ARTICLE
Auteur(s) : Jean-Marc
Alessandri, Bénédicte Langelier, Marie-Hélène Perruchot, Audrey
Extier, Fabien Pifferi, Mélanie Jouin, Serge Delpal, Monique
Lavialle, Philippe Guesnet
Unité de Nutrition et Régulation Lipidique des Fonctions
Cérébrales (Nu.Ré.Li.Ce), Institut National de la Recherche
Agronomique (INRA), CRJ, 78352 Jouy-en-Josas cedex
Introduction
Docosahexaenoic acid (DHA, 22:6n-3) is the major n-3
polyunsaturated fatty acid (PUFA) in the brain and retinal cell
membranes [1]. It has been shown in rodents and non-human primates
that inadequate supplies of n-3 PUFA during the perinatal period
result in reduced membrane accumulation of DHA and in impairments
of learning capacity, monoaminergic neurotransmission and visual
function [1, 2]. In human infants, the n-3 supplies of maternal
milk or of milk replacers also have an impact on the DHA contents
in nervous tissues and may produce transient (but significant)
outcomes on the mental development and maturation of visual
function [3]. DHA is directly provided by dietary animal fats or is
synthesized from its metabolic precursors, α-linolenic acid (ALA,
18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3) or docosapentaenoic
acid (DPA, 22:5n-3). The conversion of ALA, the essential dietary
precursor, occurs in the endoplasmic reticulum, producing the
downstream long-chain metabolites EPA, DPA and tetracosahexaenoic
acid (THA, 24:6n-3) (figure 1). THA is not
incorporated into membranes but is transferred to the peroxisomes
to be shortened in DHA through one cycle of β-oxidation (figure 1). Increasing the
ALA intakes has been considered as a possible way of increasing the
DHA status in human. However, clinical studies in adults showed
that ALA supplementation with as much as 15 g per day did not
increase DHA in plasma phospholipids, but increased EPA and DPA
(reviewed in [4]) (figure 2). Moreover, EPA
concentration in plasma (not DHA) increased in response to dietary
EPA. These metabolic responses have been related to the low
capacity of conversion of ALA to DHA. Studies using stable isotope
tracers in healthy adults have estimated that the conversion of ALA
to EPA is around 5% of the ALA intake whereas that of ALA to DHA
accounts for much less than 0.5% [5]. The reasons for this low
conversion rate are not clearly elucidated. The first hypothesis is
that ALA itself may mitigate DHA synthesis by competing with
tetracosapentaenoic acid (24:5n-3) for Δ6 desaturation and
preferential production of 18:4n-3 over that of THA [6]. Besides,
active channeling of ALA towards mitochondrial β-oxidation would
reduce its entry into the pathway of long-chain synthesis [5].
However, the two hypothesis seem contradictory since they mean that
the entry of ALA either into the conversion pathway or into the
oxidative pathway both will result in reduction of DHA synthesis.
The third hypothesis is that the peroxisomal pathway could form a
limiting step: the peroxisomal β-oxidation of THA requires complex
movements of the 22C- and 24C-PUFAs on both sides of the
endoplasmic reticulum and peroxisomal compartments, and it needs
specific peroxisomal enzymes which could be low in activity [7].
To address these questions, we studied the n-3 PUFA metabolism
in a neural cell model, the SH-SY5Y neuroblastoma cells which are
derived from the neural crest of the human embryo and exhibit
neuronal morphological features (figure 3). In a first
approach, we compared the dose-dependent incorporation of preformed
DHA into the phospholipids of SH-SY5Y cells, of rat brain areas
(frontal cortex and hippocampus) and of rat brain endothelial
cells, and we evaluated the capacity of SH-SY5Y cells to produce
neoformed DHA from its metabolic precursors, ALA, EPA and DPA. We
studied the potential of SH-SY5Y cells to express genes involved in
the fatty acid metabolism by quantifying the mRNAs of key enzymes
located at the endoplasmic reticulum level, the FA-CoA ligase
(FACL3), the Δ6 desaturase (Δ6-D) and the elongase of very long
chain fatty acids (ELOVL4), and at the peroxisomal level, the
acyl-CoA oxidase (AOX) and the two bifunctional proteins, LBP and
DBP. The mRNAs of cognate transcription factors which regulate the
transcription of lipid metabolism genes, the peroxisome
proliferator-activated receptors (PPARs) and the retinoid X
receptor (RXR), and the mRNAs of enzymes not directly involved in
the DHA synthesis but in the synthesis of ether-phospholipids or of
phospholipid classes (peroxisomal
dihidroxyacetonephosphate-acyltransferase, DHAP-AT, and
phosphatidylethanolamine N-methyltransferase, PEMT) or in the
intracellular trafficking of fatty acids (epidermal-fatty acid
binding protein, E-FABP), were also quantified in the SH-SY5Y
model.
Material and methods
Cell cultures
Neuroblastoma cells from the SH-SY5Y line were cultured in Falcon
75-cm2 tissue culture flasks at 37 °C in a 5% (by
vol) CO2 atmosphere in DMEM supplemented with 10%
heat-inactivated fetal bovine serum [7]. In parallel, two types of
brain endothelial cells were used: RBEC, which is a primary culture
of endothelial cells isolated from the rat brain, and RBE4, an
immortalized cell line issued from rat brain endothelial cells.
RBEC were purified using the puromycin-based method described by
Perriere et al. [8]. The microvessel fragments isolated from rat
cerebral cortex were seeded on Petri dishes coated with type IV
collagen (5 μg/cm2) and incubated at 37 °C in a
saturated 5% CO2 atmosphere in DMEM-F12 with 20% bovine
plasma-derived serum. The RBEC were then purified using a specific
treatment with puromycine (4 μg/mL) for 4 days. The RBE4 line is
issued from immortalized RBEC [9]. RBE4 cells were grown at
37 °C in a 5% CO2 atmosphere in Falcon
75-cm2 tissue culture flasks coated with rat-tail
collagen in α-MEM + glutamate enriched HAM’s F10 supplemented with
10% heat-inactivated fetal calf serum.
The n-3 PUFAs, ALA, EPA, DPA or DHA, were directly added to the
medium (containing 10% fetal calf serum) under the form of sodium
salts. Graded concentrations (7 to 70 μM) of each fatty acid were
used. The SH-SY5Y cells were cultured for 72 h in the
supplemented medium. The RBEC and RBE4 cells were exposed to the
fatty acid on the first day of culture and the supplemented medium
was refreshed every 3 days throughout the culture. At the end of
the cultures, the cells were trypsinised (SH-SY5Y cells) or washed
twice with PBS-0.5% BSA, scraped off (RBEC and RBE4) and collected
by centrifugation.
Animal study
Wistar pregnant female rats deficient in n-3 PUFA (first
generation) were assigned to experimental groups supplemented with
graded doses of dietary DHA (from 0 to 400 mg/100 g diet)
[10]. These doses were obtained by adding different amounts of a
microalgal oil containing about 43% of DHA (DHASCO; Martek
Biosciences Corporation, Columbia, MD). At weaning of the pups
(2nd generation, 3 wk of age), the males were retained
and fed for 5 wk the same diet as that of their dam’s group. At 8
wk of age, the rats were sacrificed and their frontal cortex and
hippocampus were dissected.
Fatty acid analysis
The total lipids were extracted from cells and from brain samples
with 4 ml chloroform-methanol containing 0.02 g/100 mL
butylhydroxytoluene as antioxidant [7]. The ethanolamine
phosphoglycerolipids (EPG), the main phospholipid class rich in
DHA, were isolated from total lipids by solid-phase extraction on a
single-use 500-mg aminopropyl-bonded silica column [11]. The EPG
fraction was dried under a nitrogen flux and its fatty acids were
converted to methyl esters by reaction with methanol and boron
trifluoride at 90 °C for 20 min (transmethylation). The
fatty acid methyl esters were separated and quantified by gas
liquid chromatography [11]; the fatty acid composition was
expressed as a weight percentage of total fatty acids in EPG.
Quantification of mRNA expression in the SH-SY5Y cells
The mRNAs encoding nuclear receptors or lipid metabolism genes were
quantified by retrotranscription of total RNAs into cDNAs; the
target-cDNAs were amplified by quantitative real time-PCR. Total
RNAs were prepared using the Rneasy lipid tissue Midi kit
(Quiagen). Aliquots of total RNA (6 μg) were reverse-transcribed at
25 °C for 10 min and at 37 °C for 2 h using the
High Capacity cDNA Archive Kit (Applied Biosystems). Primer pairs
for target genes were designed with the assistance of Primer
Express 2.0 (Applied Biosystems). The SYBR Green fluorescence
method was used to detect the amplicons, except for E-FABP, Δ6-D
and DHAP-AT whose cDNAs were amplified and detected using sets of
specific primers and the TaqMan probe (SYBR Green master mix and
TaqMan Assays-on-Demand, Applied Biosystems). Primer pairs were
validated under the condition that their PCR efficiency was above
95% (therefore, differences in abundance of different
retrotranscripts are not due to differences in PCR efficiency). The
housekeeping gene (GAPDH) was quantified using both SYBR Green and
TaqMan to normalize the abundance of all retrotranscripts. The mean
value (n = 3 triplicates per flask, 3 flasks per treatment) of the
threshold cycle number (Ct) for detecting the appearance of each
amplicon was compared to that of GAPDH. The amount of mRNA of each
target gene was expressed relative to the GAPDH abundance according
to the equation:
with n = ΔCt.Log 2 and ΔCt = Ct target gene - Ct
GAPDH
Results
DHA incorporation in the membrane phospholipids of cells and
rat brain areas – Development of a linear model for comparing the
dose-dependent responses of living tissues and of cultured cells to
graded doses of preformed DHA
Neuroblastoma cells cultured in standard conditions, i.e. with 10%
fetal calf serum and without PUFA supplementation, have a low
content of DHA (around 5% in EPG) (figure 4). The
dose-response curve for incorporation of preformed DHA was
hyperbolic: the DHA content in EPG gradually increased from the
basal value of 5% of total fatty acids to the plateau-value of 30%
in cells grown in a medium supplemented with 70 μM DHA (figure 4). The
dose-response curve for DHA incorporation in the EPG of rat frontal
cortex and hippocampus produced a similar pattern of incorporation
with 26-28% of DHA at the upper dietary dose (400 mg DHA/100 g
diet) (data not shown). We used a linear model to determine and
compare the theoretical plateau-values (DHAmax) in the in vitro and
in vivo models [10]. It consists in plotting the reciprocal of the
DHA content (1/DHA) in EPG against the reciprocal of the dose
(1/dose) expressed in (μM DHA)–1 or in (mg DHA/100 g
diet)–1(figure 4). The theoretical
DHAmax content in EPG is drawn from the reciprocal value of the
ordinate at the origin (equal to 1/DHAmax) of the straight line.
Moreover, the reciprocal value of the slope (1/slope) defines the
ratio of DHAmax to DHA50, the DHA50 being the dose of preformed DHA
required to match half the value of DHAmax in EPG. Thus, comparison
between cell models and living tissues is possible through the
comparison of the DHAmax and of the DHAmax/DHA50 ratio, reflecting
the ‘avidity’ of cells and tissues for preformed DHA [10]. The data
showed that the avidity of SH-SY5Y cells for preformed DHA was
algebraically comparable to that of the rat frontal cortex and
hippocampus, with the condition that concentrations of DHA in the
medium and in the diet were expressed in μM and in μmol DHA/10 g
diet, respectively (table 1). Thus, we
showed that the SH-SY5Y cells are able to use preformed DHA, as do
living tissues, for their membrane biogenesis, indicating that the
uptake of DHA from the medium, the acyl-CoA synthesis and the
acyltransferase activities are fully active in these cells. The
same model of preformed DHA incorporation was applied to the rat
brain endothelial cells. The dose-response curve of RBEC was
different from that of neuroblastoma cells, leading to lower DHAmax
and avidity, while the RBE4 response superimposed that of SH-SY5Y
cells (data not shown). The corresponding parameters for DHA
incorporation in RBEC and RBE4 are reported in table 1.
Table 1 Comparison of the theoretical maximal DHA
concentration (DHAmax) and the avidity for DHA in ethanolamine
phosphoglycerides (EPG) from cell models and rat tissues. Data are
the mean of 3 (cell models) or 4 (rats) individual determinations.
DHA50 values in the cells and the rat tissues are expressed in
μmol/L and μmol/10 g diet, respectively.
|
DHAmax (% of total fatty acids)
|
Avidity (DHAmax/DHA50)
|
|
Neuroblastoma human cell line SH-SY5Y
|
32.7
|
5.2
|
|
Rat frontal cortex
|
28.8
|
5.7
|
|
Rat hippocampus
|
26.2
|
5.1
|
|
Primary rat brain endothelial cells (RBEC)
|
30.3
|
2.8
|
|
Rat brain endothelial cell line (RBE4)
|
33.7
|
5.0
|
Membrane incorporation of neoformed n-3 long-chain PUFAs in
SH-SY5Y cells
The SH-SY5Y cells were incubated with graded doses of each of the
metabolic precursors of DHA (ALA, EPA or DPA) and the incorporation
of the neoformed n-3 PUFA in EPG was analysed. The resulting
dose-response curves are shown in figure 5. It appeared that
increasing the doses of ALA in the medium gradually increased the
membrane incorporation of neoformed EPA and DPA. This pattern
clearly indicates that the endoplasmic reticulum pathway of
synthesis (up to DPA) is active in these cells. Regarding neoformed
DHA, the response of cells to low concentrations of ALA (< 15
μM) showed an initial increase of incorporation which peaked at 10%
DHA in EPG (i.e. a gain of 30% DHA compared to control value)
followed by a decreased incorporation at concentrations of ALA
greater than 15 μM (figure 5). The same
pattern was observed with EPA supplementation (data not shown),
consisting in a gradual increase of EPA and of its elongation
product DPA in EPG, whereas the neoformed DHA followed a
bell-shaped curve with a peak of incorporation (9% of total fatty
acids) at 15 μM EPA [7]. When the cells were cultured with graded
doses of DPA, both the neoformed EPA (produced through the
retroconversion pathway) and DPA gradually increased in EPG. The
bell-shaped curve of neoformed DHA peaked at 30 μM DPA with a
maximum of DHA incorporation equal to 12% of total fatty acids
(figure 5).
mRNA expression of lipid metabolism genes in SH-SY5Y cells
The basal relative abundances of the mRNAs encoding proteins and
enzymes of the endoplasmic reticulum- and peroxisomal-pathways, and
those of the mRNAs encoding the cognate nuclear receptors, are
reported in figure
6. The SH-SY5Y cells constitutively express the mRNA of
these lipid metabolism genes, whose relative abundances (normalized
to that of GAPDH) range from 100/00 (PEMT,
ELOVL4, DBP) to 850/00 (Δ6 D). The basal
levels of the DBP and LBP transcripts were among the lowest that we
determined, suggesting that the transcription of peroxisomal
enzymes may be constitutively limiting in these cells. The SH-SY5Y
cells also express transcription factors known to be involved in
the regulation of lipid metabolism, i.e. the PPARs, mainly the α−
and β/δ-isotypes, and RXRα.
Discussion
We have examined the capacity of a neural cell model, the human
neuroblastoma cell line SH-SY5Y, to produce the long-chain n-3
PUFAs issued from the metabolic conversion of ALA, the essential
precursor. Our data show that the SH-SY5Y model is particularly
appropriate for studying the regulation of DHA synthesis. These
cells avidly incorporate exogenous (preformed) DHA following a
dose-dependent response that is very similar (in terms of substrate
avidity and plateau-value) to that of rat brain areas. On the other
hand, the DHA synthesis from its upstream precursors is efficient
but limited in the SH-SY5Y cells. We suggest that the production of
neoformed DHA is ‘bottlenecked’ in these cells, resulting in the
membrane accumulation of neoformed EPA and DPA at high
concentration of supplemental ALA. These data on this cell model
may have a physiological significance, since a similar effect
(accumulation of EPA and DPA) is observed in the blood lipids of
humans receiving high doses of ALA (figure 2) [4].
It must be emphasized that cells cultured in standard conditions
have very low DHA content in their phospholipids, which is
equivalent to the physiological state of a chronical deficiency in
dietary n-3 PUFAs [10, 12, 13]. Low DHA contents have been reported
from miscellaneous neural cell models [7, 11, 14-16]. Thus, it
appears that the different serum-based culture media do not provide
enough DHA, and that supplementation of the medium with preformed
DHA is needed to sustain physiological levels of DHA into the
membrane phospholipids of cultured cells. Our data showed that the
incorporation of exogenous DHA into the membrane phospholipids
(EPG) of SH-SY5Y cells supplied with graded amounts of preformed
DHA followed a dose-response hyperbolic curve from a minimum
content to a plateau value, thereby reproducing the restauration of
the DHA status in the rat brain [17]. The pattern of DHA
incorporation was featured (in vivo and in vitro) by calculating
two parameters, DHAmax and DHA50. The DHAmax values (expressed in %
by weight of total fatty acids in EPG) of neuroblastoma cells
(32.7), RBEC (30.3) and RBE4 cells (33.7) were similar to those of
the frontal cortex (28.8) and of the hippocampus (26.2). The ratios
of DHAmax to DHA50 were identical in neuroblastoma (5.2) and RBE4
(5.0) cells indicating that these two lines have the same avidity
for preformed DHA. The DHAmax/DHA50 ratio value of 2.8 in RBEC
indicated that the avidity of these primary culture cells for DHA
is around 1.8-time lower than that of the two cell lines (RBE4 and
SH-SY5Y). Differential avidity of RBEC and RBE4 for DHA may reflect
the physiological specificity of RBEC to preferentially use
arachidonic acid (20:4n-6), not DHA, for membrane biogenesis.
The expression of the dietary dose in μmol DHA/10 g diet led to
a DHAmax/DHA50 ratio value of 5.7 and 5.1 in the rat cortex and
hippocampus, respectively, that may be compared with the value of
5.0-5.2 in cultured cells (table 1).
This algebraic identity suggests that a physiological equivalence
(μmol DHA/liter vs μmol DHA/10 g diet) can be established between
the DHA concentration in the culture medium and in the diet. From
this model, it was also inferred that a dietary supply of 10-fold
the DHA50 of brain EPG should be considered as being the dose
required to reach 90% of the DHAmax in the brain [10].
The capacity of SH-SY5Y cells to produce long-chain metabolites
was evidenced by the incorporation of EPA, DPA and DHA into the EPG
fraction. Their membrane incorporation is the final outcome of the
global process of n-3 PUFA metabolism, including long-chain
conversion, acylation, phospholipid metabolism, and turnover [7,
11]. Accumulation of DPA clearly indicated that the metabolic
process is operating in SH-SY5Y cells, and their mRNA profile
supports this conclusion. We emphasize that the Δ6-desaturase,
which catalyses the first step of the long-chain PUFA synthesis
[18], was expressed to a relatively high mRNA level. Both the
accumulation of neoformed DPA and the mRNA relative abundance of
Δ6-desaturase suggest that Δ6-desaturation is not rate-limiting in
these cells. The SH-SY5Y cells cultured with ALA, EPA or DPA also
produced DHA, although the production of neoformed DHA was
restricted to a critical window of precursor concentration. Thus,
these cells have the capacity to complete the synthesis of DHA at
low concentration of precursors, through the endoplasmic reticulum
and peroxisomal pathways. This finding is notable since it is
generally thought that the synthesis of DHA from n-3 PUFAs is
limited in cultured brain cells at the elongation of DPA [16].
However, increasing the concentrations of supplemental n-3
precursors above 15 μM (ALA and EPA) or above 30 μM (DPA) resulted
in a sharp decrease of the newly formed DHA while EPA and DPA
continued to increase. The membrane accumulation of EPA and DPA at
high concentration of precursor is compatible with the hypothesis
that the downstream pathway of DHA synthesis is ‘bottlenecked’.
Therefore, we assume that production of membrane DHA by SH-SY5Y
cells is limited at the stage of DHA terminal synthesis, probably
at the peroxisomal step, resulting in the overflow in membranes of
its parent fatty acids, EPA and DPA. Since the mRNA abundances of
the peroxisomal LBP and DBP are particularly low in standard
conditions of culture, we suggest that the low expression of
proteins or enzymes specifically related to the peroxisomal
function could form a ‘metabolic bottleneck’. We conclude that the
SH-SY5Y cells provide an appropriate in vitro model for studying
the regulation of the PUFA metabolism, especially at the level of
the gene transcription of peroxisomal enzymes. The stimulation of
the peroxisomal pathway could be the key for completing the
synthesis of DHA from its dietary precursors.
Acknowledgements
The authors thank Françoise Roux and Nicolas Perrière (INSERM
U705-CNRS UMR 7157, Hôpital Fernand Widal, Paris) for the gift of
RBE4 cells and help for RBEC purification.
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