receptor superfamily that has been described as a key regulator in the
control of plasma triglyceride levels
(16). The expression of FXR is
restricted to the liver, intestine, kidney, and adrenal cortex
(17,
18). FXR is a bile
acid-activated receptor that alters transcription by binding as a heterodimer
with RXR to response elements (FXREs) within the regulatory regions of target
genes
(16–21).
With two exceptions, FXREs consist of an inverted repeat of the canonical
hexanucleotide core motif PuGGTCA spaced by one base pair (IR1)
(16). Recently, an inverted
repeat with no spacer (IR0) in the dehydroepiandrosterone sulfotransferase
gene (22) and an everted
repeat of the core motif separated by 8 nucleotides (ER8) in the multidrug
resistance-associated protein 2 (MRP2) gene
(23) have also been reported
as elements required for FXR-dependent transcriptional activation. FXR acts as
a bile acid sensor in the liver, where it mediates the negative feedback of
bile acid biosynthesis via induction of small heterodimer partner (NR0B2) and
subsequent repression of cholesterol 7-hydroxylase gene
(CYP7A1) expression
(24–27),
the rate-limiting enzyme in this pathway, and the sterol 12-hydroxylase
gene (CYP8B1), the specific enzyme required for cholic acid synthesis
(28). FXR also controls the
transport of bile acids in the liver and intestine. It promotes the excretion
of bile acids from hepatocytes into the bile by induction of bile salt export
pump gene expression (29) and
stimulates the enterohepatic circulation of bile acids by activation of the
transcription of ileal-bile acid-binding protein (I-BABP)
(30). In addition, recent
findings have shown that FXR regulates lipoprotein metabolism
(16). Phospholipid transfer
protein and apoAI, two major players in plasma high density lipoprotein
metabolism, have been described as FXR target genes
(31–33).
Studies in rodents have revealed the role of FXR in regulating triglyceride
levels. The administration of either a synthetic FXR ligand (GW4064)
(34) or a natural FXR ligand
(cholic acid) (35) to rodents
resulted in at least a 50% decrease in plasma triglyceride levels. Consistent
with these observations, FXR-deficient mice exhibited a 150% increase in
plasma triglyceride levels
(36). Importantly, Edwards and
co-workers (35) have reported
that activated FXR induces the expression of apoCII, an obligate cofactor for
LPL, which in turn would result in the hydrolysis of triglycerides in
chylomicrons and very low density lipoprotein. These data provide a mechanism
to explain the hypotriglyceridemic effects of the natural FXR activator
chenodeoxycholic acid (CDCA) when administered to patients with
cholesterol-rich gallstones
(37).
Recently, a novel apolipoprotein, designated apoAV, was suggested to play a significant role on plasma triglyceride metabolism. Pennacchio et al. (38) described the discovery of apoAV when 200 kb of orthologous sequences spanning the apoAI/CIII/AIV gene cluster were compared in humans and mice. At the same time, van der Vliet et al. (39) identified apoAV as a protein associated with an early phase of liver regeneration. Interestingly, the predicted amino acid sequence showed appreciable homology with apoAI and apoAIV. Evidence for the involvement of apoAV in triglyceride metabolism was obtained in mice, in which serum triglyceride concentrations were decreased to one-third when a human apoAV transgene was overexpressed (38) or adenoviral vectors expressing mouse apoAV were injected (40). Conversely, plasma triglyceride levels were 4-fold elevated in knockout mice lacking apoAV compared with their wild-type littermates (38). Furthermore, single nucleotide polymorphisms (SNPs) across the locus of the apoAV gene were found to be significantly associated with plasma triglyceride and very low density lipoprotein mass levels in several ethnic groups (38, 41–45). Notwithstanding, the specific biological functions of apoAV in triglyceride metabolism or liver regeneration are still unknown. Pennacchio et al. (38) reported a genomic organization of human apoAV with four exons. In contrast, van der Vliet et al. (39) reported three exons. Both groups showed that the expression of apoAV mRNA was restricted to the liver.
The current studies were undertaken to provide a comprehensive analysis of
the regulation of human apoAV gene expression. We presently describe the
genomic structure, the transcription start site, and the promoter of the human
apoAV gene. Inasmuch as PPAR and FXR play key roles in the regulation
of genes involved in triglyceride metabolism, we sought to evaluate their
potential effects on human apoAV gene expression. We show that a novel FXRE is
present in the promoter of human apoAV and demonstrate that human apoAV is a
bona fide PPAR target gene. Our findings provide new insights
into the mechanisms whereby PPAR and its ligands lower plasma
triglyceride levels.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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RNA Ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE)—The start of transcription of human apoAV was identified by using GeneRacerTM (Invitrogen). This kit provides a modification of the RACE method that ensures the capture of only full-length 5' ends of specific transcripts via elimination of truncated messages from the amplification process. Human liver total RNA (Clontech) was treated with calf intestinal phosphatase and tobacco acid pyrophosphatase according to the instructions of the kit. After the selective ligation of an RNA oligonucleotide to the 5'-ends of decapped mRNA, first strand cDNA synthesis was carried out with the ThermoScriptTM RNase H– reverse transcriptase (Invitrogen; not supplied in the GeneRacerTM kit) at 50 °C for 50 min and a reverse human apoAV-specific primer (JCR81; see Table I). The cDNA 5'-end was amplified by using PlatinumTM TaqDNA Polymerase High Fidelity (Invitrogen; not supplied in the GeneRacerTM kit) and primers GeneRacerTM 5' and JCR80 (Table I). The PCR conditions were as follows: 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 65 °C for 30 s, and 68 °C for 1 min. The sample was then diluted 20-fold, and 1 µl was used in a second PCR with nested primers (GeneRacerTM 5' Nested and JCR79). DNA products were cloned into pCR4-TOPOTM vector (Invitrogen) and then sequenced.
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Plasmids—Several constructs containing the 5'-flanking
region of human apoAV (–2455/+18, –617/+18, –617/+529,
–535/+18, –437/+18, –242/+18, and –82/+18, relative to
the transcription start site reported in this paper) were obtained by PCR from
human bacterial artificial chromosome-RPCI-11–442E11 DNA (BACPAC
Resources, Children's Hospital Oakland Research Institute) with
PlatinumTM TaqDNA Polymerase High Fidelity (Invitrogen). Forward
primers were tailed with a KpnI restriction site. Reverse primers
were tailed with an NheI site. The PCR products were digested with
KpnI and NheI and cloned into the corresponding sites of the
promoterless firefly (Photinus pyralis) luciferase reporter plasmid
pGL3-basic (Promega), generating p–2455/+18hAvLUC,
p–617/+18hAvLUC, p–617/+529hAvLUC, p–535/+18hAvLUC,
p–437/+18hAvLUC, p–242/+18hAvLUC, and p–82/+18hAvLUC.
Site-directed mutagenesis of the constructs p–2455/+18hAvLUC,
p–617/+18hAvLUC, and p–242/+18hAvLUC were accomplished using the
QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the
recommendations of the manufacturer. The vector pGL3-TK contains a fragment
corresponding to nt –109 to +20 of the thymidine kinase (TK) gene
promoter of herpes simplex virus
(46) subcloned into the
BglII/HindIII sites of the pGL3-basic vector. The reporter
plasmids p(AvIR8)n-TK (n = 1–4) were
generated by insertion of 1–4 copies of a double-stranded
oligonucleotide (obtained by annealing JCR139 and JCR140; see
Table I) containing the
sequences spanning nt –109 to –80 into the BglII site of
pGL3-TK. Similarly, the four-copy mutant IR8 constructs
p(mt1AvIR8)4-TK, p(mt2AvIR8)4-TK, and
p(mt3AvIR8)4-TK were generated by annealing oligonucleotides JCR159
with JCR160, JCR161 with JCR162, and JCR163 with JCR164, respectively, before
ligation into BglII-digested pGL3-TK. The luciferase reporter plasmid
p(ER8)4-TK contains four copies of the rat MRP2 ER8 element
(23) in front of TK promoter
and was provided by L. Moore (GlaxoSmithKline, Research Triangle Park, NC).
Plasmids expressing human cDNAs for PPAR, RXR, and FXR were
provided by J. A. Holt and J. M. Maglich (GlaxoSmithKline, Research Triangle
Park, NC). The backbone of those plasmids was the mammalian expression vector
pSG5 with a modified polylinker. Plasmid DNA was prepared using the Qiagen
endotoxin-free maxipreparation method and quantitated spectrophotometrically.
The integrities of all plasmids were verified by DNA sequencing.
Cell Transfection and Reporter Assays—Human hepatoblastoma
Hep3B and monkey kidney CV-1 cell lines were cultured in Eagle's basal medium
supplemented with nonessential amino acids, 2 mM L-glutamine, 100
units/ml penicillin, and 100 µg/ml streptomycin sulfate (medium A) and 10%
(v/v) fetal calf serum (FCS). On day 0, cells were seeded on 24-well plates at
a density of 3 x 105 or 5 x 104 cells/well
for Hep3B or CV-1, respectively. On day 1, cells were transfected with FuGENE
6 reagent (Roche Applied Science) according to the manufacturer's
instructions. Typically, each well of a 24-well plate received 100 ng of
firefly luciferase reporter plasmid and, when indicated, 200 ng of plasmids
expressing human PPAR or FXR. Effector plasmid dosage was kept constant
by the addition of appropriate amounts of the empty expression vector pSG5.
100 ng/well of a sea pansy (Renilla reniformis) luciferase plasmid
pRL-TK (Promega) was included in all transfections as an internal control for
transfection efficiency. After 5 h, cells were switched to medium A
supplemented with 10% delipidated calf serum (Sigma) in the presence, when
indicated, of either 1 µM GW9003 (GlaxoSmithKline), 50 or 100
µM CDCA, 1 µM GW4064 (GlaxoSmithKline), or the
vehicle (Me2SO). On day 3, cell lysates were prepared by shaking
the cells in 200 µlof1x Promega lysis buffer for 15 min at room
temperature. Firefly and Renilla luciferase activities were measured
using a Dual-Luciferase® Reporter Assay System (Promega) and a Lumistar
luminometer (BMG Lab Technologies). Firefly luciferase activity values were
divided by Renilla luciferase activity values to obtain normalized
luciferase activities. To facilitate comparisons within a given experiment,
activity data were presented either as relative luciferase activities or as
-fold induction over the normalized activity of the reporter plasmid in the
absence of nuclear receptor cotransfection and agonist supplementation. All
transfection experiments were performed at least three times and with each
experimental point done in triplicate. The data are expressed as the means
± S.D. Statistic significance analysis were done with Student's
t test.
Treatment of HepG2 Cells—On day 0, human hepatoblastoma HepG2 cells were plated on 24-well plates at 3.5 x 105 cells/well in medium A supplemented with 10% (v/v) FCS. On day 1, the medium was replaced with fresh medium A supplemented with 1% (v/v) FCS and 1 µM GW9003, 1 µM GW4064, or vehicle (Me2SO). On day 3, the cells were washed twice with phosphate-buffered saline and harvested for isolation of RNA.
Treatment of Primary Cultured Cynomolgus Monkey Hepatocytes— Cynomolgus monkey hepatocytes (batch L-1811; Biopredict) were seeded on day 0 at a density of 6 x 105 cells/well on 12-well plates in Williams' medium E supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin sulfate, 4 µg/ml insulin (medium B), and 10% (v/v) FCS. On day 1, cells were switched to medium B containing 100 nM dexamethasone, 1% (v/v) FCS and 1 µM GW9003, 1 µM GW4064, 30 µM lithocholic acid (LCA), or vehicle (Me2SO). After 24 h, a fresh experimental medium was provided. On day 3, the cells were washed twice with PBS and harvested for isolation of RNA.
Real Time PCR Quantification of mRNAs—Total RNA was prepared from primary cynomolgus hepatocytes, human THP-1 monocytes differentiated to macrophages, HepG2, Hep3B, Huh-7, and Caco-2 cells with the RNeasyTM Mini kit, the QIAshredderTM homogenizers, and the RNase-Free DNase set (Qiagen) according to the manufacturer's instructions. A 1-µg aliquot was used as a template for cDNA synthesis, employing the TaqManTM Reverse Transcription Reagents kit (Applied Biosystems). Specific primers for each gene were designed with Primer Express Software (PerkinElmer Life Sciences). The sequences of forward and reverse primers are shown in Table I. The specificity of the primers was verified by showing that the real time reverse transcriptase (RT)-PCR reaction product generated a single band after agarose gel electrophoresis. In addition, each couple of primers was tested in successive dilutions of cDNA to analyze and validate its efficiency. The reactions contained, in a final volume of 25 µl, 5 µl of diluted (1:10) cDNA, a 300 nM concentration of the forward and reverse primers, and 2x SYBRTM Green PCR Master Mix (Applied Biosystems). Real time PCRs were carried out in 96-well plates by using the ABI PRISMTM 7700 sequence detection system (Applied Biosystems). Since there is a high level of sequence identity for apoAV between humans and monkeys, the primers were valid for RNA from both primates. Levels of apoAV were normalized to 18 S to compensate for variations in input RNA amounts (18 S levels were unaffected by the treatments). All points were performed in triplicate during two independent experiments, and the RT-PCRs were carried out in duplicate for each sample. The relative amounts of mRNAs were calculated using the Comparative CT method as described in Ref. 55.
In Vitro Transcription/Translation and Electrophoretic
Mobility Shift Assay (EMSA)—Human PPAR, RXR, and FXR
proteins were synthesized in vitro from the corresponding expression
plasmids in rabbit reticulocyte lysate by using the TNT® Quick Coupled
transcription/translation system (Promega) according to the instructions of
the manufacturer. In order to obtain an unprogrammed lysate as a negative
control for EMSA, a reaction was performed with the empty vector pSG5.
Double-stranded oligonucleotides were radiolabeled by fill in with the Klenow
fragment of DNA polymerase I and used as probes. For competition experiments,
increasing -fold molar excesses of unlabeled probes were included during a
15-min preincubation on ice. Protein-DNA binding assays were performed as
described (47). AvDR1 is a
double-stranded oligonucleotide corresponding to nt –275 to –247
of human apoAV promoter. mtAvDR1 is a modified version of AvDR1 that contains
mutations corresponding to nt –263(G
C)/–262(G
A)/–261(T A). The control probe used in EMSA with
PPAR-RXR contains the PPRE of the rat mitochondrial
3-hydroxy-3-methylglutaryl-CoA synthase (mitHMGS) gene promoter
(48). The probe AvIR8 contains
the sequence spanning nt –109 to –80 of the human apoAV promoter.
The mutations present in the modified versions of AvIR8 (mt1AvIR8, mt2AvIR8,
and mt3AvIR8) are shown in Fig.
10B. The probe used as a control for competition
experiments with FXR-RXR contains the FXRE of the human I-BABP gene
promoter (30). Samples were
electrophoresed at 4 °C on a 4.5% polyacrylamide gel in 0.5x TBE
buffer (45 mM Tris, 45 mM boric acid, 1 mM
EDTA, pH 8.0). Gels were dried and analyzed using an Amersham Biosciences
PhosphorImager STORM 860 and ImageQuant software (Amersham Biosciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Characterization of the Structure of the Human ApoAV Gene, Identification of an Alternatively 5'-Untranslated Region, and Determination of the Transcription Start Site—Two previous reports coincided to show by Northern blot analysis that apoAV has transcripts of
1.3 and 1.9 kb in the human liver, which are likely to result from
alternative polyadenylation
(38,
39). However, the number of
exons and the details of the exon/intron organization in the 5' region
of the human apoAV gene have been contradictory. In order to delineate the
structure of the gene, we performed RLM-RACE. This powerful technique is a
modification of the RACE method that ensures the capture of only full-length
specific transcripts via elimination of truncated messages from the
amplification process. Thus, using antisense primers from the 3' region
of mRNA, it is possible to amplify all of the upstream exons and hence to
detect the presence of alternatively spliced exons. A primer was designed at
the last exon of human apoAV, and a specific reverse transcription was carried
out from human liver RNA. In order to avoid secondary structures of mRNA, we
made the first strand cDNA synthesis at 50 °C with a reverse transcriptase
having a high thermal stability (see "Experimental Procedures").
Subsequently, specific PCRs were performed using additional apoAV nested
primers (Table I). The PCR
products were cloned and sequenced. Based on the genomic sequence spanning
part of human chromosome 11q23 present in the GenBankTM data bank
(accession number AC007707
[GenBank]
), we obtained an exon/intron organization of apoAV
with two distinct 5'-untranslated regions
(Fig. 1A). As shown in
Fig. 1B, the four-exon
structure of apoAV is also present in apoAI and apoCIII genes (as deduced from
the analysis of sequences in NM_000039
[GenBank]
, NM_000040
[GenBank]
, X01038
[GenBank]
, X03120
[GenBank]
, and J00098
[GenBank]
)
but not in apoAIV (structure taken from Ref.
50). The most important
feature of the RLM-RACE method is that it permits the determination of the
transcription start site with more precision than the primer extension and the
classic 5'-RACE procedures. As shown in
Fig. 1C, a single
human apoAV transcription start site was identified. The newly identified
first exon, which was named exon 1, has only 12 untranslated nt and contains a
stop codon in frame with the first ATG, which is located in the following exon
(Fig. 1C).
Interestingly, a sequence of 24 nt was observed just after exon 1 in 4 of 20
clones. This novel sequence, which we named exon 2a, was flanked on the
5' side by an ag consensus splice acceptor site on the first intron and
on the 3' side by the exon previously described in the literature as
exon 1, which we renamed exon 2b (Fig.
1C). Therefore, the second exon has either 57 nt (exon
2b) or 81 nt (exon 2a plus exon 2b), depending on the utilization of an
internal acceptor site (Fig.
1A). No novel exons were observed, so the coding region
is distributed in three exons, in agreement with earlier results
(39). Considered together, the
human apoAV gene consists of four exons, the second exon with two alternative
acceptor sites and the last exon with two alternative polyadenylation sites.
On the other hand, we noticed the presence of an SNP (A G) 3 nt upstream
from the predicted translation start codon
(Fig. 1C).
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Expression of ApoAV in Human Cell Lines—Previously published Northern blots with RNA from several different human tissues have shown that apoAV expression is restricted to the liver (38, 39). We carried out real time PCR quantification assays with RNA from several human cell lines with different origins. We found that apoAV is expressed in HepG2 and Hep3B hepatoma cells with levels comparable with the liver, but not in hepatic Huh-7 cells. Consistent with the hepatic specific expression, intestinal Caco-2 cells and THP-1 monocytes differentiated to macrophages showed no detectable expression of apoAV (data not shown).
Cloning and Characterization of the Human ApoAV Gene
Promoter—Analysis of the 5'-flanking region of the human
apoAV gene identified a TATA box 30 nt upstream of position +1
(Fig. 1C), further
confirming the mapped transcription start site. No consensus Sp1 binding sites
were found within the first 3 kb, although several putative nuclear receptor
response elements containing imperfect repeats of the half-site PuGGTCA were
detected (see below). To investigate whether this 5'-flanking region
could drive transcription of a reporter gene, we subcloned 0.6 kb of the
human apoAV sequence upstream of the transcription start site into the firefly
luciferase pGL3-basic vector. The resulting construct, p–617/+18hAvLUC,
was transiently transfected into HepG2 and Hep3B cells. The activity of this
construct was about 5-fold the activity of the parental promoterless vector in
both cell lines (Fig. 2). Next,
we observed that neither the addition of a more upstream region, in construct
p–2455/+18hAvLUC, nor the inclusion of the first intron, in
p–617/+529hAvLUC, substantially altered promoter activity (data not
shown).
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Overexpression of the Nuclear Receptor PPAR Enhances
the Activity of the Human ApoAV Gene Promoter—ApoAV is an important
determinant of plasma triglyceride levels
(38,
40–45).
Inasmuch as PPAR regulates the expression of genes involved in
triglyceride metabolism (10),
we evaluated whether this nuclear receptor modulates human apoAV gene
expression. For this purpose, transient transfection studies were performed in
Hep3B cells. Cotransfection of a human PPAR expression plasmid resulted
in a significant increase of the activity of the firefly luciferase reporter
gene driven by the –617/+18 sequence of the human apoAV promoter
(Fig. 2). The effect of
PPAR overexpression was promoter-dependent, because it was not observed
with the promoterless pGL3-basic vector. In line with previous studies in
hepatic cells (48,
49), transiently transfected
PPAR showed substantial constitutive activity, possibly due to
activation by endogenous ligands. Nevertheless, we observed that it could be
further activated upon the exogenous addition of the specific PPAR
ligand GW9003 (Fig. 2).
Mapping of the Human ApoAV Promoter Site Conferring Responsiveness to
PPAR—In order to localize the region required for
PPAR activation, a series of progressively larger 5'-deletion
human apoAV promoter constructs from nt –2455 to +18 were transiently
transfected into Hep3B cells together with a human PPAR expression
plasmid in the presence or absence of GW9003. As shown in
Fig. 3, the sequence upstream
to position –437 can be deleted without loss of the response to
PPAR. However, further deletion up to nt –242 eliminated the
PPAR responsiveness. Analysis of the sequence in the
–437/–242 fragment revealed two potential hexamer binding sites
separated by a single nucleotide between nt –271 and –259
(Fig. 1C), thereby
conforming to the DR1 response element for PPAR (PPRE).
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To unequivocally characterize the DR1 as a PPRE, Hep3B cells were
cotransfected with a human PPAR expression vector and an apoAV
promoter-luciferase reporter plasmid in which the DR1 sequence was mutated in
the 3'-hexamer (Fig.
4A). In contrast to the native promoter construct,
PPAR and GW9003 had only a modest residual effect on the luciferase
gene expression of the construct bearing the mutated DR1
(Fig. 4B). These
results demonstrate that this DR1 indeed confers PPAR response to the
apoAV promoter.
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Binding Analysis of PPAR-RXR
Heterodimers to the apoAV DR1 Element—PPAR binds to DNA
as a heterodimer with RXR
(10). To investigate whether
PPAR-RXR heterodimers directly bind to the human apoAV gene promoter,
radiolabeled double-stranded oligonucleotides corresponding to portions of the
–617/+18 fragment of the apoAV promoter were used as probes in EMSA
experiments (Fig. 5). As
expected, the addition of in vitro translated PPAR and
RXR proteins resulted in the appearance of a robust retarded complex
when using a –275/–247 fragment containing the apoAV DR1
(Fig. 5A, lane
4), but not when –617/–593, –169/–147,
–144/–117, and –52/–31 probes
(Fig. 5A, lanes 2,
6, 8, and 10, respectively) or –484/–462,
–399/–374, –298/–263, and –109/–80
fragments were employed (data not shown). Furthermore, the specificity of the
RXR-PPAR heterodimerapoAV DR1 interaction was demonstrated by
competition analysis. The formation of the retarded complex was inhibited by
the addition of increasing concentrations of either the unlabeled apoAV DR1
probe (Fig. 5B,
lanes 5–7) or a cold probe containing the PPRE of the mitHMGS
gene (Fig. 5B,
lanes 11–13). In contrast, a cold double-stranded
oligonucleotide that is equivalent to the apoAV DR1 probe but harbors point
mutations in the 3' half-site (nt –263G C/–262G
A/–261T A in Fig.
4A) could not displace the labeled wild-type element
(Fig. 5B, lanes
8–10). In addition, it is worth noting that a more retarded band of
the labeled apoAV DR1 containing probe was visible upon the addition of
RXR alone (Fig.
5B). Further investigations will be done to elucidate the
nature of this band. Taken together, these results show that this apoAV DR1 is
a genuine PPRE.
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PPAR Agonists Induce ApoAV mRNA Levels in Human
Hepatoma HepG2 Cells and Primary Cultures of Cynomolgus
Hepatocytes—To analyze whether apoAV mRNA expression is regulated
by PPAR agonists, primary hepatocytes isolated from cynomolgus liver
and human hepatoma HepG2 cells were incubated for 48 h in medium containing
GW9003 or vehicle. As determined by quantitative real time RT-PCR, treatment
with the PPAR agonist markedly increased apoAV mRNA levels
(Fig. 6).
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Bile Acids Induce Human apoAV Gene Promoter Activity via the Nuclear Receptor FXR—FXR regulates lipoprotein metabolism (16), and FXR agonists have hypotriglyceridemic effects on rodents and humans (34, 35, 37). To determine whether FXR and bile acids are able to modulate the apoAV gene promoter activity, transient transfection assays were performed with a firefly luciferase reporter gene expression vector driven by the –617/+18 sequence of the human apoAV promoter. In Hep3B, the bile acid CDCA alone induced apoAV promoter activity to some extent (Fig. 7A). Cotransfection of a human FXR expression plasmid significantly enhanced CDCA-induced promoter activity. To further confirm the role of FXR in the bile acid-induction of the apoAV promoter, monkey kidney CV-1 cells, a cellular model frequently used in order to avoid the background of endogenous FXR (19–21, 32), were also employed. As shown in Fig. 7B, promoter activity was unaffected by CDCA in the absence of FXR supplied exogenously. By contrast, cotransfection of FXR led to a more than 10-fold induction of apoAV promoter activity in the presence of 100 µM CDCA. In both cell lines, the transactivation of the apoAV promoter-luciferase reporter plasmid by CDCA via FXR was dose-dependent (Fig. 7, A and B). Moreover, the effects of CDCA and FXR were promoter-dependent, because they were not observed with the promoterless pGL3-basic vector. Since bile acids may also exert their actions through FXR-independent pathways, we also tested the synthetic nonsteroidal specific FXR ligand GW4064 (34). As expected, treatment of Hep3B cells with GW4064 further enhanced the transactivation of the apoAV promoter by FXR (Fig. 7C). These data clearly demonstrated the existence of an FXR response element in the promoter of the human apoAV gene.
|
A Novel Element in the Human ApoAV Promoter Is Required for Transcriptional Activation by FXR—To localize the region within the human apoAV promoter that confers transcriptional responsiveness to FXR and bile acids, a series of constructs containing sequential 5'-deletions from nt –2455 to +18 of the apoAV promoter in front of the firefly luciferase reporter gene were transiently transfected into CV-1 cells together with a human FXR expression plasmid in the presence or the absence of 100 µM CDCA (Fig. 8). The results showed that the nucleotide sequence between –2455 and –242 could be removed without preventing strong activation of the reporter gene by bile acid-activated FXR. In contrast, deletion of the fragment between nt –242 and –82 completely abolished the induction of apoAV promoter activity by CDCA-activated FXR, indicating that this region mediates the effects of bile acids.
|
Surprisingly, analysis of the sequence in the –242/–82 fragment did not reveal any of the previously described FXR response element (IR0, IR1, or ER8) motifs (16, 22, 23). The only repeat of the hexanucleotide core motif PuGGTCA with a low degree of degeneration present within this region corresponds to an inverted repeat separated by 8 nucleotides (IR8) between nt –103 and –84 (Fig. 1C). Although a response element with this kind of organization has never been reported before, we mutated both halves of the IR8 site (Fig. 9A) in the context of the apoAV promoter on both the longest plasmid (Fig. 9B) and the shortest construct that still responds to FXR (Fig. 9C) and performed transient transfections in Hep3B cells. Whereas cotransfection of an FXR expression plasmid significantly enhanced wild-type apoAV promoter activity, FXR failed to induce the activity of the mutated constructs. These results unequivocally show that this IR8 element is required for the response to FXR and that there is no other FXRE in the promoter of human apoAV.
|
The ApoAV IR8 Element Confers FXR Responsiveness to Heterologous Promoters—To evaluate whether this IR8 element could confer FXR responsiveness to a heterologous promoter, we linked the apoAV IR8 site upstream of the TK promoter and the luciferase reporter gene. Reporter constructs containing 1–4 copies of the apoAV IR8 element were transiently transfected into Hep3B cells along with a human FXR expression plasmid in the presence or the absence of CDCA. As demonstrated in Fig. 10A, bile acid-activated FXR enhanced the activity of apoAV IR8-driven promoter constructs, whereas the reporter construct with the TK promoter alone was not stimulated at all. Indeed, an induction of about 50-fold was reached with four copies, and the response was manifested in a copy number-dependent manner, thereby confirming that the IR8 element functions as an FXRE.
Next, the effects of selective mutations of each half-site in the IR8
element (Fig. 10B)
were examined in Hep3B cells transfected with reporter constructs driven by
four copies of the apoAV IR8 in front of the TK promoter. The FXR-dependent
induction was abrogated when mutations were introduced in any half-site
(Fig. 10C). These
observations confirm the organization of this FXR response element as an
inverted repeat of the canonical hexanucleotide separated by 8 nt. Finally,
the ability of the apoAV IR8 to respond to FXR and bile acids was assessed by
comparison with the ER8 element described in the promoter of MRP2 gene
(23). The FXR-dependent
response of the MRP2 ER8 in front of TK was 2.5-fold higher than the
apoAV IR8 (Fig.
10A).
The FXR-RXR Heterodimer Binds Specifically to the ApoAV
IR8 Element—Direct binding of FXR/RXR heterodimers to the
human apoAV IR8 element was examined. For this purpose, gel shift assays were
performed using in vitro translated human FXR and RXR and a
radiolabeled double-stranded oligonucleotide containing the apoAV IR8 element.
The addition of FXR resulted in the appearance of a weak protein-DNA complex
band (Fig. 11, lane
2), a phenomenon that we also perceived in previously published gel shift
analysis performed with other FXREs (Fig. 5B in Ref.
23, Fig. 3B in Ref.
29, and Fig. 5A in
Ref. 30). Nevertheless, this
faint band disappeared, and a robust and more shifted band emerged when both
FXR and RXR were present (Fig.
11, lane 4). This RXR-FXR-IR8 binding was
specific, as demonstrated by competition of an excess of either unlabeled
wild-type apoAV IR8 oligonucleotide (Fig.
11, lanes 5–7) or a cold probe containing the IR1
sequence of the human I-BABP FXRE (Fig.
11, lanes 17–19). Furthermore, mutation of either
half-site (Fig. 10B)
abolished the ability of the probe to compete
(Fig. 11, lanes
8–16). These results demonstrate that FXR-RXR heterodimers
are able to directly and specifically interact with the apoAV IR8 and not with
cryptic binding motifs embedded within this site. We conclude that this novel
IR8 is a bona fide FXR response element.
|
ApoAV mRNA Levels Are Not Induced by Treatments with a FXR Agonist or a Bile Acid—To analyze whether apoAV mRNA expression is regulated by FXR agonists and bile acids, primary hepatocytes isolated from cynomolgus liver and human hepatoma HepG2 cells were incubated for 48 h in medium containing GW4064, LCA, or vehicle. Whereas the treatment of hepatocytes with the FXR agonist or LCA increased the expression of small heterodimer partner and bile salt export pump genes, no significant induction of apoAV expression was observed (Fig. 12).
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The present study aimed to comprehend the mechanisms controlling the human apoAV gene expression and to identify factors capable of positively modulating its transcription. Our data reveal that two nuclear receptors involved in the regulation of triglyceride metabolism, namely PPAR
and FXR, may control
the human apoAV gene.
The apoAV gene resides 27 kb distal to apoAIV in the apoAI/CIII/AIV
gene cluster on human chromosome 11q23
(38,
39). Our results showed that
the human apoAV gene contains four exons, with the first intron within the
5'-untranslated regions (Fig.
1A). With the exception of apoAIV
(50), both features are also
present in the rest of the apolipoprotein genes
(Fig. 1B). This
similarity gives further insight into the understanding of the process of
evolution of the apolipoprotein genes from a common ancestor
(51). In addition to the
previously reported alternative 3'-untranslated regions
(39), we found that the second
exon may have alternative length depending on the use of two distinct acceptor
sites. Interestingly, alternative transcripts have not been reported for other
members within this gene cluster. However, although some variation in terms of
mRNA stability may be inferred, none of the alternative transcripts contains a
different coding sequence. Strikingly, we came across an SNP (A G) 3 nt
upstream from the predicted translation start codon
(Fig. 1C). The
incidence of this SNP (called c.–3A G) and its association with
higher triglyceride levels were recently reported
(42). There is another ATG in
frame 9 nt downstream of the start codon
(Fig. 1C). The
nucleotide sequences surrounding both methionine codons conform to the Kozak
consensus sequence (RNNATGG), with a G at +4 and a purine (R), preferably A, 3
nucleotides upstream (52). It
remains as an interesting question for future research to determine whether
this c.–3A G polymorphism could modify the start of translation or
reduce the rate of apoAV protein synthesis. On the other hand, a T C SNP
in the 5'-region of the human apoAV gene, called SNP3, has also been
associated with elevated plasma triglyceride levels
(38,
41–45).
Luciferase reporter assays revealed that the change of the common allele T to
C, which is located at position –600 relative to the transcription start
site (Fig. 1C), has no
significant effect on the basal activity of the apoAV promoter. Similarly, the
induction by either PPAR or FXR of the apoAV promoter activity was not
affected by this change of T to C (data not shown). Analysis of the sequence
surrounding SNP3 reveals no apparent transcription factor binding sites. In
addition, EMSA showed no binding of nuclear receptors to this region
(Fig. 5A and data not
shown). Considered together, these data suggest that this polymorphism may not
be functional but, as proposed by others
(43), may act as a marker for
another functional change elsewhere in the gene cluster.
The presence of a TATA box 30 nt upstream of the start of transcription identified by RLM-RACE along with the results from reporter experiments showing transcriptional activity and responsiveness to nuclear receptors validate this proximal 5'-flanking region as the promoter of the human apoAV gene. The expression of apoAI, apoCIII, and apoAIV is controlled by a common enhancer sequence located at the distal regulatory region of the apoCIII gene (53). Based on the notion that apoAV and these apolipoprotein genes evolved from a common ancestral gene, it is tempting to speculate that a distal enhancer also regulates the apoAV gene. If this is the case, however, then the enhancer for apoAV should be elsewhere within a 26-kb XhoI fragment that contains only apoAV, which was able to induce specific expression of human apoAV when injected into mouse eggs (38). Further work will be required to fully assess this hypothesis.
Deletion, site-directed mutagenesis, and binding analysis demonstrated unequivocally that the apoAV gene promoter responds to bile acids and FXR via a novel response element (IR8). Furthermore, the IR8 element was capable of conferring FXR responsiveness on a heterologous promoter in a copy number-dependent manner, and selective mutation of each half-site in the IR8 abolished the nuclear receptor response. However, despite exhaustive efforts, we have not been able to demonstrate a significant induction of apoAV mRNA levels by either bile acids or the FXR ligand GW4064 in hepatocytes (Fig. 12). This paradox suggests that other factors might potentially interfere in the regulation by FXR. Recent studies are beginning to establish the notion that the DNA sequences of hormone response elements for several nuclear receptors, once thought to be specific, can be quite varied. The MRP2 ER8, for example, has been shown to function as a pregnane X receptor (NR1I2), constitutive androstane receptor (NR1I3), and FXR response element (23). It is possible that the novel IR8 could also act as response element for the convergence of alternative transcription factors present in the systems that we have analyzed, and the response to bile acids depends on the ratio between FXR and these factors. Alternatively, sequences in the 5'-flanking region of human apoAV upstream to position –2455 might respond negatively to bile acids and thereby counteract the effect through the proximal FXRE. Further studies will be necessary to determine whether potential activation of the apoAV gene by bile acids is dependent on a particular environmental or physiological cue.
The current study shows that treatment of hepatocytes with a PPAR
activator increased apoAV mRNA levels. Consistent with this observation,
induction of apoAV mRNA levels by fibrates has recently been reported
(54). Furthermore,
cotransfection of a PPAR expression plasmid induced the activity of the
human apoAV promoter, suggesting that the increase by PPAR activators
occurs, at least in part, via transcriptional mechanisms. Using
5'-deletion, mutagenesis, and gel shift analysis, a functional PPRE was
identified. A reporter construct bearing a mutated version of this PPRE still
showed a minimal transactivation by PPAR. The basis for this residual
response is unclear, and the presence of an additional, weak response element
between nt –617 and –271 should not be excluded.
It is well established that PPAR activators lower plasma
triglyceride levels by modulating the transcription of several lipid-related
genes (10). PPAR
induces the expression of enzymes involved in the uptake, transport, and
metabolism of fatty acids. As a result, the substrate availability for
triglyceride synthesis and very low density lipoprotein production is
diminished, which will ultimately decrease plasma triglyceride-rich
lipoprotein concentrations
(11). In addition, activated
PPAR may increase LPL-mediated lipolysis and clearance of
triglyceride-rich lipoprotein particles by stimulating LPL expression in the
liver and by reducing hepatic production of apoCIII
(12–15).
Our results demonstrate that apoAV is de facto a PPAR target
gene, which is consistent with the triglyceride-lowering role proposed for
apoAV. These findings shed new light on the mechanisms whereby PPAR
agonists lower plasma triglyceride levels.
|
FOOTNOTES |
|---|
* This work was supported by a Marie Curie Fellowship of the European Community program Quality of Life under contract number QLK5-CT-2000-60009. The costs of publication of this article were defrayed in part by the payment of page charges. This 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.: 33-1-69-29-61-22; Fax:
33-1-69-07-48-92; E-mail:
jcrodrig{at}freesurf.fr.
1 The abbreviations and trivial name used are: PPAR, peroxisome
proliferator-activated receptor ; apoAI, apoAIV, apoAV, apoCII, and
apoCIII, apolipoprotein AI, AIV, AV, CII, and CIII, respectively; CDCA,
chenodeoxycholic acid; DR, direct repeat; FXR, farnesoid X-activated receptor;
ER, everted repeat; FXRE, farnesoid X-activated receptor response element;
I-BABP, ileal-bile acid-binding protein; IR, inverted repeat; LCA, lithocholic
acid; LPL, lipoprotein lipase; mitHMGS, mitochondrial
3-hydroxy-3-methylglutaryl-CoA synthase; MRP2, multidrug resistance-associated
protein 2; nt, nucleotide(s); PPRE, peroxisome proliferator-activated receptor
response element; RACE, rapid amplification of cDNA ends; RLM-RACE, RNA
ligase-mediated RACE; RT-PCR, reverse transcriptase-PCR; RXR, retinoid
X receptor ; SNP, single nucleotide polymorphism; TK, thymidine kinase;
FCS, fetal calf serum; GW9003, propanoic acid,
2-[4-[[[[2-(4-chlorophenyl)-4-methyl-5-thiazolyl]carbonyl]
amino]methyl]phenoxy]-2-methyl-(9Cl).
|
ACKNOWLEDGMENTS |
|---|
We thank J. Kirilovsky and M. Walker for encouragement of this work; D. Grillot and P. Martres for scientific discussions; J. Pilot, V. Baudet, and G. Krysa for technical assistance; P. A. Wilson for bioinformatics analysis; E. Nicodeme for help with cynomolgus hepatocytes; J. Chapman (U551 INSERM, France) for critical reading of the manuscript; and A. Brewster for manuscript corrections.
|
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T. Degenhardt, M. Matilainen, K.-H. Herzig, T. W. Dunlop, and C. Carlberg The Insulin-like Growth Factor-binding Protein 1 Gene Is a Primary Target of Peroxisome Proliferator-activated Receptors J. Biol. Chem., December 22, 2006; 281(51): 39607 - 39619. [Abstract] [Full Text] [PDF]
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S. F. C. Vaessen, F. G. Schaap, J.-A. Kuivenhoven, A. K. Groen, B. A. Hutten, S. M. Boekholdt, H. Hattori, M. S. Sandhu, S. A. Bingham, R. Luben, et al. Apolipoprotein A-V, triglycerides and risk of coronary artery disease: the prospective Epic-Norfolk Population Study J. Lipid Res., September 1, 2006; 47(9): 2064 - 2070. [Abstract] [Full Text] [PDF]
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E. A. Ruiz-Narvaez, Y. Yang, Y. Nakanishi, J. Kirchdorfer, and H. Campos APOC3/A5 haplotypes, lipid levels, and risk of myocardial infarction in the Central Valley of Costa Rica J. Lipid Res., December 1, 2005; 46(12): 2605 - 2613. [Abstract] [Full Text] [PDF]
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M. Ishihara, T. Kujiraoka, T. Iwasaki, M. Nagano, M. Takano, J. Ishii, M. Tsuji, H. Ide, I. P. Miller, N. E. Miller, et al. A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration J. Lipid Res., September 1, 2005; 46(9): 2015 - 2022. [Abstract] [Full Text] [PDF]
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P. J. Talmud, J. Palmen, W. Putt, L. Lins, and S. E. Humphries Determination of the Functionality of Common APOA5 Polymorphisms J. Biol. Chem., August 5, 2005; 280(31): 28215 - 28220. [Abstract] [Full Text] [PDF]
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A. E. Schultze, W. E. Alborn, R. K. Newton, and R. J. Konrad Administration of a PPAR{alpha} agonist increases serum apolipoprotein A-V levels and the apolipoprotein A-V/apolipoprotein C-III ratio J. Lipid Res., August 1, 2005; 46(8): 1591 - 1595. [Abstract] [Full Text] [PDF]
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X. Prieur, T. Huby, H. Coste, F. G. Schaap, M. J. Chapman, and J. C. Rodriguez Thyroid Hormone Regulates the Hypotriglyceridemic Gene APOA5 J. Biol. Chem., July 29, 2005; 280(30): 27533 - 27543. [Abstract] [Full Text] [PDF]
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A. Lookene, J. A. Beckstead, S. Nilsson, G. Olivecrona, and R. O. Ryan Apolipoprotein A-V-heparin Interactions: IMPLICATIONS FOR PLASMA LIPOPROTEIN METABOLISM J. Biol. Chem., July 8, 2005; 280(27): 25383 - 25387. [Abstract] [Full Text] [PDF]
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S.-O. Olofsson ApoA-V: The Regulation of a Regulator of Plasma Triglycerides Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1097 - 1099. [Full Text] [PDF]
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A. Genoux, H. Dehondt, A. Helleboid-Chapman, C. Duhem, D. W. Hum, G. Martin, L. A. Pennacchio, B. Staels, J. Fruchart-Najib, and J.-C. Fruchart Transcriptional Regulation of Apolipoprotein A5 Gene Expression by the Nuclear Receptor ROR{alpha} Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1186 - 1192. [Abstract] [Full Text] [PDF]
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J. Gao, Y. Wei, Y. Huang, D. Liu, G. Liu, M. Wu, L. Wu, Q. Zhang, Z. Zhang, R. Zhang, et al. The Expression of Intact and Mutant Human apoAI/CIII/AIV/AV Gene Cluster in Transgenic Mice J. Biol. Chem., April 1, 2005; 280(13): 12559 - 12566. [Abstract] [Full Text] [PDF]
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P. J. O'Brien, W. E. Alborn, J. H. Sloan, M. Ulmer, A. Boodhoo, M. D. Knierman, A. E. Schultze, and R. J. Konrad The Novel Apolipoprotein A5 Is Present in Human Serum, Is Associated with VLDL, HDL, and Chylomicrons, and Circulates at Very Low Concentrations Compared with Other Apolipoproteins Clin. Chem., February 1, 2005; 51(2): 351 - 359. [Abstract] [Full Text] [PDF]
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C.-Q. Lai, S. Demissie, L. A. Cupples, Y. Zhu, X. Adiconis, L. D. Parnell, D. Corella, and J. M. Ordovas Influence of the APOA5 locus on plasma triglyceride, lipoprotein subclasses, and CVD risk in the Framingham Heart Study J. Lipid Res., November 1, 2004; 45(11): 2096 - 2105. [Abstract] [Full Text] [PDF]
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H. Jakel, M. Nowak, E. Moitrot, H. Dehondt, D. W. Hum, L. A. Pennacchio, J. Fruchart-Najib, and J.-C. Fruchart The Liver X Receptor Ligand T0901317 Down-regulates APOA5 Gene Expression through Activation of SREBP-1c J. Biol. Chem., October 29, 2004; 279(44): 45462 - 45469. [Abstract] [Full Text] [PDF]
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I. Pineda Torra, L. P. Freedman, and M. J. Garabedian Identification of DRIP205 as a Coactivator for the Farnesoid X Receptor J. Biol. Chem., August 27, 2004; 279(35): 36184 - 36191. [Abstract] [Full Text] [PDF]
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F. G. Schaap, P. C. N. Rensen, P. J. Voshol, C. Vrins, H. N. van der Vliet, R. A. F. M. Chamuleau, L. M. Havekes, A. K. Groen, and K. W. van Dijk ApoAV Reduces Plasma Triglycerides by Inhibiting Very Low Density Lipoprotein-Triglyceride (VLDL-TG) Production and Stimulating Lipoprotein Lipase-mediated VLDL-TG Hydrolysis J. Biol. Chem., July 2, 2004; 279(27): 27941 - 27947. [Abstract] [Full Text] [PDF]
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C.-Q. Lai, E-S. Tai, C. E. Tan, J. Cutter, S. K. Chew, Y.-P. Zhu, X. Adiconis, and J. M. Ordovas The APOA5 locus is a strong determinant of plasma triglyceride concentrations across ethnic groups in Singapore J. Lipid Res., December 1, 2003; 44(12): 2365 - 2373. [Abstract] [Full Text] [PDF]
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