# Differences in Transcriptional Activation by the Two Allelic (L162V Polymorphic) Variants of PPARα after Omega-3 Fatty Acids Treatment

Authors: Iwona Rudkowska; Mélanie Verreault; Olivier Barbier; Marie-Claude Vohl
Journal: PPAR Research
 (2009)

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## Abstract

Omega-3 fatty acids (FAs) have the potential to regulate gene
expression via the peroxisome proliferator-activated receptorα (PPARα);
therefore, genetic variations in this gene may
impact its
transcriptional activity on target genes. It is hypothesized that
the transcriptional activity by wild-type L162-PPARα is enhanced
to a greater extent than the mutated variant (V162-PPARα) in the
presence of eicosapentaenoic acid (EPA), docosahexaenoic acid
(DHA) or a mixture of EPA:DHA. To examine the functional
difference of the two allelic variants on receptor activity,
transient co-transfections were performed in human hepatoma HepG2
cells activated with EPA, DHA and EPA:DHA mixtures. Results
indicate that the addition of EPA or DHA demonstrate potential to
increase the transcriptional activity by PPARα with respect to
basal level in both variants. Yet, the EPA:DHA mixtures enhanced
the transcriptional activity to a greater extent than individual
FAs indicating possible additive effects of EPA and DHA.
Additionally, the V162 allelic form of PPARα demonstrated
consistently lower transcriptional activation when incubated with
EPA, DHA or EPA:DHA mixtures than, the wild-type variant. In
conclusion, both allelic variants of the PPARα L162V are activated
by omega-3 FAs; however, the V162 allelic form displays a lower
transcriptional activity than the wild-type variant.


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## Body

## 1. Introduction

Higher intake of long-chain n-3
fatty acids (FAs) eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) has been recommended to decrease plasma triglyceride (TG) levels. Conventionally, the mechanism of
action after n-3 FAs intake focused on plasma membrane fluidity; however,
recently the emphasis shifted to regulation of gene expression [1]. In
particular, FAs and their derivatives are physiological ligands of peroxisome
proliferator-activated receptor α (PPARα). As such they activate the PPARα-retinoid X receptor (RXR) heterodimer-dependent gene transcription by
binding to the peroxisome proliferator response elements (PPRE) in the promoter
region of target genes [2]. Target genes include lipoprotein lipase (LPL) involved in
plasma TG clearance [2].Several
polymorphisms within the PPARα gene and the encoded
proteins have been identified including L162V
and V227A, which are the most common PPARα polymorphisms reported [3].
Of particular interest, the PPARα
L162V polymorphism has
been associated with obesity indices and plasma lipid levels in numerous
studies [4–8].
Additionally, Robitaille et al. in 2004 found that the interaction
between the PPARα
L162V polymorphism and fat intake
estimated from a food frequency questionnaire (FFQ) explains a significant
percentage of the variance observed in waist girth in a sample of 260
French-Canadians [9].
Tai et al. in 2005 [10] also established
that the effect of the PPARα
L162V polymorphism
on plasma TG and apolipoprotein (apo)-CIII
concentrations depends on the dietary polyunsaturated FA (PUFA), with a high intake triggering
lower TG in carriers of the V162-PPARα variant.  Finally, Paradis
et al. in 2002 [11] demonstrated that the interindividual variations in total
cholesterol, apo A-I, and cholesterol concentrations in small low-density
lipoprotein (LDL) particles observed after modification of the
polyunsaturated/saturated FA ratio of the diet is partly attributable to the PPARα
L162V polymorphism. Clearly, both epidemiological and
interventional studies demonstrate a relation between the PPARα L162V polymorphisms, metabolic
parameters, and FAs intake; yet, only two functional studies examined the
receptor activity of the L162V polymorphic variants
activated with synthetic agonists-fibrates [4, 12]. It was demonstrated that the effect of the L162V polymorphic variants on the transcriptional activation
was associated with the concentration of the ligand to which it is exposed
[12].For that reason, the aim
of this functional study was to determine whether the transcriptional activity by the wild-type
variant, L162-PPARα,
is enhanced in the presence of natural PPARα
agonists-omega-3 FAs, mimicking the action of synthetic PPARα
agonists, comparatively to the variant, V162-PPARα.


## 2. Laboratory Methods

### 2.1. Plasmid Construction

The wild-type L162-PPAR expression
plasmid (pSG5-hPPARα vector) was a kind gift from Pr. B. Staels (Unité
INSERM 545, Institut Pasteur de Lille, France). The pSG5-mRXRα
plasmid was described previously [13]. The V162-PPARα
expression plasmid was derived from the wild-type, through site-directed
mutagenesis (QuickChange site-directed mutagenesis kit, Stratagene, La Jolla, Calif,
USA) using the
5′-CGATTTCACAAGTGCGTTTCTGTCGGGATG-3′
oligonucleotide  (the nucleotide in boldface type denotes the C→G change mutated base). Variant cDNAs were
directly sequenced to confirm that no spurious base changes have been
introduced during the procedure. As a response element representative of the
vast variety of human PPREs, we choose the consensus artificial direct repeat
(DR)1 sequence for analyzing the functional consequences of L162V variation on
PPARα activation by omega-3
FAs. Thus, a reporter plasmid (DR1x6-PPRE)
was
generated by cloning in front of the thymidine kinase promoter-driven luciferase
reporter gene (TKpGL3 vector), six
copies of the
5′-AAAACT
AGGTCAAAGGTCA
CGG-3′
sequence where underlined nucleotides correspond to the direct repeat of the
AGGTCA hexamer.


### 2.2. Transient Transfection and N-3 Fatty Acids Activation

Human
hepatoma HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented by 10% fetal bovine serum (FBS), 1% of streptomycin penicillin, 1%
of sodium pyruvate and 1% of glutamine. HepG2 cells were plated at a density of75×103 cells/well of 24-well plates which were then transfected using the ExGen
reagent (Invitrogen, Burlington, Canada) with 50 ng of the DR1x6-PPRE reporter plasmid, 10 ng of the PPARα (wild-type or mutant) and RXR expression plasmids and 30 ng of the pRL-NULL expression vector (Promega) for 6 hours 37°C. All samples were complemented with pBS-SK+
plasmid (Stratagene) to an identical amount (500 ng/well). Similar experiments were performed with a
negative control consisting of the empty TK-pGL3-basic plasmid (Promega). After
transfection, cells were cultured in DMEM supplemented by 0.2% FBS for 24 hours to strengthen
cell membrane before addition of FAs. Afterwards, cells were transactivated for 24 hours in absence or
presence of omega-3 FAs in concentrations varying between 1–15 μM to reflect
biological plasma or red blood cells concentration of FAs [14]. Cells were treated with either
solvent (dimethyl sulfoxide (DMSO), 0.01% final concentration), or treatments of EPA and/or DHA (Sigma-Aldrich,
Oakville, ON, Canada).
Briefly, pure EPA or DHA was dissolved by serial dilution to 1, 5, 10,
and 15 μM in DMSO. For mixtures of 5:5, 15:5, and 5:15 μM EPA:DHA, the dissolved individual omega-3 FAs at appropriate concentrations were
mixed together. The luciferase
activity was quantified with a luminometer (Bertholus, LB956V) and expressed as
fold induction in the presence of variable doses of omega-3 FAs over control.
Ciprofibrate (250 μM) (Sigma-Aldrich) was used as a positive control of
induction. The assays were performed in triplicates. The experiment was
conducted in duplicate.


### 2.3. Data Analysis

Firefly luciferase activities were
normalized with the corresponding Renilla luciferase reporter activity as
internal control. Fold induction was calculated by taking the control  DMSO (Sigma-Aldrich) as baseline.


## 2.1. Plasmid Construction

The wild-type L162-PPAR expression
plasmid (pSG5-hPPARα vector) was a kind gift from Pr. B. Staels (Unité
INSERM 545, Institut Pasteur de Lille, France). The pSG5-mRXRα
plasmid was described previously [13]. The V162-PPARα
expression plasmid was derived from the wild-type, through site-directed
mutagenesis (QuickChange site-directed mutagenesis kit, Stratagene, La Jolla, Calif,
USA) using the
5′-CGATTTCACAAGTGCGTTTCTGTCGGGATG-3′
oligonucleotide  (the nucleotide in boldface type denotes the C→G change mutated base). Variant cDNAs were
directly sequenced to confirm that no spurious base changes have been
introduced during the procedure. As a response element representative of the
vast variety of human PPREs, we choose the consensus artificial direct repeat
(DR)1 sequence for analyzing the functional consequences of L162V variation on
PPARα activation by omega-3
FAs. Thus, a reporter plasmid (DR1x6-PPRE)
was
generated by cloning in front of the thymidine kinase promoter-driven luciferase
reporter gene (TKpGL3 vector), six
copies of the
5′-AAAACT
AGGTCAAAGGTCA
CGG-3′
sequence where underlined nucleotides correspond to the direct repeat of the
AGGTCA hexamer.


## 2.2. Transient Transfection and N-3 Fatty Acids Activation

Human
hepatoma HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented by 10% fetal bovine serum (FBS), 1% of streptomycin penicillin, 1%
of sodium pyruvate and 1% of glutamine. HepG2 cells were plated at a density of75×103 cells/well of 24-well plates which were then transfected using the ExGen
reagent (Invitrogen, Burlington, Canada) with 50 ng of the DR1x6-PPRE reporter plasmid, 10 ng of the PPARα (wild-type or mutant) and RXR expression plasmids and 30 ng of the pRL-NULL expression vector (Promega) for 6 hours 37°C. All samples were complemented with pBS-SK+
plasmid (Stratagene) to an identical amount (500 ng/well). Similar experiments were performed with a
negative control consisting of the empty TK-pGL3-basic plasmid (Promega). After
transfection, cells were cultured in DMEM supplemented by 0.2% FBS for 24 hours to strengthen
cell membrane before addition of FAs. Afterwards, cells were transactivated for 24 hours in absence or
presence of omega-3 FAs in concentrations varying between 1–15 μM to reflect
biological plasma or red blood cells concentration of FAs [14]. Cells were treated with either
solvent (dimethyl sulfoxide (DMSO), 0.01% final concentration), or treatments of EPA and/or DHA (Sigma-Aldrich,
Oakville, ON, Canada).
Briefly, pure EPA or DHA was dissolved by serial dilution to 1, 5, 10,
and 15 μM in DMSO. For mixtures of 5:5, 15:5, and 5:15 μM EPA:DHA, the dissolved individual omega-3 FAs at appropriate concentrations were
mixed together. The luciferase
activity was quantified with a luminometer (Bertholus, LB956V) and expressed as
fold induction in the presence of variable doses of omega-3 FAs over control.
Ciprofibrate (250 μM) (Sigma-Aldrich) was used as a positive control of
induction. The assays were performed in triplicates. The experiment was
conducted in duplicate.


## 2.3. Data Analysis

Firefly luciferase activities were
normalized with the corresponding Renilla luciferase reporter activity as
internal control. Fold induction was calculated by taking the control  DMSO (Sigma-Aldrich) as baseline.


## 3. Results

Transient transfection assays in
human hepatoma HepG2 cells were done to compare L162-PPARα to V162-PPARα transcriptional activity. In sum, two
independent transients’
transfection assays were performed with similar results for transcriptional
activity. The V162-PPARα variant showed similar basal transcriptional activity after treatment
with DMSO compared with L162-PPARα on the DR1x6-PPRE. For
positive control, the presence of the PPAR synthetic ligand, ciprofibrate,
showed enhanced transactivation activity in V162-PPARα
compared with L162-PPARα
(Figure 1). Most importantly, the results from this functional study demonstrate
that increase in activity in the V162-PPARα variant did not reach the same
level of extent of transcriptional activity as the L162-PPARα variant in all replicates and doses of omega-3
FAs.Figure 1
Transcriptional
activity by L162-PPARα and V162-PPARα
in HepG2 cells supplemented with EPA, DHA, and mixtures of EPA:DHA. The DR1-PPRE-TKpGL3
reporter construct (100 ng) was cotransfected with the pRL-NULL plasmid (30 ng)
in HepG2 cells in presence of 10 ng pSG5-hPPARα wild-type (black bars) or mutated (white bars)
and pSG5-mRXRα
(10 ng) plasmids. Cells were subsequently treated or not with ciprofibrate (250 μM) or varying concentrations and mixtures of EPA and/or DHA for 24 hours. Values were normalized
to internal Renilla luciferase activity as described in materials and methods
and expressed as fold-induction relative to the control (TK-pGL3) set at 1.
Values are representative of 2 independent experiments realized in triplicates.In more details, the addition of 5 and 15μM EPA resulted
in an increased in activity with respect to basal level of EPA of 1 μM in L162-PPARα variant, yet only 15 μM EPA resulted in a
slight increase in transcriptional rate compared to DMSO (Figure 1). In the same way, the
addition of 5, 10, and 15 μM EPA resulted in an higher activity, with respect
to basal level in V162-PPARα variant (Figure 1).
Nevertheless, transcriptional activity by the L162-PPARα variant compared to V162-PPARα variant was 9%,
11%, 4% and 6% consistently
greater with 1, 5, 10, and 15 μM of EPA (Figure 1)
representing functional differences between the variants.Similarly, the addition
of DHA enhanced transcriptional activity at most concentrations in both the L162-PPARα and V162-PPARα variant compared to basal level of
DHA (Figure 1). However, only 10 or 15 μM of DHA in the L162-PPARα increased activity compared to DMSO. Likewise,
the addition of 1, 5, 10, or 15 μM of DHA increased to a greater extent the
transcriptional activity by the L162-PPARα variant compared to the V162-PPARα variant (17%, 5%, 15%, and 3%;
resp.) (Figure 1).In addition, EPA:DHA mixtures tested showed a marked
increase in transcriptional activity that was higher with respect to individual FA
transcriptional activity or basal activity (Figure1). Again in the V162-PPARα variant, the ratios of EPA:DHA
increased the receptor activity but to a lesser degree than in L162-PPARα (Figure
1). The disparities in transcriptional activity between the L162-PPARα and V162-PPARα variants were even more important:
24%, 28%, and 17% for 5:5, 5:15 and 15:5 μM EPA:DHA ratios,
respectively. Overall, even if the individual FAs show a smaller
transcriptional activity by PPARα with a larger standard deviation,
this transcriptional activity is consistently lower in the V162-PPARα than L162-PPARα. Further, this information is supported by the
results of the mixtures of EPA:DHA, where there is clearly an increased
transcriptional activity and this effect is of lesser magnitude in V162-PPARα than L162-PPARα.


## 4. Discussion

The present study represents the first examination of the variation in
transcriptional activity after
omega-3 FA activation in the L162V polymorphic variant. Overall,
the use of natural PPARα agonists, such as omega-3 FAs, may
influence the activation of PPARα at higher doses.
Nevertheless, differences exist in the rates of transcriptional activity
by the V162-PPARα and the L162-PPARα variant of
the PPARα
L162V polymorphism. In addition, the additive effects of EPA and DHA mixtures on
transcription rates may reveal supplementary benefits compared to the
individual omega-3 FAs.The results clearly
reveal that the V162-PPARα has lower transcriptional activity than the
L162-PPARα. Previous research has demonstrated
the impact of PPARα on the clearance of TG-rich lipoproteins in humans
after treatment with PPARα agonist, fibrates [15]. The plasma TG lowering
effect of fibrates can be duplicated in animal studies [16]. In contrast,
plasma TGs are elevated in animals lacking PPARα [17]. This data
suggest that PPARα adjusts LPL-dependent TG lypolysis by altering
expression of pro- and antilipolytic factors [18]. Thus, the current results demonstrate that
individuals carrying a V162-PPARα variant may potentially have elevated TG levels due to lower
transcription rate of target genes, such as LPL.
These in vitro results support
the numerous human studies in which the PPARα
L162V polymorphism exhibited associations with total
cholesterol, LDL-cholesterol, apo B, TG, and high-density lipoproteins (HDL)-cholesterol [4–9].  In general, from the current and previous
human studies, the V162 allele appears to be associated with a more harmful
lipid profile potentially due to a lower transcription rate of target genes
with PPREs.The improve transactivation of both allelic variants following an
omega-3 FA activation reveals the importance of stratifying
individuals according to their dietary fat intakes including omega-3 FA to
demonstrate the influence of PPARα
L162V polymorphism on lipid parameters and other metabolic
factors. Since the
mutation is located in the DNA binding domain, this single nucleotide
polymorphism is thought to have an impact on the receptor’s ability to bind DNA
[12]. While receptors coregulators (i.e., coactivators and corepressors)
generally interact with the ligand binding domain of nuclear receptors [19], we
cannot exclude that the L162V amino acid substitution affects the PPARα's ability to adequately separate from cytoplasmic corepressor, transit
to the nuclei and/or recruit coactivators, as it was demonstrated for the V227A
variant of this receptor [20]. To the best of our knowledge functional studies
have never been performed for the PPARα
L162V mutation, and the mechanisms at the basis of
differential omega-3 FAs-dependent activation
of the wild-type and mutated receptors remain to be
elucidated. Yet, it appears that the V162-PPARα
has the potential to reach comparable transcription rates as L162-PPARα with
higher intakes of individual or mixtures of omega-3 FAs. Therefore,
the influence of the L162V polymorphic variant may be more apparent in
individuals who consume a lower intake of omega-3 FAs. These results are in
accordance with previous human studies [10, 11, 21] which examined the effect of the PPARα
L162V polymorphism in relation to diet.  These
previous researchers determined
that a high intake of dietary PUFA can lower TG in carriers of the V162-PPARα allele [10, 11] due to higher n-3 FA intakes that may lead to increased activation of PPARα. Finally, a recent study by Caron-Dorval et al. in 2008 [21] demonstrated
that plasma TG levels decreased similarly between a group of 28 young men with
or without the L162V polymorphism
after an intense omega-3 FA supplementation for 4 weeks. These results confirm
that dietary modifications including higher amounts of EPA and DHA, which
activate PPARs to a greater level, may be an effective method in reducing
metabolic risk in those with high-risk allele, such as V162. However, this
point requires further investigation to ascertain a precise nutritional
recommendation.An
additional purpose of this study was to determine whether EPA, DHA, and
combinations of EPA:DHA have differential roles in transcriptional activity. Most studies regarding the effects
of n-3 PUFA on blood lipids were conducted with fish oils that contain
a mixture of EPA and DHA [22, 23]. Yet, a number of studies have been
conducted with EPA and DHA individually. In vitro [24] and animal [25–28] studies suggest that EPA rather than DHA may be a hypotriglyceridemic agent.
However, divergent findings have been reported in human studies [29, 30].
Results from the current study with individual FAs indicate that a higher dose
of either EPA or DHA can increase transcriptional rate of target genes.
However, our results demonstrated that DHA may have a slightly higher
transcriptional activity than EPA. A recent study by
Sanderson et al. in 2008 [31] showed that DHA behaved
as a highly potent inducer of PPARα dependent gene expression
compared to other FAs, although they did not examine the effects of EPA or
mixtures of these FAs. On the other hand, investigators who examined the
effects of oleic acid, EPA, and DHA on intestinal gene expression in mice
identified 19, 46, and 41 genes, respectively, that were activated with these
fatty acids versus 74 genes with the PPARα
agonist [32]. In addition in the current study, all tested concentrations of combination of EPA and DHA induced slightly higher transcription rates than individual FAs. However, it still remains unclear whether EPA and DHA have similar TG
lowering potential. Further studies are needed to
determine whether EPA and DHA in combination, as they are found
naturally in fish oils, have an additive effects on gene expression
rates; hence, potentially reducing TG concentrations to a greater extent than
individual FAs.In conclusion, these
results indicate that the V162-PPARα variant has lower transcriptional
activity than L162-PPARα variant in response
to omega-3 FAs; therefore, clearly demonstrating that a nutrient-gene
interaction exists between PPARα
L162V polymorphism
and omega-3 FAs. Further studies are needed to
confirm whether this difference in transcriptional activity by PPARα is translated into differences in gene
expression levels of physiological target genes. Overall, the functional understanding of omega-3 FAs in relation to PPARα
L162V genotypes may allow
more targeted individualized dietary advice to maximising the benefit gained by
the individual.



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*Source: 369602-2009-02-25.xml*