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Metabolism and functions of docosahexaenoic acid-containing membrane glycerophospholipids

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Omega-3 (ω-3) fatty acids (FAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are known to have important roles in human health and disease. Besides being utilized as fuel, ω-3 FAs have specific functions based on their structural characteristics. These functions include serving as ligands for several receptors, precursors of lipid mediators, and components of membrane glycerophospholipids (GPLs). Since ω-3 FAs (especially DHA) are highly flexible, the levels of DHA in GPLs may affect membrane biophysical properties such as fluidity, flexibility, and thickness. Here, we summarize some of the cellular mechanisms for incorporating DHA into membrane GPLs and propose biological effects and functions of DHA-containing membranes of several cell and tissue types. This article is protected by copyright. All rights reserved.
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REVIEW ARTICLE
Metabolism and functions of docosahexaenoic
acid-containing membrane glycerophospholipids
Daisuke Hishikawa
1
, William J. Valentine
1
, Yoshiko Iizuka-Hishikawa
1
, Hideo Shindou
1,2,3
and
Takao Shimizu
1,4
1 Department of Lipid Signaling, National Center for Global Health and Medicine, Shinjuku-ku, Tokyo, Japan
2 Department of Lipid Science, The University of Tokyo, Bunkyo-ku, Japan
3 AMED, Chiyoda-ku, Tokyo, Japan
4 Department of Lipidomics Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Japan
Correspondence
D. Hishikawa, National Center for Global
Health and Medicine, Tokyo 162-8655,
Japan
Fax:+81-3-3202-7364
Tel: +81-3-5273-5351
E-mail: dhishikawa@ri.ncgm.go.jp
(Received 22 July 2017, revised 13 August
2017, accepted 17 August 2017, available
online 7 September 2017)
doi:10.1002/1873-3468.12825
Edited by Wilhelm Just
Omega-3 (x-3) fatty acids (FAs) such as docosahexaenoic acid (DHA) and
eicosapentaenoic acid (EPA) are known to have important roles in human
health and disease. Besides being utilized as fuel, x-3 FAs have specific func-
tions based on their structural characteristics. These functions include serving
as ligands for several receptors, precursors of lipid mediators, and compo-
nents of membrane glycerophospholipids (GPLs). Since x-3 FAs (especially
DHA) are highly flexible, the levels of DHA in GPLs may affect membrane
biophysical properties such as fluidity, flexibility, and thickness. Here, we
summarize some of the cellular mechanisms for incorporating DHA into mem-
brane GPLs and propose biological effects and functions of DHA-containing
membranes of several cell and tissue types.
Keywords: lysophospholipid acyltransferase; membrane remodeling;
omega-3 fatty acid
Fatty acids (FAs) have a wide range of physiological
roles as an energy source, membrane phospholipid
component, donor substrate for protein acylation,
and lipid mediators (or precursor) [13]. FAs can be
classified based on several structural features.
From their carbon chain lengths, FAs are defined as
short-chain FAs (SCFAs; fewer than six carbons),
medium chain FAs (MCFAs; from 6 to 12 carbons),
long-chain FAs (LCFAs; from 14 to 20 carbon
chains), and very long-chain FAs (VLCFAs; over 22
carbon chains). FAs are also classified by double
bond number as saturated FAs (SFAs; no dou-
ble bonds), monounsaturated FAs (MUFAs; one dou-
ble bond), and polyunsaturated FAs (PUFAs; two or
more double bonds). Unsaturated FAs are also classi-
fied by the position of the first double bond from
their methyl(x) end and are commonly designated
as being x-9, x-6, or x-3 FAs [4].
Omega-3 FAs, including alpha-linolenic acid
(ALA), eicosapentaenoic acid (EPA), and
Abbreviations
AA, arachidonic acid; ACS, acyl-CoA synthetase; AdipoR1, adiponectin receptor 1; ADP, adenosine diphosphate; ALA, alpha-linolenic acid;
AMPK, adenosine monophosphate-activated protein kinase; Ab, amyloid-beta; BBB, bloodbrain barrier; BRB, bloodretinal barrier; DHA,
docosahexaenoic fatty acid; ELOVL, elongation of very long-chain fatty acid; EPA, eicosapentaenoic acid; FA, fatty acid; FABP, fatty acid-
binding protein; FADS, fatty acid desaturase; GPL, glycerophospholipid; LCFA, long-chain fatty acid; LPAAT, lysophosphatidic acid acyltrans-
ferase; LPC, lysophosphatidylcholine; LPLAT, lysophospholipid acyltransferase; MCFA, medium chain fatty acid; Mfsd2a, major facilitator
domain-containing protein 2a; MUFA, monounsaturated fatty acid; OS, outer segment; PA, phosphatidic acid; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PGC1, peroxisome proliferator-activated receptor gamma coactivator-1 alpha; PLA, phospholipase; PPAR, peroxi-
some proliferator-activated receptor; PUFA, polyunsaturated fatty acid; RPE, retinal pigmented epithelium; SCFA, short-chain fatty acid; SFA,
saturated fatty acid; TBC, tubulobulbar complex; VLCFA, very long-chain fatty acid.
2730 FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
docosahexaenoic acid (DHA), are essential nutrients
for normal development and health [5]. Mammals
cannot synthesize x-3 FA de novo because they lack
the enzymes to produce double bonds at the x-3
position and therefore must obtain x-3 FAs from
diet [6]. x-3 FAs are initially formed in the chloro-
plasts of green plants, algae, and plankton, and they
are concentrated and reach relatively high levels in
fish and seafoods. Health benefits of dietary x-3 FA
intake attracted much interest by a study in 1978
which demonstrated a low incidence of acute
myocardial infarction in Greenland Eskimos who
have diets very rich in x-3 FAs [7]. To date, numer-
ous studies have demonstrated the importance of x-3
FAs in various tissues including heart, brain, retina,
and testes [811].
The biological roles of x-3 FAs consist of four dis-
tinct categories: lipid mediator precursor, transcrip-
tional regulators, modulator of membrane protein
functions, and component of glycerophospholipids
(GPLs). As lipid mediators, it is reported that EPA-
and DHA-derived lipid mediators are less potent to
stimulate platelet aggregation than arachidonic acid
(AA)-derived lipid mediators, which they displace
[12,13], and more recent studies revealed that EPA and
DHA are also converted to proresolving lipid media-
tors, such as protectins, resolvins, and maresins
[14,15]. As transcriptional regulators, EPA and DHA
act as ligands for several nuclear receptors/transcrip-
tional factors, including peroxisome proliferator-
activated receptor alpha (PPARa)[16]. By binding
PPARa, EPA and DHA reduce circulating triglyceride
levels, and EPA and DHA supplements are clinically
used to treat hyperlipidemia [16]. As modulators of
membrane proteins, x-3 FAs are reported to directly
bind to several ion channels and thereby modulate
their activities [1719]. This activity occurs in myocar-
dium and may be important for some of the cardio-
protective effects associated with fish oil consumption.
In addition to these effects, since x-3 FAs, especially
DHA, are highly flexible, their incorporation into
membrane GPLs affects membrane physicochemical
properties [20,21]. However, compared to the other
biological roles of PUFAs, the mechanisms of incorpo-
ration and significance of DHA in membranes have
remained obscure. In recent years, studies utilizing
gene knockout strategies and molecular dynamics sim-
ulations have begun to clarify the importance of x-3
PUFA incorporated into membrane GPLs as well as
the cellular mechanisms by which DHA incorporation
occurs. Here, we review recent progress to understand
the roles of x-3 PUFAs, especially DHA, in membrane
GPLs.
Production of x-3 PUFA-containing
GPLs
Cellular membranes are composed of several types of
GPLs which are classified by their polar head groups,
such as phosphatidic acid (PA), phosphatidylcholine
(PC), phosphatidylethanolamine (PE), phosphatidylser-
ine, phosphatidylglycerol, and phosphatidylinositol
[22]. GPLs are also classified by their FA linkages to
the hydroxyl group of the glycerol backbone as diacyl
type, alkyl-acyl type, and alkenyl-acyl type (plasmalo-
gens) [23]. Due to the many combinations of FAs at
sn-1 and sn-2 positions combined with the variation in
linkages and classes, cellular membranes may contain
over 1000 distinct species of GPLs [24].
Diversity of FA compositions in membrane GPLs is
mainly generated by the action of lysophospholipid
acyltransferases (LPLATs) and occurs through two
different pathways: the de novo pathway (Kennedy
pathway) and the remodeling pathway (Lands’ cycle;
Fig. 1)[25]. Glycerol-3-phosphate (G3P) acyltrans-
ferases and lysophosphatidic acid acyltransferases
(LPAATs) function in the Kennedy pathway.
LPAATs, which use lysophosphatidic acid as an acyl
acceptor, regulate FA compositions of GPLs during
production of PA, a common intermediate for GPLs
and neutral lipid synthesis. In the Lands’ cycle, phos-
pholipase (PLA)- and LPLAT-mediated deacylation
reacylation reactions ensure remodeling of membranes
and contribute to diversity of cellular GPLs. The coor-
dinated actions of both the Kennedy pathway and the
Lands’ cycle are required to generate the full diversity
of FA compositions of membrane GPLs. These com-
positions differ among tissues and cell types, and may
be further modified to in response to physiological
cues. In the regulation of AA level in GPLs, FA
remodeling by LPCAT3 regulates levels of AA-con-
taining GPLs and is required for triglyceride transport
in liver and small intestine [2628]. It is suggested that
AA incorporation is dependent on both the Kennedy
pathway and Lands’ cycle [29]. In contrast, DHA
incorporation into GPLs may occur primarily in the
Kennedy pathway, because it is reported that
cytidine diphosphate-ethanolamine:diacylglycerol
(DAG) ethanolaminephosphotransferase, which con-
verts DAG to PE, and PE N-methyltransferase, which
converts PE to PC, prefer DHA-containing species as
substrates [30,31]. In addition, a recent study compar-
ing in vitro LPLAT activities and tissue PC composi-
tions also suggested that levels of DHA-containing
GPLs may be primarily regulated by LPAAT activities
functioning in the Kennedy pathway rather than other
LPLATs functioning in the Lands’ cycle [29]. Recently,
2731FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
D. Hishikawa et al. Roles of DHA-containing membrane GPLs
we also reported that in LPAAT3 KO mice DHA-con-
taining GPLs were greatly decreased [32,33], consistent
with the idea that DHA-containing GPLs are primar-
ily produced through the Kennedy pathway.
Lysophospholipid acyltransferases require acyl-CoA
as donors in their acylation reactions, therefore acyl
donor substrate supply is also an important determi-
nant of FA compositions of cellular GPLs. In cells,
FAs are activated to acyl-CoA by acyl-CoA syn-
thetases (ACSs) [34] and are subsequently either used
as substrates by LPLATs or else further elongated
and/or desaturated by the actions of FA elongases and
desaturases [22,35]. Alternatively, acyl-CoAs are
subjected to b-oxidation when cells require energy
[36,37]. Although mammals cannot synthesize x-3 FAs
de novo, DHA and EPA can be formed from dietary
a-linolenic acid (x-3, C18:3) through FA elongation,
desaturation, and beta-oxidation steps [38]. Fatty acid
desaturases (FADS1 and FADS2) and elongation of
very long-chain fatty acid enzymes (ELOVL2 and
ELOVL4) are required for this formation of DHA-
and EPA-CoA [39]. Genetic deletion of ELOVL2 in
mice leads to the dramatic reduction of DHA in hep-
atic GPLs and triglycerides [40], indicating that DHA
formed from precursor x-3 FAs provides an important
source of donor substrate for acylation reactions and
Fig. 1. The synthetic pathways of DHA-containing GPLs (DHA-GPLs) in mammalian cells. Since mammalian cells cannot synthesize x-3
FAs, dietary uptake of DHA or other x-3 FAs such as ALA is required to generate DHA-containing GPLs. In the cells, DHA-containing GPLs
are formed by two independent pathways, the de novo pathway (Kennedy pathway) and the remodeling pathway (Lands’ cycle). In the
Kennedy pathway, DHA is mainly incorporated into the sn-2 position of PA by the action of LPAATs. PA is a common intermediate for all
GPL synthesis, and DHA-containing PA is converted to other DHA-containing GPLs. Alternatively, DHA may be incorporated into GPLs by
the concerted actions of PLAs and LPLATs in the Lands’ cycle. DHAP, dihydroxyacetone phosphate.
2732 FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
Roles of DHA-containing membrane GPLs D. Hishikawa et al.
also affects the diversity of FA compositions in mem-
brane GPLs.
Effects of GPL compositions on the
function of cellular membrane
Glycerophospholipids are major components of bio-
logical membranes, and their FA compositions may
profoundly affect cellular processes. Based on their
physiochemical properties, PUFAs and especially
DHA are predicted to impart unique characteristics to
biological membranes.
Membrane fluidity
Compared to SFAs, the structure of unsaturated FAs
tends to be more curved due to that cis double bonds
make a ‘kicked’ structure [41]. Since this curvature
decreases the molecular interaction between FA chains
of neighboring GPLs, enrichment of PUFA-containing
GPLs increases membrane fluidity and affects many
cellular functions that are dependent on membrane
dynamics (Fig. 2A) [41,42].
Membrane flexibility
In addition to the effect on membrane fluidity, molec-
ular dynamics simulations reveal a lowered barrier for
rotation about vinyl-methylene bonds of FAs imparts
an increased propensity for flexibility to PUFAs, espe-
cially DHA, over SFAs or MUFAs [20,21], and DHA
incorporated into GPLs accordingly affects membrane
flexibility (Fig. 2B) [43]. Indeed, high DHA incorpora-
tion is reported to facilitate membrane deformation in
processes such as membrane fusion and fission [44].
Polar head groups of GPLs are also known to affect
membrane fusion and fission. As opposed to cylinder-
shaped GPLs with large polar head groups (e.g., PC),
cone-shaped GPLs with small polar head groups (e.g.,
PA and PE) form inverted hexagonal-II phases and
are reported to promote membrane fusion and fission
[45]. In this regard, enrichment of DHA in PE rather
than in PC might be important for affecting cellular
functions involving membrane fusion and fission in
several tissues. Additionally, highly flexible DHA-con-
taining membranes may influence or promote confor-
mational changes of membrane proteins (Fig. 2B) [46].
Lipid packing defects
In highly curved membrane, as occurs during mem-
brane fission, the hydrophobic FA tails of GPLs are
partially extruded from the hydrophobic interior of the
bilayer and physically exposed on the hydrophilic sur-
face. This phenomenon is termed ‘lipid packing defect’
(Fig. 2C) [47]. Several proteins are known to sense and
bind lipid packing defects via hydrophobic domains or
amphipathic helices. FA compositions of membranes
also affect generation and biophysical properties of
lipid packing defects, and high PUFA content is sug-
gested to favor generation of more shallow defects.
Depending on the molecular interactions between these
defects and the defect-sensing motifs, it is possible that
the sensing proteins may detect and respond differently
to the packing defects depending on the PUFA con-
tent of very highly curved membranes (Fig. 2C) [48].
Membrane thickness
Fatty acid chain length and cis double bonds of GPLs
are known to affect lipid bilayer thickness (Fig. 2D)
[49,50]. Liposome lipid bilayers composed of PUFA-
containing GPLs are thinner than those composed of
SFAs, and it is thought that DHA-enriched membrane
may be thin, as has been reported for membranes of
rod photoreceptor disks, which have high amounts of
DHA-containing GPLs and are very thin [51]. Since
transmembrane domain lengths of integral membrane
proteins have correlations with membrane thickness,
enrichment of DHA-containing GPLs may affect the
localization and trafficking of membrane proteins [52].
Also, thinner and loosely packed DHA-enriched mem-
branes are reported to have increased permeability to
small polar molecules (Fig. 2D) [53]. This is also an
important property of DHA in cellular membranes.
Roles of DHA-containing GPLs in
various tissues
Fatty acid compositions of GPLs vary among tissues
and cell types, suggesting the compositions are tightly
regulated and impart specific biochemical properties to
membranes based upon physiological requirements.
DHA-containing GPLs are abundant in retina, testes,
brain, heart, and skeletal muscle and may have impor-
tant functions in each of these tissues [810,29,54].
The roles of DHA-containing GPLs in the retina
It has long been known that retinal membrane con-
tains a high amount of DHA [55]. In the retina, DHA-
containing GPLs are abundant in outer segment (OS)
disks of rod photoreceptor cells [33,56]. OS disk is a
unique organelle specialized in phototransduction.
Rhodopsin, a dim light-sensitive G protein-coupled
receptor, is a major protein component (>90%) of
2733FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
D. Hishikawa et al. Roles of DHA-containing membrane GPLs
rod photoreceptor OS disks [57]. The interaction
between rhodopsin and DHA in GPL was observed by
several approaches including molecular dynamics sim-
ulations and nuclear magnetic resonance studies
[58,59] Those studies suggest that the flexible property
of DHA permits very rapid conformational changes of
rhodopsin after photoactivation in the membrane
(Fig. 3A).
Since photoreceptors are segregated from blood flow
by bloodretinal barrier (BRB) composed of retinal
capillary endothelial cells and retinal pigmented epithe-
lium (RPE) [60], it has been a long-standing mystery
how photoreceptors obtain such a high amount of
DHA. Recent studies identified two important mole-
cules involved in the transport of DHA from circula-
tion into the retina. One is adiponectin receptor 1
(AdipoR1) [61] and the other is major facilitator
superfamily domain-containing protein 2a (Mfsd2a)
[62]. AdipoR1 is a receptor for adiponectin, an
adipocyte-derived hormone implicated in insulin sensi-
tivity [63]. Besides the well-studied function of adipo-
nectin in metabolic tissues, Rice et al. [61] reported the
expression and function of AdipoR1 in the retina. Adi-
poR1 KO mice showed progressive retinal degenera-
tion with a dramatic reduction of unesterified DHA,
DHA-containing GPLs, and very long-chain PUFA-
containing GPLs in the retina. Surprisingly, adiponec-
tin KO mice did not show retinal degeneration, indi-
cating the functions of AdipoR1 in retina may be
independent of cognate ligand binding. AdipoR1 is
expressed both in RPE and photoreceptor cells, there-
fore AdipoR1 may be involved in trafficking of DHA
from circulation into RPE as well as from RPE into
photoreceptor cells. Rice et al. [61] also reported Adi-
poR1 overexpression-enhanced DHA incorporation
into human spontaneously transformed RPE cells, and
an association reported for a single nucleotide poly-
morphism on the AdipoR1 locus and age-related
Fig. 2. The roles of DHA-containing GPLs in membranes. (A) Enrichment of PUFA, including DHA, in membrane GPLs may impart fluidity to
membranes. Highly fluid cellular membrane may enhance membrane dynamics to facilitate processes such as lateral diffusion of membrane
proteins. (B) Highly flexible and DHA-containing GPLs facilitate the fusion and fission of membranes. Highly deformable DHA-rich
membranes also support rapid conformational changes of membrane proteins. (C) Strong curvature of membrane leads to partial exposure
of hydrophobic tails of GPLs to the cytosolic surface, a phenomenon termed lipid packing defect. DHA-containing GPLs may promote
formation of more shallow defects than MUFA-containing GPLs, and this depth of lipid packing defect may affect the recruitment of several
lipid packing defect-sensing proteins. (D) Lipid bilayers composed of DHA-containing GPLs are thinner compared to those composed of
disaturated phospholipids and cholesterol. Loosely packed thin membranes may have increased permeability to ions and small molecules.
Membrane thickness is also a determinant of membrane protein localization and activity.
2734 FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
Roles of DHA-containing membrane GPLs D. Hishikawa et al.
macular degeneration in humans further supports an
important role for AdipoR1 in human vision [64]. In
addition to AdipoR1, Mfsd2a, which is highly
expressed in RPE, was also shown to be involved in
DHA incorporation across BRB. DHA-containing
GPLs in Mfsd2a KO mouse retina were reduced by
half compared to wild-type, resulting in OS disorgani-
zation and rhodopsin mislocalization [62]. Unlike Adi-
poR1, Mfsd2a transports DHA as DHA-containing
lysophosphatidylcholine (LPC) rather than unesterified
DHA in a sodium-dependent manner [65]. Taken
together, these studies indicate retinas may obtain
DHA by at least two independent mechanisms. It is
intriguing that although DHA-containing GPLs are
decreased in both AdipoR1- and Mfsd2a-KO mice,
dramatic decreases of VLCFA species occur only in
AdipoR1 KO mice [61,62]. These results suggest that
the uptake mechanism and form of the DHA may also
affect its incorporation into GPLs and metabolism.
Very recently, novel VLCFA-derived neuroprotective
lipid mediators, termed ‘elovanoids’, have been
reported [66]. These VLCFA-derived lipid mediators
A
B
Fig. 3. The proposed functions of DHA-containing GPLs in retinal rod photoreceptor cells (A) and mature sperm (B). (A) DHA-containing
GPLs are enriched in OS disks. OS disks are formed by plasma membrane evagination, and DHA-rich flexible membranes may be critical for
this process. Highly flexible DHA-rich membranes might also support rapid conformational changes of rhodopsin after light stimulation. The
phenotypes of Mfsd2a- and LPAAT3-deficient mice suggest that DHA-containing GPLs are required for the OS disk organization and
maintenance, as well as rhodopsin trafficking. CC, connecting cilium. (B) Defective spermatogenesis in LPAAT3 KO mice indicates the
critical role of DHA-containing GPLs in the final step of sperm formation. In this step, excess sperm cytoplasm and plasma membranes are
removed with spermSertoli cell junctions through apical TBC. LPAAT3 KO sperm possess an excess of cytoplasm and Sertoli cell
membranes around the sperm head, suggesting that DHA-containing GPLs promote clathrin-mediated endocytosis and/or rapid membrane
migration through the actin-lined narrow tubules.
2735FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
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D. Hishikawa et al. Roles of DHA-containing membrane GPLs
may contribute to the phenotypic differences between
AdipoR1- and Mfsd2a-KO mice.
The enzyme responsible for DHA incorporation into
retinal GPLs was reported recently by our group.
LPAAT3 KO mice showed a specific and almost com-
plete lack of DHA-containing GPLs in the retina,
while AA-containing GPLs were dramatically
increased [33]. LPAAT3 KO mice showed retinal
degeneration with abnormal morphology/organization
of photoreceptor OS disks, indicating the critical
requirement of DHA in retina and suggesting that
increased AA-containing GPLs cannot rescue the func-
tions of DHA-containing species. Abnormal disk mor-
phology/disorganization in Mfsd2a- and LPAAT3-KO
mice raise the possibility that enrichment of DHA-con-
taining GPLs is required not only for rhodopsin acti-
vation but also photoreceptor OS disk organization
(Fig. 3A). Recent studies clearly showed that nascent
rod photoreceptor OS disks are formed by evagination
of plasma membrane at a neck region of OS (Fig. 3A)
[6769]. OS disks are thin, and DHA-containing GPLs
may impart flexibility to the membranes that con-
tributes to formation of strong curvatures at the edge
of disk membranes in photoreceptor cells. Mislocaliza-
tion of rhodopsin in the perinuclear region was seen in
both Mfsd2a and LPAAT3 KO mice (Fig. 3A) [33,62].
As DHA-containing GPLs may also promote mem-
brane fusion and fission, another possible function for
DHA-containing GPLs in photoreceptor cells might be
to promote trafficking of rhodopsin from endoplasmic
reticulum to the plasma membrane via the Golgi appa-
ratus (Fig. 3A) [70].
The roles of DHA-containing GPLs in the testes
The testes are also known to possess high amounts of
DHA in their membranes, especially in mature sper-
matids [9,71]. The importance of PUFAs in the testes
was demonstrated using FADS2 KO mice by two
independent groups [72,73]. FADS2 KO mice, deficient
in highly unsaturated FAs (possessing four or more
double bonds), showed multiple abnormalities includ-
ing male infertility. FADS2 KO mice lacked elongated
spermatids due to a failure in acrosome formation, a
process essential for maturation of round to elongating
spermatids [7274]. Acrosome formation requires two
membrane-associated steps where highly fluid and flex-
ible membrane may be required: the budding and fis-
sion of acrosomal vesicles from Golgi apparatus, and
the fusion of acrosomal vesicles [75]. A subsequent
study further demonstrated that the defect of sper-
matid maturation in FADS2 KO mice could be com-
pletely rescued by dietary DHA supplementation,
while rescue by AA supplementation was partial [10].
These studies indicate an essential role of DHA in
spermatid maturation.
Why is DHA, but not AA, essential for the sper-
matid maturation? Further evidence for the specific
requirement of DHA in spermatid maturation has
been demonstrated using LPAAT3 KO mice. LPAAT3
was originally identified as an LPAAT enzyme highly
expressed in the testes which showed preference to uti-
lize PUFA-CoA in in vitro assays [76]. LPAAT3
expression increased in testes during sexual matura-
tion, suggesting a role in male reproduction. Male
LPAAT3 KO mice showed complete infertility due to
abnormal sperm formation [32]. Similar to the GPL
compositions in LPAAT3 KO retinas, DHA-contain-
ing GPLs in LPAAT3 KO testes and isolated testicular
mature spermatids were dramatically decreased, while
AA-containing GPLs were compensatorily increased.
In contrast to FADS2 KO mouse spermatids,
LPAAT3 KO spermatids are able to mature into elon-
gated spermatids and form acrosomes, suggesting that
in these processes other PUFA-containing species may
compensate for the lack of DHA-containing species.
However, LPAAT3 KO mice failed to form fully
mature fertile spermatids due to a defect in elimination
of excess cytoplasmic component. At the final step of
spermatogenesis, surrounding Sertoli cells uptake
excess spermatid cytoplasm and plasma membrane
along with spermatidSertoli cell junctions via tubu-
lobulbar complexes (TBCs) and release fully developed
spermatids into seminiferous tubules (Fig. 3B) [77].
TBCs are composed of actin-lined narrow (~50 nm in
diameter) tubules, endoplasmic reticulum-surrounded
bulbous portions, and clathrin-coated pits (Fig. 3B).
At the tips of TBCs, spermatid and Sertoli cell mem-
branes along with spermatid cytoplasm are internalized
by Sertoli cells via clathrin-mediated endocytosis and
degraded by lysosomes-dependent mechanisms. The
defect in spermatogenesis in LPAAT3 KO mice sug-
gests that DHA-containing GPLs may impart the
required flexibility to sperm membranes necessary for
efficient endocytosis through TBC tubules, and substi-
tution of other PUFAs for DHA is not able to provide
the membrane properties required for this process. In
this regard, the mechanisms underlying abnormal pho-
toreceptor disk morphology and spermatid maturation
in LPAAT3 KO mice might be similar. Together with
the fact that cone-shaped GPLs such as PE facilitate
membrane fusion and fission processes [45,78], the
higher proportion of DHA-containing species in PE
(~40% in total PE) than in PC (~10% in total PC) in
elongated spermatids may also be important for their
maturation.
2736 FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
Roles of DHA-containing membrane GPLs D. Hishikawa et al.
Sperm undergo further maturation during passing
through the epididymis. In this process, sperm mem-
brane is reported to be more enriched in PUFAs
including DHA [79], suggesting DHA-containing
GPLs are also important for the sperm functions in
addition to their required role in spermatogenesis. It is
reported that genetic deletion of group III secreted
PLA
2
(sPLA
2
-III), which is highly expressed in caput
epididymis, caused decreased DHA- and DPA-contain-
ing GPLs of sperm in cauda epididymis, but not in
caput epididymis [80]. Since the sPLA
2
-III KO sperm
have defects in fertility, enrichment of DHA- and
DPA-containing GPLs during passage through the epi-
didymis is also required for the sperm maturation.
The roles of DHA-containing GPLs in the brain
The brain is also abundant in DHA-containing GPLs
[8]. DHA has been linked to brain development, func-
tions, and diseases [81,82]. DHA may be preferentially
supplied into the brain through the bloodbrain bar-
rier (BBB) as DHA-containing LPC [83,84]. In support
of this, loss of Mfsd2a, which is expressed in endothe-
lial cells of the BBB, resulted in a dramatic reduction
of DHA-containing GPLs in the brain [65]. This sug-
gests that both brain and retina depend upon the Mfs-
d2a-mediated transport of DHA-containing LPC to
incorporate DHA through BBB and BRB, respectively.
Mfsd2a KO mice showed brain dysfunctions, such as
cognitive deficits, severe anxiety, and microcephaly,
suggesting the importance of DHA in brain [65], and
homozygous inactivating mutations in Mfsd2a have
been likewise found in patients with severe micro-
cephaly [85,86]. In addition to the decrease of DHA-
containing GPLs in Mfsd2a KO mouse brain, it is also
reported that BBB of these mice are leaky [87]. A
recent study revealed that Mfsd2a-mediated DHA
incorporation controls the BBB permeability by sup-
pressing the caveolae-mediated transcytosis [88]. It is
important that Mfsd2a and Caveolin-1 double KO
could rescue the leaky BBB but not microcephaly phe-
notype in Mfsd2a KO mice [88]. This suggests that
BBB leakage is not related to the microcephaly in Mfs-
d2a KO mice. Therefore, further studies are required
to clarify the molecular mechanisms underlying the
behavioral abnormalities and microcephaly in Mfsd2a
KO mice. Recently, fatty acid-binding protein 5
(FABP5) was also reported to incorporate DHA into
the brain through BBB but in unesterified form [89].
Although the DHA incorporation into BBB is reduced
in FABP5 KO mice, the contribution of FABP5 to
supply DHA across the BBB may be minor compared
to Mfsd2a.
A variety of effects of DHA in the brain, such as
enhanced neurotransmission, neuronal outgrowth, and
synaptic plasticity, have been demonstrated using vari-
ous approaches [90]. These effects may be exerted, at
least in part, by altering membrane physical properties
that affect fusion/fission and membrane-involved sig-
nal transduction processes. However, the precise mech-
anisms by which DHA-containing GPLs contribute to
brain functions are largely unknown. An imaging mass
spectrometric study demonstrated that PUFA-contain-
ing GPLs are higher in axon tips than cell bodies of
cultured neurons [91]. This suggests that PUFA-con-
taining GPLs may have a role in effective release of
neurotransmitters via impacting membrane physical
properties.
Decreased DHA levels in the brain are reported
under several conditions including aging and Alzhei-
mer’s disease [92]. A biochemical study utilizing lipo-
somes suggesting that gamma-secretase produced the
more pathologic amyloid-beta
42/43
(Ab
42/43
) from amy-
loid precursor protein in thicker liposomes, containing
longer acyl chains, than in thinner liposomes [9395].
This observation suggests that DHA-containing thin
membranes may inhibit production of Ab
42/43
and the
onset of Alzheimer’s disease. However, the effects of
DHA on Alzheimer’s disease and cognitive functions
differs among the studies and remains controversial
[96,97].
The roles of DHA-containing GPLs in heart
Clinical and epidemiological studies support that x-3
FA consumption promotes cardiovascular health and
may prevent or improve coronary heart disease [11].
The precise roles of x-3 FAs in cardiac physiology are
not well understood, and x-3 FA consumption may
affect cardiac functions by both indirect and direct
mechanisms. Indirect mechanisms include effects on
plasma lipid profiles, the vascular system, autonomic
functions, and whole-body metabolism, while several
direct effects of fish oil may be mediated by x-3 FAs
that are incorporated into myocardial membranes.
These direct effects may include improved heart rate
and resistance to ischemic stress, arrhythmia, and con-
gestive heart failure [98].
Elevated resting heart rate is a major risk factor for
cardiovascular mortality, and regular x-3 supplemen-
tation lowers intrinsic heart rate and protects against
arrhythmias. x-3 FAs might alter heart rate by several
potential mechanisms, and studies of heart transplant
patients [99], isolated rat hearts [100], and isolated rab-
bit pacemaker cells [101] indicate a direct effect inde-
pendent of central autonomic functions [98,102].
2737FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
D. Hishikawa et al. Roles of DHA-containing membrane GPLs
Although the physiological functions of x-3 FAs
within the myocardial membranes are not clear, GPL-
incorporated x-3 FAs might alter membrane fluidity,
permeability, or electrophysiological properties and
thereby modulate ion channel functions and impact
heart rate; similar mechanisms might also provide pro-
tection from Ca
2+
overload and irregular cytosolic
Ca
2+
fluctuations, causing antiarrhythmic effects [102].
Although both DHA and EPA are abundant in fish
oil, they may be differentially incorporated into
myocardial GPLs and also differ in their effects on
cardiac physiology [98]. In rats, levels of DHA are
higher than EPA in myocardial membrane GPLs, and
following fish oil supplementation DHA is preferen-
tially incorporated into myocardial phospholipids over
EPA regardless of DHA and EPA levels in the supple-
ment [103]. In humans, DHA-containing GPLs were
higher than EPA-containing species in right atrial
appendages removed during bypass surgery [104], and
DHA-containing GPL was preferentially increased by
preoperative dietary fish oil supplementation [105].
Functional outcomes between supplementation with
DHA or EPA may also differ. DHA but not EPA sup-
plementation was efficacious to lower heart rate in
humans [106,107]. In spontaneous hypertensive rats,
DHA but not EPA supplementation inhibited ische-
mia-induced arrhythmia [108]. Further studies are
required to uncover the cellular mechanisms underly-
ing the selective incorporation and protective effects of
DHA in myocardial membranes.
The roles of DHA-containing GPLs in skeletal
muscles
Skeletal muscle undergoes metabolic adaptation in
response to physiological cues such as physical activity
and nutritional status. Adaptation of skeletal muscle
to regimes of endurance exercise training includes a
switching of glycolytic to more oxidative fiber types
and is associated with whole-body metabolic improve-
ments such as increased endurance and resistance to
obesity [109]. The adaption to endurance exercise is
also associated with changes in the FA compositions
of GPLs, and several lines of evidence support a role
for increased incorporation of DHA into GPLs.
In several studies in mice, rats, and humans, DHA-
containing GPLs were increased by endurance exercise
training independently of diet, and DHA-containing
GPL levels showed positive correlations with oxidative
capacity of the skeletal muscle [110114]. In mice,
exercise-induced increases of DHA in PE were
observed in extensor digitorum longus muscles,
whereas more oxidative soleus muscle already had high
DHA in PE even without training [110]. In rats,
DHA-containing PE content was higher in oxidative
than in glycolytic vastus lateralis [114]. In humans,
DHA-containing GPL levels were higher in vastus lat-
eralis muscles of endurance-trained young men and
correlated with increased type I oxidative fiber percent-
age [113]. In another study in humans, 4 weeks of one-
leg exercise training led to increased DHA-containing
GPLs as well as increased citrate synthase activity
compared to the untrained leg [112].
Reports also indicate that dietary DHA supplemen-
tation may promote endurance even without training.
In untrained rats, 9 weeks of dietary DHA supplemen-
tation resulted in increased skeletal muscle DHA-con-
taining GPL levels as well as increased capacity for
endurance exercise. The DHA supplementation altered
several metabolic parameters in isolated and permeabi-
lized skeletal muscle myofibers, indicating improved
mitochondrial functions [115]. Also in birds, 6 weeks
of DHA or EPA supplementation strongly increased
oxidative enzyme activities in flight muscles of con-
fined quail concurrent with enhanced DHA- or EPA-
containing GPL contents [116].
The mechanisms and functions of enhanced DHA-
containing GPL in endurance-trained skeletal muscle
require further elucidation. PPARccoactivator-1 a
(PGC1a) is activated by exercise downstream of ade-
nosine monophosphate-activated protein kinase
(AMPK) and is a key regulator of mitochondrial bio-
genesis and cellular metabolism. In one study, mice
overexpressing PGC1ain skeletal muscle had
enhanced levels of several DHA-containing GPL
species [110]. DHA incorporation into the same GPL
species was also increased by endurance training in a
PGC1a-dependent manner, indicating that training-
induced increases in DHA-containing GPLs may be
transcriptionally regulated downstream of exercise-
induced signaling pathways. The functional signifi-
cance of the enhanced DHA-containing GPLs is not
clear but may affect cellular metabolism through sev-
eral mechanisms. Increased DHA content incorpora-
tion into membrane GPLs may alter biophysical
properties of the membranes to affect cellular bioener-
getics. In support of this theory, fish oil supplementa-
tion to active men resulted in increased DHA and
EPA in skeletal muscle mitochondrial GPLs that was
accompanied by improvements in several mitochon-
drial respiratory parameters including adenosine
diphosphate (ADP) sensitivity [117]. Enhanced DHA
content in membrane GPLs may also affect levels of
free DHA or its metabolites which are known ligands
for PPAR nuclear receptors and may alter their activi-
ties to transcriptionally regulate metabolic gene
2738 FEBS Letters 591 (2017) 2730–2744 ª2017 The Authors. FEBS Letters published by John Wiley &Sons Ltd
on behalf of Federation of European Biochemical Societies
Roles of DHA-containing membrane GPLs D. Hishikawa et al.
expressions [118120]. Further identification of the
enzymes and mechanisms that regulate DHA-contain-
ing GPL levels in skeletal muscle is required to clarify
many of the metabolic functions of DHA.
Future perspectives and conclusion
remarks
Omega-3 PUFAs have important functions on human
health, and recent studies have clarified some of the
molecular mechanisms underlying the effects of DHA
in various tissues. Several important molecules
involved in the incorporation of DHA into cells and
production of DHA-containing GPLs have been iden-
tified, including LPAAT3, Mfsd2a, and AdipoR1
[32,33,61,65]. However, very basic questions still
remain as to how DHA is incorporation is regulated
and the precise functions of DHA in membrane GPLs.
The studies using LPAAT3 KO mice demonstrated
the essential role of LPAAT3 to generate DHA-con-
taining GPLs in retinas and testes [32,33]. As LPAAT3
is not ubiquitously expressed and other enzymes such
as LPAAT4 may be involved in DHA-containing GPL
production [121], further studies are required for a
comprehensive understanding of how DHA-containing
GPLs are generated and metabolized in the body. It is
possible that FA remodeling in the Lands’ cycle is also
important in the production of DHA-containing GPLs
in various tissues. It is intriguing that Mfsd2a-
mediated DHA incorporation is important into brain
and retina but not liver and skeletal muscle [62,65].
This suggests that Mfsd2a is specifically required to
incorporate DHA into GPL in areas isolated from
blood flow. However, a dramatic induction of Mfsd2a
mRNA expression during cold exposure and fasting in
brown adipose tissue and liver is also reported
[122,123], suggesting these tissues may also use the
DHA-containing LPC as a source of DHA under
specific situations. It is unknown why these tissues
obtain DHA as LPC in a sodium-dependent manner.
The production mechanisms of DHA-containing LPC
and its sources also remain open questions.
Although DHA in GPLs may have a specific role in
various cellular processes by imparting membrane flex-
ibility, it is also easily oxidized under oxidative stress
and generates toxic lipid peroxides [124]. Lipid peroxi-
dation is known to cause apoptosis [125], and recent
studies have reported a novel iron-dependent cell
death, termed ‘ferroptosis’, that is also caused by lipid
peroxidation [126]. Thus, cells with PUFA-enriched
membranes may require protective mechanisms against
various oxidative stresses. In this regard, it is proposed
that PUFA incorporation into plasmalogens may be
important because the vinyl double bond linkage is
preferentially oxidized, preventing oxidation of the
PUFA and possibly mitigating cellular lipid peroxida-
tion reactions [127]. Further studies are required to
understand the cellular mechanisms that protect
PUFA-enriched membranes from oxidative stresses.
In addition to understanding their biological effects
and functions, how cells are able to distribute and reg-
ulate PUFA-enriched membranes also needs to be
addressed. In the case of neurons, accumulation of
PUFA-containing PC species in axon tips is actin
dependent, suggesting PUFA-containing GPL-enriched
vesicles may be selectively sensed and trafficked [111].
Incorporated PUFAs are also freed from the mem-
branes, and GPL-esterified DHA is not only a mem-
brane structural component but also a potential
precursor for lipid mediators and other metabolites.
Comprehensive understanding of how DHA-enriched
membranes are generated, trafficked, and metabolized
will help clarify biological functions and patho-physio-
logical roles of DHA.
Acknowledgements
The authors are grateful to all members of our labora-
tories for valuable suggestions (National Center for
Global Health and Medicine and The University of
Tokyo).
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Roles of DHA-containing membrane GPLs D. Hishikawa et al.
... Among the different lipids, polyunsaturated fatty acids (PUFA) and omega-3 fatty acids have attracted a particularly great interest for their many different associated health benefits, including prevention or treatment of neurological diseases [9,10], relief of symptoms of inflammatory disorders [11][12][13], improvements in whole body metabolism [14,15], and prevention of the progress of certain cancers [16,17]. Omega-3 fatty acids were proposed to exert their biological activities through different mechanisms, such as: acting as lipid mediator precursors, transcriptional regulators, modulators of membrane protein functions, and, importantly, by shaping the membranes as either free molecules or components ("apolar tails") of glycerophospholipids [18,19]. The last point is directly linked to the peculiar chemical structure of omega-3; their long carbon chain with multiple double bonds-for example 20 carbon atoms and 5 double bonds in the case of eicosapentaenoic acid (EPA) and 22 carbon atoms and 6 double bonds in the case of docosahexaenoic acid (DHA), the two most representative members of this family [20]-allows a great degree of conformational flexibility that inevitably affects the physico-chemical and structural properties of the membranes in which they are embedded [21]. ...
... The last point is directly linked to the peculiar chemical structure of omega-3; their long carbon chain with multiple double bonds-for example 20 carbon atoms and 5 double bonds in the case of eicosapentaenoic acid (EPA) and 22 carbon atoms and 6 double bonds in the case of docosahexaenoic acid (DHA), the two most representative members of this family [20]-allows a great degree of conformational flexibility that inevitably affects the physico-chemical and structural properties of the membranes in which they are embedded [21]. However, the mechanisms and significance of omega-3 incorporation in membranes have remained obscure, compared to the other biological roles of these molecules [18]. In this respect, we have recently indicated that small amounts of the di-DHA phospholipid 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine (22:6-22:6PC) are able to perturb liquid disordered bilayers by increasing their fluidity slightly but sufficiently to promote morphological rearrangements [22]. ...
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Lipid structural diversity strongly affects biomembrane chemico-physical and structural properties in addition to membrane-associated events. At high concentrations, cholesterol increases membrane order and rigidity, while polyunsaturated lipids are reported to increase disorder and flexibility. How these different tendencies balance in composite bilayers is still controversial. In this study, electron paramagnetic resonance spectroscopy, small angle neutron scattering, and neutron reflectivity were used to investigate the structural properties of cholesterol-containing lipid bilayers in the fluid state with increasing amounts of polyunsaturated omega-3 lipids. Either the hybrid 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine or the symmetric 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine were added to the mixture of the naturally abundant 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and cholesterol. Our results indicate that the hybrid and the symmetric omega-3 phospholipids affect the microscopic organization of lipid bilayers differently. Cholesterol does not segregate from polyunsaturated phospholipids and, through interactions with them, is able to suppress the formation of non-lamellar structures induced by the symmetric polyunsaturated lipid. However, this order/disorder balance leads to a bilayer whose structural organization cannot be ascribed to either a liquid ordered or to a canonical liquid disordered phase, in that it displays a very loose packing of the intermediate segments of lipid chains.
... Lipid supplementations was used to increase embryo developmental rates in cattle. Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) with a 22 carbon chain (C22:6) [6,7]. DHA supplied in the cow diet modified the lipid composition of bovine oocytes [1]. ...
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Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100μM) coupled with carnosine (5mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation.
... The upward trend of unsaturated fatty acids is indeed believed to be a common mechanism for dealing with oxidative damage in aquatic animals exposed to environmental stressors (Rocchetta et al., 2006;Signa et al., 2015). This assumption was herein confirmed by the increase of some particular PUFAs such as DHA, ARA, and EPA, which have been proven to play an important anti-oxidant role, strongly influences membrane flexibility and fluidity, and modulate immunological and inflammatory responses (Vance and Vance, 2008;Fokina et al., 2013;Hishikawa et al., 2017). Of particular note, an unanticipated substantial increase of C16:2 was recorded in both gills and digestive gland of exposed scallops. ...
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Glyphosate is the most sprayed pesticide across the globe. Its toxicity to non-target marine organisms has recently piqued the scientific community's interest. Therefore, the purpose of this study is to investigate the potentially toxic effects of glyphosate on scallops, an ecologically and economically important bivalve group. To do that, specimens of the smooth scallop Flexopecten glaber were exposed to different concentrations (10, 100, and 1000 μg L⁻¹) of the technical-grade glyphosate acid (GLY) for 96 h. The detrimental effects of this pollutant were assayed at cellular and tissular levels. The obtained results showed that the GLY was able to induce oxidative stress in the gills and the digestive gland of F. glaber as revealed by the enhanced hydrogen peroxide (H2O2), protein carbonyls (PCO), malondialdehyde (MDA), and lipid peroxides (LOOH) levels and the altered antioxidant defense system (the glutathione GSH content and the superoxide dismutase (SOD) activity). Additionally, GLY was found to alter the fatty acid profile, to exert a neurotoxic effect through the inhibition of the acetylcholinesterase (AChE) activity, and to provoke several histopathological damages in the two organs studied. The obtained results revealed that the pure form of GLY may exert toxic effects on F. glaber even at relatively low concentrations.
... The increased PCs containing two SFAs are associated with biologically related physiochemical properties, such as membrane fluidity and thickness (Chiantia and London, 2012), cell permeability (Davis and Silbert, 1974), and cholesterol solubility (Ringel et al., 1998). A study showed that increased levels of DHA-containing PCs and PEs promote Gprotein receptor coupled signaling (Mitchell et al., 2003) and sustain normal development and function of the brain (Hishikawa et al., 2017), besides being structural elements. Moreover, the total content of LPCs and ether-PCs shared the same change pattern with that of PCs, which was in accordance with a study showing that the reduced total content of LPCs and ether-PCs was mainly attributed to a great decrease in concentration in the yolk of zebrafish embryos (Fraher et al., 2016). ...
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