Progress in Lipid Research 82 (2021) 101095

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Progress in Lipid Research

journal homepage: www.elsevier.com/locate/plipres

Review

Regulation of long-chain polyunsaturated fatty acid biosynthesis in

teleost fish
Dizhi Xie a, 1, Cuiying Chen b, 1, Yewei Dong c, 1, Cuihong You c, Shuqi Wang b, *, Óscar Monroig d, *,
Douglas R. Tocher b, e, Yuanyou Li a, *
a
College of Marine Sciences of South China Agricultural University & Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China
b
Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou 515063, China
c
Animal Science & Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510642, China
d
Instituto de Acuicultura Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC), 12595 Castellón, Spain
e
Institute of Aquaculture, Faculty of Natural Sciences, University of Stirling, Stirling FK94LA, Scotland, United Kingdom

A R T I C L E I N F O A B S T R A C T

Keywords: Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA, C20-24), including eicosapentaenoic acid (EPA,
LC-PUFA biosynthesis 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are involved in numerous biological processes and have a
Transcriptional regulation range of health benefits. Fish have long been considered as the main source of n-3 LC-PUFA in human diets.
Post-transcriptional regulation
However, the capacity for endogenous biosynthesis of LC-PUFA from C18 PUFA varies in fish species based on the
Aquaculture
presence, expression and activity of key enzymes including fatty acyl desaturases (Fads) and elongation of very
long-chain fatty acids (Elovl) proteins. In this article, we review progress on the identified Fads and Elovl, as well
as the regulatory mechanisms of LC-PUFA biosynthesis both at transcriptional and post-transcriptional levels in
teleosts. The most comprehensive advances have been obtained in rabbitfish Siganus canaliculatus, a marine
teleost demonstrated to have the entire pathway for LC-PUFA biosynthesis, including the roles of transcription
factors hepatocyte nuclear factor 4α (Hnf4α), liver X receptor alpha (Lxrα), sterol regulatory element-binding
protein 1 (Srebp-1), peroxisome proliferator-activated receptor gamma (Pparγ) and stimulatory protein 1
(Sp1), as well as post-transcriptional regulation by individual microRNA (miRNA) or clusters. This research has,
for the first time, demonstrated the involvement of Hnf4α, Pparγ and miRNA in the regulation of LC-PUFA
biosynthesis in vertebrates. The present review provides readers with a relatively comprehensive overview of
the progress made into understanding LC-PUFA biosynthetic systems in teleosts, and some insights into
improving endogenous LC-PUFA biosynthesis capacity aimed at reducing the dependence of aquafeeds on fish oil
while maintaining or increasing flesh LC-PUFA content and the nutritional quality of farmed fish.

1. Introduction basket providing, not only high-quality protein, but also n-3 (or “omega-
3”) long-chain (C20-24) polyunsaturated fatty acids (n-3 LC-PUFA) such
Fish and seafood are important components of the human food as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid

Abbreviations: ALA, α-linolenic acid (18:3n-3); ARA, arachidonic acid (20:4n-6); PUFA, polyunsaturated fatty acid; C/EBP, CCAAT/enhancer binding protein;
DBD, DNA binding domain; DHA, docosahexaenoic acid (22:6n-3); DPA, docosapentaenoic acid (22:5n-3); DR-1, direct repeat 1; EFA, essential fatty acid; Elovl,
elongation of very long-chain fatty acids; EMSA, electrophoretic mobility shift assay; EPA, eicosapentaenoic acid (20:5n-3); FA, fatty acid; Fads, fatty acyl desaturase;
FO, fish oil; Hnf4α, hepatocyte nuclear factor 4α; LA, linoleic acid (18:2n-6); LBD, ligand binding domain; LC-PUFA, long-chain (C20-24) polyunsaturated fatty acid;
lncRNA, long non-coding RNA; Lxr, liver X receptor; LRE, LXR response element; miRNA, microRNA; MUFA, monounsaturated fatty acid; ncRNA, non-coding RNA;
NF-1, nuclear factor 1; NF-Y, nuclear factor Y; Ppar, peroxisome proliferator activated receptor; PPRE, peroxisome proliferator response element; PUFA, poly­
unsaturated fatty acid (≥ 2 double bonds); RXR, retinoid X receptor; SCHL, Siganus canaliculatus hepatocyte line; siRNA, small interfering RNA; Sp1, stimulatory
protein 1; SRE, sterol response element; Srebp-1, sterol-regulatory element binding protein 1; TF, transcription factor; TSS, transcription start site; UTR, untranslated
region; VLC-PUFA, very long-chain (>C24) polyunsaturated fatty acid; VO, vegetable oil.
* Corresponding authors.
E-mail addresses: sqw@stu.edu.cn (S. Wang), oscar.monroig@csic.es (Ó. Monroig), yyli16@scau.edu.cn (Y. Li).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.plipres.2021.101095
Received 6 November 2020; Received in revised form 24 February 2021; Accepted 12 March 2021
Available online 16 March 2021
0163-7827/© 2021 Published by Elsevier Ltd.
D. Xie et al.
(DHA, 22:6n-3). n-3 LC-PUFA are physiologically important compounds
required for normal growth and development of vertebrates including
humans, and can play key roles in mitigating inflammatory, cardiovas­
cular and cerebrovascular diseases, and are beneficial in some types of
cancer [1,2]. Seafood in general and oily fish in particular are unique
sources of n-3 LC-PUFA for humans, unlike terrestrial animals and their
products (e.g. milk and eggs) in which these essential nutrients are ab­
sent or at very low levels. While annual global fish production has
increased from 19 million tons in 1950 to 179 million tons in 2018 [3],
capture fisheries production has remained stagnant for some decades,
while provision of seafood from aquaculture has been steadily
increasing and currently accounts for almost 53 % of total production
[3], with prospects to reach up to 60–70 % by the year 2030 [4].
In addition to being important for human health, n-3 LC-PUFA are
also essential nutrients ensuring normal growth and development of fish
themselves. The presence of n-3 LC-PUFA in fish derives both from their
diet and endogenous production (biosynthesis), with the latter varying
substantially with species [5]. Historically, it has been widely accepted
that freshwater and salmonid species have significant LC-PUFA
biosynthetic capacity and so they can convert the C18 polyunsaturated
fatty acids (PUFA), α-linolenic acid (ALA, 18:3n-3) and linoleic acid (LA,
18:2n-6), to the biologically active LC-PUFA including the n-3 EPA and
DHA, and the n-6 arachidonic acid (ARA, 20:4n-6). In contrast, marine
teleosts were believed to have generally very limited or no capacity to
biosynthesize LC-PUFA from C18 PUFA precursors. The dichotomy of
freshwater/salmonid fish vs. marine fish having high and low LC-PUFA
biosynthetic capacity, respectively, is now perceived as too simplistic
according to recent discoveries on the repertoire and function of key
enzymes involved in these pathways, as well as the mechanisms regu­
lating their activity. While this will be covered in detail in the present
review, it is important to note that, regardless of the species’ habitat,
understanding the ability of farmed fish for LC-PUFA biosynthesis has
practical implications for fish health and wellbeing.
For fish species with high LC-PUFA biosynthesizing capacity, phys­
iological demand for essential fatty acid (EFA) can be met through di­
etary supply of the C18 biosynthetic precursors LA and ALA present in
vegetable oils (VO) used widely in aquafeeds. However, endogenous
production of LC-PUFA from dietary precursors typically leads to lower
levels of n-3 LC-PUFA in the fillet and, to compensate lower nutritional
and commercial value, fish fed VO-rich diets during most of the pro­
duction cycle can be fed a “finishing diet” with high inclusion of fish oil
(FO) before harvest [6]. On the contrary, for species with low capacity of
LC-PUFA biosynthesis, supply of pre-formed physiologically important
LC-PUFA is required, which is achieved practically with inclusion of FO
rich in LC-PUFA and, to a lesser extent, fishmeal in feed formulations
[7]. Algal products, particularly microalgal oils, can contain high con­
tents of n-3 LC-PUFA and thus have potential to replace FO in aquafeeds
[7]. Additionally, owing to rapidly changing environmental factors (e.g.
temperature and salinity), fish species with LC-PUFA biosynthesis ability
also have specific requirements for n-3 LC-PUFA for maintaining mem­
brane functionality and cellular osmoregulation [8–10]. The finite na­
ture of FO as a resource and its high price associated with an increasing
demand to satisfy a growing industry has resulted in VO being
increasingly used in aquafeeds. As mentioned above, such a strategy
represents a metabolic challenge for species with low LC-PUFA biosyn­
thetic capacities and this has prompted considerable interest in under­
standing the regulatory mechanisms involved in LC-PUFA biosynthesis
of fish, especially farmed species, so as to develop farming strategies that
enable efficient and effective utilization of dietary VO [7,10–12].
The present paper reviews the substantial progress made recently on
the regulatory mechanisms of LC-PUFA biosynthesis at both transcrip­
tional and post-transcriptional levels in teleosts. As the pathways of LC-
PUFA biosynthesis in vertebrates including fish have been reviewed in
detail recently [5,13], these are only summarized to provide the
appropriate context while the focus of the review is on providing readers
with knowledge of the molecular mechanisms underpinning the control
2
Progress in Lipid Research 82 (2021) 101095
and regulation of the biosynthetic pathways. This will provide readers
with a comprehensive understanding of the state-of-the-art of LC-PUFA
biosynthesis in teleost fish. The research reviewed in this article is key to
gain insight into the capacity that farmed fish have to utilize dietary oil
sources currently used in aquafeed formulations to satisfy both their
own physiological demands and produce food with high nutritional
value (i.e. n-3 LC-PUFA rich) for humans.
2. Pathways of LC-PUFA biosynthesis in teleost fish
Fish, like all vertebrates, cannot synthesize LA or ALA de novo due to
the lack of Δ12 and Δ15 desaturases, respectively, enzymes found only
in plants, marine microbes including microalgae, heterotrophic protists
and bacteria, and invertebrates [14–16]. Therefore, LA and ALA are
essential dietary nutrients for all vertebrates. The biosynthesis of LC-
PUFA from C18 precursors requires a series of desaturation and elon­
gation reactions catalyzed by fatty acyl desaturase (Fads)2 enzymes and
elongation of very long-chain fatty acid (Elovl) proteins (Fig. 1). As
described below, several members of the Fads and Elovl protein families
participate in these pathways. It is largely accepted that LC-PUFA
biosynthesis from LA and ALA is usually initiated by a ∆6 desatura­
tion, followed by an elongation and subsequent ∆5 desaturation leading
to the production of ARA and EPA, respectively (Fig. 1). An alternative
pathway, termed the “Δ8 pathway”, starts with a chain elongation,
followed by Δ8 and then Δ5 desaturations to similarly enable ARA and
EPA production from LA and ALA, respectively [24,25].
Two distinct pathways allow vertebrates to biosynthesize DHA from
EPA. Arguably, the more widespread pathway for DHA biosynthesis
among vertebrates is the so-called “Sprecher pathway”, consisting of
two consecutive elongations of EPA to produce 24:5n-3, followed by a
∆6 desaturation to form 24:6n-3, which then undergoes partial
β-oxidation to DHA [18]. This pathway was first described in rats [26]
and rainbow trout Oncorhynchus mykiss [27], and subsequently
described in other teleosts [12,28,29]. Alternatively, DHA can be also
synthesized through a more direct pathway termed the “∆4 pathway” by
which docosapentaenoic acid (DPA, 22:5n-3) is directly ∆4 desaturated
to DHA (Fig. 1). This pathway was first reported in vertebrates in the
marine teleost Siganus canaliculatus [19], and was subsequently shown
to be present in several other teleosts (Table 1) [12,30–33].
2.1. Fads enzymes
Fads enzymes introduce unsaturation (double bonds) between a pre-
existing double bond and the carboxyl end of the fatty acyl chain, and
thus they are also termed “front-end” desaturases. Compared to mam­
mals, which possess two distinct Fads-like desaturases including FADS1
(∆5 desaturase) and FADS2 (∆6 desaturase) [34], teleosts have a rather
different fads gene complement (Fig. 1) [20]. With the exception of the
Elopomorpha species like Japanese eel Anguilla japonica, which has both
Fads1 and Fads2 desaturases [20], virtually all teleosts possess only
Fads2 as the sole Fads-like desaturase in their genomes [5,13]. However,
unlike mammals, teleosts can have a varied number of fads2 genes, such
as one (e.g. zebrafish Danio rerio), two (e.g. rabbitfish S. canaliculatus),
three (e.g. Nile tilapia Oreochromis niloticus), or even four (e.g. Atlantic
salmon Salmo salar) genes, while other species such as the pufferfish
2
Gene/protein nomenclature: In this review, the gene/protein symbols used
for human, mouse and teleosts follow the nomenclature guidelines of human
(Homo sapiens), mouse (Mus musculus) and zebrafish (Danio rerio), respectively.
Using as example “Fads2”, the human gene is referred to as “FADS2” and its
protein as “FADS2”; mouse genes are also italicized but lower case other than
the first letter “Fads2”, while proteins are the same as for human proteins
“FADS2”; for zebrafish and other fish, genes are entirely lower case and itali­
cized "fads2", while proteins are also lower case other than the first letter and
not italicized "Fads2".
D. Xie et al. Progress in Lipid Research 82 (2021) 101095

Fig. 1. Biosynthetic pathways of LC-PUFA in teleosts. General characteristics of LC-PUFA biosynthetic capability and the key enzymatic genes in teleosts are as
follow: 1) freshwater fish, salmonids and a few marine teleosts such as rabbitfish Siganus canaliculatus have LC-PUFA biosynthetic capacity, whereas most marine
teleosts such as Atlantic cod Gadus morhua, grouper Epinephelus coioides and golden pompano Trachinotus ovatus either lack or have only weak capability due to the
absence of Δ5 Fads2 activity [5,13,17]; 2) There are two distinct pathways of DHA biosynthesis from DPA: the widespread “Sprecher pathway” [18], and the “∆4
pathway” that exists in a few teleosts such as S. canaliculatus [19]; 3) Several Fads types were found in teleosts, e.g. both ∆5 Fads1 and ∆6 Fads2 in Japanese eel
Anguilla japonica [20], three ∆6 Fads2 and one ∆5 Fads2 in Atlantic salmon (Salmo salar) [12,21,22], three Fads2 (∆6∆5 Fads2, ∆4 Fads2, and one unidentified Fads2)
in tilapia (Oreochromis niloticus) [12], two Fads2 (∆6∆5 Fads2 and ∆4 Fads2) in rabbitfish S. canaliculatus [19], and one ∆6∆5 Fads2 in zebrafish (Danio rerio) [23].
“∆x” denotes reactions catalyzed by fatty acyl desaturases, Fads; “Elo” indicates reactions catalyzed by elongation of very long-chain fatty acid proteins, Elovl.

Table 1
Fatty acyl desaturases identified in teleosts.
Fads type Habitat Fish species References

Δ6 Fads2 Freshwater fish Common carp Cyprinus carpio; Eurasian perch Perca fluviatilis; grass carp Ctenopharyngodon idellus; mandarin fish Siniperca [35–38]
chuatsi; pirarucu Arapaima gigas; rainbow trout Oncorhynchus mykiss
Diadromous fish Atlantic salmon Salmo salar; Japanese eel Anguilla japonica; meagre Argyrosomus regius; pike eel Muraenesox cinereus [21,39–41]
Marine fish Atlantic bluefin tuna Thunnus thynnus; Atlantic cod Gadus morhua; Asian sea bass/barramundi Lates calcarifer; black [35,42–54]
seabream Acanthopagrus schlegelii; Chu’s croaker Nibea coibor; cobia Rachycentron canadum; gilthead seabream Sparus
aurata; grouper Epinephelus coioides; Japanese flounder Paralichthys olivaceu; Nibe croaker Nibea mitsukurii; salema Sarpa
salpa; blackspotted croaker Nibea diacanthus; European sea bass Dicentrarchus labrax; spotted scat Scatophagus Argus; thick-lip
grey mullet Chelon labrosus; turbot Psetta maxima
Amphidromous Boddart’s goggle-eyed goby Boleophthalmus boddarti [55]
fish
Δ5 Fads2 Freshwater fish O. mykiss [56]
Diadromous fish A. japonica (Δ5 Fads1) [20]
Marine fish C. labrosus; Senegalese sole Solea senegalensis; S. salpa [30,33]
Δ6Δ5 Freshwater fish African catfish Clarias gariepinus; Mexican silverside Chirostoma estor; silver barb Barbonymus gonionotus; striped snakehead [12,23,31,57–61]
Fads2 Channa striata; tambaqui Colossoma macropomum; tench Tinca tinca; Nile tilapia Oreochromis niloticus; zebrafish Danio rerio
Diadromous fish S. salar [22]
Marine fish Rabbitfish Siganus canaliculatus; sand sole Pegusa lascaris [19,33]
Δ4 Fads2 Freshwater fish C. estor; C. striata; medaka Oryzias latipes; O. niloticus [12,31,32]
Marine fish Golden pompano Trachinotus ovatus; P. lascaris; S. canaliculatus; S. senegalensis; sand smelt Atherina presbyter [19,30,42,62]

Takifugu rubripes and Tetraodon nigroviridis have no fads genes [5,13].
Likely compensating for the lack of fads1 in their genomes, teleost
Fads2 have functionally diversified during evolution [13] and a sum­
mary of the Fads desaturase activities reported in freshwater, diadro­
mous and marine teleost species can be found in Table 1. Some fish
species, such as zebrafish [23], African catfish (Clarias gariepinus) [57]
and silver barb (Barbonymus gonionotus) [58], possess one fads2 with
bifunctional Δ6Δ5 desaturation activity. Other species including
rainbow trout have two distinct fads2 with separate Δ5 and Δ6 desa­
turation activities [35,56]. Other teleosts including rabbitfish [19], pike
silverside (Chirostoma estor) [31] and striped snakehead (Channa striata)
[32,61] also have two fads2 genes that were characterized as coding for
enzymes with Δ6Δ5 and Δ4 desaturase activities. Three copies of fads2
genes have been isolated from Nile tilapia, with two encoding Δ6Δ5
bifunctional and Δ4 monofunctional desaturases, respectively, and one
with unknown function [12]. Furthermore, four fads2 genes have been
found in Atlantic salmon, with three exhibiting Δ6 desaturase activity
and one being essentially a Δ5 desaturase [12,21,22]. The varying
numbers of fads2 genes have likely arisen as a result of both whole
genome duplication and tandem duplication events that have occurred
in teleosts, which provided “spare” genetic material for the adaptive
mutation of fads2-type genes, and the regaining of Δ5 activity in some
species [5,13].
Since its first report in S. canaliculatus [19], the ∆4 Fads2 has been
found in several other fish species (Table 1) [12,30–33], which may
indicate it is more widespread in teleosts than initially believed. Inter­
estingly, Δ8 desaturation activity has been reported for almost all the
presently analyzed Fads2, which indicates that Δ8 desaturation is likely
an intrinsic enzymatic ability within vertebrate Fads2 [25]. A general
conclusion from the above studies is that carnivorous marine fish have a
limited LC-PUFA biosynthetic capacity due to having only a single fads2
with Δ6 desaturase activity and, thus, they lack the ability for the Δ5
desaturation required for biosynthesis of EPA and ARA. The acquisition
of several copies of fads2 with functional diversification has enabled an
enhanced capacity for endogenous production of DHA in some teleosts.
Thus, a recent study demonstrated that this process enabled some
sticklebacks to successfully colonize habitats that are naturally depleted
in DHA [63]. Furthermore, it was also shown that the acquisition of
various DHA biosynthetic pathways enabled flatfishes to colonize
freshwater environments [64].
2.2. Elongation of very long-chain fatty acids (Elovl) proteins
Elovl catalyze the usually rate-limiting step in the multi-enzyme
complex enabling the two-carbon elongation of pre-existing fatty acyl
chains. Seven members of the ELOVL family (ELOVL1-7) have been

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D. Xie et al. Progress in Lipid Research 82 (2021) 101095

identified in mammals based on their protein motif sequences and Table 2

substrate specificity [34,65]. Generally, ELOVL1, ELOVL3, ELOVL6 and Elongation of very long-chain fatty acids (Elovl) proteins identified in teleosts.
ELOVL7 have the ability to elongate saturated fatty acids (SFA) and Elovl Habitat Species References
monounsaturated fatty acids (MUFA), while the preferred substrates of type
ELOVL2, ELOVL4 and ELOVL5 are PUFA [65–68]. Since the first report Elovl5 Freshwater fish African catfish [32,36,58–60,69–74]
of the molecular and functional characterization of an Elovl5 in a teleost, Clarias gariepinus;
zebrafish [69], many studies have reported the characterization of PUFA Eurasian perch
elongases including Elovl2, Elovl4 and Elovl5, from a large variety of Perca fluviatilis;
grass carp
fish species (see [5,13]). Table 2 provides a list of teleost species in Ctenopharyngodon
which at least one PUFA Elovl has been functionally characterized. idellus; mandarin
Briefly, Elovl5 is present in virtually all teleosts, and is responsible for fish Siniperca
the elongation of primarily C18 and C20 PUFA substrates (e.g., chuatsi; Northern
pike Esox Lucius;
[32,41,52,75]). Some Elovl5 from euryhaline herbivorous species
rainbow trout
including rabbitfish, spotted scat (Scatophagus argus) [85], salema (Sarpa Oncorhynchus
salpa) [33], and the carnivorous fish, Chinese perch (Siniperca chuatsi) mykiss; striped
[33] and sand sole (Pegusa lascaris) [33], have been reported to also have snakehead Channa
some elongation capacity towards C22 PUFA. Elovl2 has a preference for striata; tambaqui
Colossoma
C20 and C22 PUFA, with some residual capability to elongate C18 PUFA macropomum; silver
[13], and has been identified in freshwater zebrafish [94], African cat­ barb Barbonymus
fish [57], tench (Tinca tinca) [60], salmonids S. salar [75] and O. mykiss gonionotus; tench
[74], and the catadromous Japanese eel [95]. The expansion of teleosts Tinca tinca; Nile
tilapia Oreochromis
during evolution has been accompanied by a loss of the elovl2 gene in the
niloticus; zebrafish
more recently emerged lineages that include the majority of marine Danio rerio
species [13]. Such loss of elongation capacity has been hypothesized to Diadromous Atlantic salmon [39,41,75,76]
further explain the limited capability of marine fish for LC-PUFA fish Salmo salar;
biosynthesis alluded to above. While this argument is still valid for Japanese eel
Anguilla japonica;
most marine fish, including many farmed species, a recent study re­
meagre
ported the presence of a functional elovl2 in the marine European Argyrosomus regius;
sardine (Sardina pilchardus) [96]. Along with Elovl5 and Elovl2, Elovl4 pike eel Muraenesox
has been widely studied in fish, with data confirming that teleosts cinereus
Marine fish Asian sea bass Lates [30,33,42,49,50,52,70,77–85]
generally have two distinct elovl4 genes, elovl4a and elovl4b, with
calcarifer; Atlantic
varying elongation capacities toward C18-22 PUFA substrates from which bluefin tuna
they can produce the so-called very long-chain (>C24) PUFA (VLC- Thunnus thynnus;
PUFA), with chains up to 36 carbons [29,69,81,83–87,90,92]. Impor­ Atlantic cod Gadus
tantly, beyond their role in VLC-PUFA biosynthesis, Elovl4 enzymes morhua; black
seabream
have a role in the biosynthesis of LC-PUFA such as DHA since they can
Acanthopagrus
elongate EPA and DPA to 24:5n-3, a key intermediate of the Sprecher schlegeli; Chu’s
pathway. Such elongation ability of teleost Elovl4 enzyme has been croaker Nibea
hypothesized to compensate for the absence of Elovl2 in many marine coibor; cobia
Rachycentron
teleost species alluded to above [86]. A recently uncovered elongase
canadum; gilthead
with putative roles in LC-PUFA biosynthesis is elovl8, for which two seabream Sparus
genes, elovl8a and elovl8b, exist in teleosts [97]. This novel elongase, aurata; golden
apparently absent in mammals [97], has been shown to have elongation pompano
activity toward C18 and C20 PUFA in teleosts, as demonstrated by Trachinotus ovatus;
Japanese flounder
functional assays of Elovl8 enzymes of African catfish [71] and rabbit­
Paralichthys
fish [97]. olivaceus; large
Although generally not involved in LC-PUFA biosynthesis pathways, yellow croaker
the other members of the Elovl family have also been studied in teleosts. Larimichthys crocea;
Nibe croaker Nibea
Zebrafish possess two elovl1 genes, elovl1a and elovl1b, and knockdown
mitsukurii; orange-
of elovl1 increased levels of C14 to C20 SFA, MUFA and PUFA in zebrafish spotted grouper
embryos, suggesting Elovl1 in zebrafish may also be involved in the Epinephelus coioides;
elongation of PUFA [98], different from human ELOVL1 that has high rabbitfish Siganus
activity toward C20 and C22 SFA and MUFA [99]. However, molecular canaliculatus;
salema Sarpa salpa;
characterization and nutritional regulation of Elovl6 was reported in
sand sole Pegusa
loach (Misgurnus anguillicaudatus) [100], large yellow croaker (Lar­ lascaris;
imichthys crocea) [101] and rainbow trout [102], with loach Elovl6 blackspotted
shown to elongate 16:0 and 16:1 to 18:0 and 18:1, respectively, similar croaker Nibea
to mammals [100]. While Elovl3 and Elovl7 have not yet been reported diacanthus;
Senegalese sole Solea
in fish, compared to mammalian Elovl7, Elovl7 from the marine crus­ senegalensis; spotted
tacean orange mud crab (Scylla olivacea) had elongation activity towards scat Scatophagus
16:1n-7 and, to a lower extend, C18 PUFA [103]. argus; turbot Psetta
maxima; southern
bluefin tuna
3. The transcriptional regulation of LC-PUFA biosynthesis in Thunnus maccoyii;
teleosts thick-lip grey
(continued on next page)
Based on the reported activities of their Fads and PUFA Elovl

4
D. Xie et al. Progress in Lipid Research 82 (2021) 101095

Table 2 (continued ) Table 3

Elovl Habitat Species References
Teleost species that have been reported to have a complete LC-PUFA biosyn­
type thetic pathway established by either possessing at least Fads with Δ6 and Δ5
activities and elongation capacity toward C18-22 PUFA or, alternatively, Fads
mullet Chelon
with Δ6, Δ5 and Δ4 activities and C18-20 elongation capacity.
labrosus
Amphidromous Boddart’s goggle- [55] Teleosts Fads and Elovl References
fish eyed goby
Freshwater species
Boleophthalmus
African catfish Clarias Δ6/Δ5 Fads2, Elovl5, Elovl4, [57,71]
boddarti
gariepinus Elovl2, Elovl8
Elovl4 Freshwater fish C. gariepinus; [71,86,87]
Mexican silverside Δ6Δ5 Fads2, Δ4 Fads2, Elovl5 [12,31]
D. rerio; loach
Chirostoma estor
Misgurnus
Rainbow trout Oncorhynchus Δ6 Fads2, Δ5 Fads2, Elovl5, [35,56,70,74]
anguillicaudatus;
mykiss Elovl4, Elovl2
O. mykiss
Silver barb Barbonymus Δ6Δ5 Fads2, Elovl5 [58]
Diadromous S. salar [88]
gonionotus
fish
Striped snakehead Channa Δ6Δ5 Fads2, Δ4 Fads2, Elovl5 [32,61]
Marine fish A. schlegeli; [29,50,81,83,85,89–93]
striata
E. coioides;
Tambaqui Colossoma Δ6Δ5 Fads2, Elovl5 [59]
L. crocea; Nibea
macropomum
mitsukurii;
Tench Tinca tinca Δ6Δ5 Fads2, Elovl5, Elovl2 [60]
R. canadum;
Tilapia Oreochromis niloticus Δ6Δ5 Fads2, Δ4 Fads, Elovl5 [12,70]
S. aurata; S.
Zebrafish Danio rerio Δ6Δ5 Fads2, Elovl5, Elovl4, [23,69,86,94]
canaliculatus;
Elovl2
S. argus;
S. senegalensis; Diadromous species
T. ovatus; T. thynnus Atlantic salmon Salmo salar Δ6 Fads2, Δ6Δ5 Fads2, Elovl5, [12,21,22,75,88]
Elovl2 Freshwater fish C. gariepinus; [57,59,60,74,94] Elovl4, Elovl2
C. macropomum; Japanese eel Anguilla Δ6 Fads2, Δ5 Fads1, Elovl5, [20,41,95]
D. rerio; O. mykiss; japonica Elovl2
T. tinca
Marine species
Diadromous A. japonica; Salmo [75,95]
fish solar Rabbitfish Siganus Δ6Δ5 Fads2, Δ4 Fads2, Elovl5, [19,83,97]
Marine fish Sardina pilchardus [96] canaliculatus Elovl4, Elovl8
Elovl8 Freshwater fish C. gariepinus [70,97] Salema Sarpa salpa Δ6 Fads2, Δ5 Fads2, Elovl5 [33,51]
Marine fish S. canaliculatus [97] Sand sole Pegusa lascaris Δ6Δ5 Fads2, Δ4 Fads2, Elovl5 [33,51]
Thicklip grey mullets Chelon Δ6 Fads2, Δ5 Fads2, Elovl5 [33,51]
labrosus

enzymes (Tables 1 and 2), and considering that Δ6 and Δ5 desaturation

capacity, as well as C18-22 PUFA elongation capacity, are required for
biosynthesis of LC-PUFA (ARA, EPA and DHA) from the C18 biosynthetic
precursors LA and ALA, a total of 15 fish species have been reported to
have complete LC-PUFA biosynthetic pathways (Table 3). These include
nine freshwater, two diadromous and four marine fish. In addition, the
ability of fish to biosynthesize LC-PUFA depends, not only on their
complement of desaturase and elongase genes and their corresponding
enzyme activities, but also on the levels of gene expression at both
transcriptional and post-transcriptional levels. Thus, understanding the
regulatory mechanisms of key enzymatic gene expression may be
helpful for developing methods and strategies to increase the ability for
endogenous LC-PUFA biosynthesis in farmed fish species.
It is well known that transcription is a vital step in the central dogma
of transition from genes contained in chromosomes to active proteins.
Transcription factors (TF) or “trans-acting elements” with specific acti­
vator or repressor roles bind to cis-acting elements with corresponding
nucleotide sequences in the promoter region of target genes, regulating
the initiation efficiency of RNA polymerase and gene expression. The
promoter region is defined as the upstream non-coding sequence relative
to the first base-pair of the first exon of the target gene, also called the
transcription start site (TSS), and with position set as “+1” for promoter
analysis purposes. Thus, the investigation of transcriptional regulation
of LC-PUFA biosynthesis usually follows the following main steps: (1)
cloning and characterization of fads2 and elovl promoters; (2) identifi­
cation of the key TF regulating the expression of fads2 and elovl target
genes; (3) confirmation of the transcription regulatory effects and
mechanisms of TF in LC-PUFA biosynthesis.
In the last two decades, research into the transcriptional regulation
of mammal LC-PUFA biosynthesis was mainly focused on human Δ6
FADS2 [104] and mouse Elovl5 genes [105]. Binding sites for the TF
sterol-regulatory element binding protein-1c (SREBP-1c) were identified
in the promoters of Δ6 FADS2 [104] and Elovl5 genes [105], while those
for peroxisome proliferator activated receptor α (PPARα) and
stimulatory protein 1 (SP1) were also found in the Δ6 FADS2 promoter
[106]. Activation of PPARα was shown to promote Δ6 FADS2 promoter
activity [106] and the expression of Elovl5 [105]. In addition, PPARγ
agonist troglitazone significantly decreased Δ6 FADS2 mRNA level and
enzymatic activity, which suggested that PPARγ would potentially
down-regulate LC-PUFA biosynthesis [107]. Other studies showed
human Δ6 FADS2 and mouse Elovl5 genes were not direct targets of liver
X receptor α (LXRα), but were indirectly activated through the effects of
LXRα on SREBP-1c gene by combining with the LXR response element
(LRE) on the latter promoter [104,105]. Moreover, the expression of Δ6
FADS2, Δ5 FADS1 and ELOVL5 were significantly increased by agonists
of LXRα, which indicted that an LXRα–SREBP-1c pathway plays a reg­
ulatory role in LC-PUFA biosynthesis in mammals [108]. It is only more
recently that similar studies have been performed in fish and here we
summarize the major progress made in understanding the transcrip­
tional regulation of LC-PUFA biosynthesis in teleosts.
3.1. Cloning and characterization of fads2 and elovl gene promoters
In order to investigate the transcriptional regulation of LC-PUFA
biosynthesis in teleosts, the upstream sequences from the initiation
codon ATG of fads2 and elovl genes were cloned as the candidate pro­
moters and structures characterized. To date, the promoter of Δ6 fads2
has been cloned in Atlantic salmon [109], Atlantic cod (Gadus morhua)
[109], European seabass (Dicentrarchus labrax) [110], Japanese seabass
(Lateolabrax japonicus) [111], rainbow trout [112], large yellow croaker
[112], and orange-spotted grouper (Epinephelus coioides) [113]. More­
over, the promoter of Δ6Δ5 fads2 has been cloned in rabbitfish [114]
and zebrafish [115], and Δ4 fads2 promoter has been cloned from rab­
bitfish [116] and golden pompano (Trachinotus ovatus) [62]. Regarding
elovl, the promoter of elovl5 has been cloned in Atlantic salmon [117],
large yellow croaker [81], orange-spotted grouper [82], golden pom­
pano [80], rabbitfish [118], zebrafish and snakehead [119], while the

5
D. Xie et al.
elovl4 promoter has been cloned from large yellow croaker [112] and
golden pompano [93].
After the upstream sequences of the target genes were cloned, the
core promoter regions were identified through progressive deletion
mutation and analysis by dual luciferase reporter assays. Binding sites
for TF were identified in the promoters of target genes by bioinformatic
analysis, site-directed mutation and functional assays. In teleosts, mul­
tiple TF binding sites were confirmed to be present in the core promoters
of teleost fads2 and elovl. Sp1 binding element was identified in the
promoter of Atlantic salmon Δ6 fads2 [109], rabbitfish Δ6Δ5 fads2 and
elovl5 [116,118], and C/EBP (CCAAT/enhancer binding protein)
element was identified in the Atlantic cod Δ6 fads2 promoter [109]. NF-
1 (nuclear factor 1) element was found in the promoter of rabbitfish Δ4
fads2 and Δ6Δ5 fads2 [114,116], and PPRE (peroxisome proliferator
response element, binding site for PPAR) was discovered in the promoter
of rabbitfish Δ6Δ5 fads2 [116], and golden pompano Δ4 fads2, elovl5
and elovl4a [62,80,93]. DR-1 (direct repeat 1, binding site for hepato­
cyte nuclear factor 4α, HNF4α) element was reported in the promoter of
rabbitfish Δ4 fads2, Δ6Δ5 fads2 and elovl5 [114,116,118]. The most
conserved cis-acting elements, NF-Y (nuclear factor Y) and SRE (sterol
response element, binding site for SREBP), were confirmed in almost all
the reported fads2 and elovl gene promoters, including those of the
Atlantic salmon Δ6 fads2 and elovl5 [109,117], Atlantic cod Δ6 fads2
[109], rabbitfish Δ6Δ5 fads2, Δ4 fads2 and elovl5 [114,116,118],
zebrafish Δ6Δ5 fads2 and elovl5 [115,119], and snakehead elovl5 [119].
3.2. Identification of TF regulating the expression of fads2 and elovl genes
As described above, the structural analysis of promoters of fads2 and
elovl genes from teleosts uncovered a series of functional cis elements,
such as DR-1, SRE, NF-Y, Sp1 and PPRE [114,116,118], which may play
vital roles in promoter activity and, thus, are likely to be involved in the
regulation of LC-PUFA biosynthesis in teleost fish. Subsequently, bind­
ing/interaction of the TFs with the promoters was confirmed by elec­
trophoretic mobility shift assays (EMSA) and mass spectrometry
[114–119]. Moreover, the roles of the corresponding TF, including
Hnf4α, Srebp-1, Lxr, Sp1 and Ppar, in the transcriptional regulation of
LC-PUFA biosynthesis have been extensively investigated at two anal­
ysis levels including promoter activity and gene expression [120–122].
Promoter activity was investigated through dual luciferase reporter
assay, and gene expression was examined by quantitative polymerase
chain reaction (qPCR). In addition, TF overexpression, agonist or in­
hibitor treatment, and small interfering RNA (siRNA) were also applied
in the study of promoter activity and gene expression. The main TFs
involved in the transcriptional regulation of LC-PUFA biosynthesis in
teleosts and likely vertebrates in general are as follows:
3.2.1. Hnf4α
Hnf4α is a major transcriptional regulator of lipid homeostasis. In
mammals, HNF4α contains two typical structural domains, a DNA
binding domain (DBD) and a ligand binding domain (LBD) [123]. In
teleosts, analysis of the hepatic transcriptome of Atlantic salmon showed
that high expression of hepatic lipid transport-related genes was related
to alterations of hnf4α expression [124]. Additionally, the binding site of
Hnf4α was predicted in the promoter of cptIα1b and cptIα2a genes in
grass carp (Ctenopharyngodon idella), which suggested that Hnf4α may
be involved in modulating fatty acid β-oxidation [125]. In recent years,
our group discovered that Hnf4α was involved in the regulation of LC-
PUFA biosynthesis in rabbitfish by targeting the promoters of Δ6Δ5
fads2, Δ4 fads2, and elovl5, and up-regulating the expression of these
genes [114,120,126]. Indeed, these studies were the first to indicate that
Hnf4α was involved in the regulation of LC-PUFA biosynthesis in
vertebrates.
3.2.2. Srebp-1 and Lxrα
SREBP belongs to the basic helix–loop–helix leucine zipper family
6
Progress in Lipid Research 82 (2021) 101095
and includes three isoforms (SREBP-1a, -1c and -2), and SREBP-1c was
shown to bind SRE in gene promoters and regulate target gene expres­
sion [127]. LXR, including LXRα and LXRβ, belong to the nuclear hor­
mone receptor superfamily, which can heterodimerize with retinoid X
receptor (RXR) and bind to the LXR response element (LRE) of SREBP-1c
promoter [128]. As mentioned above, an LXRα–SREBP-1c pathway
plays a regulatory role in mammalian LC-PUFA biosynthesis
[104,105,108]. In teleosts, srebp-1 and lxrα cDNAs have been isolated
from Atlantic salmon [129,130], rainbow trout [129], Japanese seabass
[131] and rabbitfish [132]. As to the regulatory roles of Srebp1 and Lxr
in LC-PUFA biosynthesis in teleosts, Lxrα was suggested to be a potential
up-regulator of elovl5 [130], and the Lxrα-Srebp-1 pathway was reported
to up-regulate the transcription of Δ6 fads2, elovl5a and elovl5b in
Atlantic salmon [133]. This pattern of regulation was also confirmed in
rabbitfish [132] and large yellow croaker [81]. Recently, the presence of
SRE in the core regulatory region of the elovl5 promoter of snakehead
and zebrafish was demonstrated, and transcriptional activation of elovl5
expression by Srebp-1 was confirmed [119]. These studies confirmed
that the Lxrα-Srebp-1 pathway is conserved between teleost and
mammals.
3.2.3. Pparα and Pparγ
PPAR, a family of ligand-activated nuclear hormone receptors with
DBD and LBD domains, includes three subtypes, namely PPARα, PPARβ
and PPARγ [134]. Similar to LXR, activated PPAR heterodimerize with
RXR, and typically bind to DNA elements containing a common
consensus response element that consists of two copies of the binding
site –AGGTCA– separated by n nucleotides (DRn) [135]. As mentioned
above, PPARα up-regulated mammalian LC-PUFA biosynthesis by pro­
moting the expression of Elovl5 and Δ6 FADS2 [105,106], while PPARγ
has a potential down-regulation effect [107]. In teleosts, the regulation
of LC-PUFA biosynthesis by Pparα and Pparγ appeared more compli­
cated than in mammals. Pparα up-regulated fads2 expression and
enhanced LC-PUFA production in rainbow trout [112], and similar re­
sults were confirmed in a series of studies in golden pompano
[62,80,93]. However, the above study on rainbow trout also indicated
that Pparα had no significant influence on fads2 expression in Japanese
seabass and large yellow croaker [112]. Consistent with this, Pparα had
no effect on key enzymes involved in LC-PUFA biosynthesis in rabbitfish,
whereas Pparγ down-regulated expression of the Δ6Δ5 fads2 gene and
decreased LC-PUFA biosynthesis in rabbitfish [121].
3.2.4. Sp1
SP1, a ubiquitous trans-activator containing three contiguous
carboxyl-terminal Cys2His2 zinc-fingers, is a basal TF maintaining the
expression of housekeeping genes lacking a TATA-box [136]. The
binding sites for SP1 often contain GC-rich motifs in the promoters of
target genes [137]. Five predicted SP1 elements were found in the
human FADS2 promoter [106]. Reed et al. [138] inferred that SP1 may
regulate mammal LC-PUFA biosynthesis by acting as a vital co-factor of
SREBP, because the SP1 binding sites are often very close to SRE [139].
Comparing the Δ6 fads2 promoter sequences of Atlantic salmon, Atlantic
cod, orange-spotted grouper, European seabass and Japanese seabass,
the Sp1 binding element was only detected in salmon and not in the
marine species [110,111], which suggested that the lack of Sp1 binding
element may account for the low promoter activities of Δ6 fads2 of those
species and, possibly, in part explain the low LC-PUFA biosynthetic
capacity of marine carnivorous fish [109,110]. Consistent with this
hypothesis, the insertion of the Sp1 binding element of rabbitfish Δ6Δ5
fads2 promoter into rabbitfish Δ4 fads2 or orange-spotted grouper Δ6
fads2 promoters significantly increased their promoter activities
[113,118]. Subsequently, the Sp1 element was discovered in the elovl5
promoter of rabbitfish, snakehead and zebrafish [118,119], and Sp1 was
demonstrated to be involved in LC-PUFA biosynthesis by upregulating
the expression of liver desaturase and elongase genes in rabbitfish [118],
and thus may be an important regulator of LC-PUFA biosynthetic genes
D. Xie et al.
in teleosts.
3.3. The transcriptional regulation of LC-PUFA biosynthesis in rabbitfish
The rabbitfish S. canaliculatus is a small commercially important
marine teleost widespread along the Indo-West Pacific coast and one of
the main species harvested (Fig. 2). In recent years, S. canaliculatus
farming has expanded rapidly in south-eastern Asia, including China,
due to its popularity with consumers and with a market size of around
150 g. It usually feeds on macroalgae and seagrasses in nature, whereas
in farming it is often fed chopped fish but can also accept formula feed
after simple training. As the first marine teleost demonstrated to have
the capability for biosynthesis of LC-PUFA from C18 PUFA precursors
[140,141] and have its complete PUFA biosynthesis pathway charac­
terized [19,25,83,97], the rabbitfish arose as a good model for the
investigation of LC-PUFA biosynthesis in teleosts. Consequently, rab­
bitfish is arguably the teleost species in which the transcriptional reg­
ulatory mechanisms of LC-PUFA biosynthesis have been most
comprehensively studied. In addition, the establishment of the
S. canaliculatus hepatocyte line (SCHL) provided an important experi­
mental tool [142]. Below summarizes the recent progress made into our
understanding of the regulation of LC-PUFA biosynthesis in rabbitfish.
3.3.1. Promoter characteristics of three key desaturase and elongase genes
for rabbitfish LC-PUFA biosynthesis
In rabbitfish, the promoter sequences of three key LC-PUFA biosyn­
thesis genes including Δ4 fads2, Δ6Δ5 fads2 and elovl5 have been cloned
and characterized. In the promoter region of Δ4 fads2, several key ele­
ments including GATA-2, C/EBP, NF-1, NF-Y, SRE and DR-1 were pre­
dicted, and the binding of Hnf4α and Nf-1 to the promoters was
confirmed using EMSA and LC-MS analyses [116]. In the core region of
the Δ6Δ5 fads2 promoter, elements such as C/EBP, NF-1, Sp1, NF-Y,
SRE, AP1, DR-1 and PPRE were predicted, and the binding of Hnf4α
and NF-1 were also confirmed [114]. Additionally, NF-Y, SRE and DR-1
elements were predicted in the elovl5 promoter, and interaction between
Hnf4α and the DR-1 element was confirmed [122]. In summary, the core
promoter regions of all three genes shared some similar structure, with
all being close to the TSS and containing binding sites for Hnf4α (DR-1)
and Srebp-1c (NF-Y, SRE), while the promoters of both Δ6Δ5 fads2 and
elovl5 genes contained the Sp1 element, and PPRE, the specific element
for PPARγ, was found only in the Δ6Δ5 fads2 promoter.
3.3.2. Stimulatory role of Hnf4α in the transcriptional regulation of
rabbitfish LC-PUFA biosynthesis
The role of Hnf4α in the transcriptional regulation of rabbitfish LC-
PUFA biosynthesis has been demonstrated by both in vitro and in vivo
experiments. Overexpression of Hnf4α in HEK 293T cells or hnf4α mRNA
in SCHL cells, significantly increased Δ4 fads2 promoter activity and the
expression level of Δ4 fads2 mRNA, which confirmed Hnf4α as an
activator of the Δ4 fads2 gene [116]. In SCHL cells, the expression level
of the Δ6Δ5 fads2 gene was also increased by Hnf4α agonists and
Fig. 2. Rabbitfish Siganus canaliculatus.
7
Progress in Lipid Research 82 (2021) 101095
inhibited by Hnf4α inhibitors or siRNA against hnf4α mRNA [114,120].
Moreover, after injection of Hnf4α agonist, inhibitor or Hnf4α siRNA
into rabbitfish abdomen to modulate hnf4α expression, transcript levels
of Δ4 fads2 and Δ6Δ5 fads2 were positively correlated with the
expression level of the hnf4α gene [126]. The effect of Hnf4α regulation
on LC-PUFA biosynthesis of SCHL cells transfected with hnf4α mRNA or
liver of rabbitfish injected with Hnf4α agonist or inhibitor was deter­
mined by analyzing fatty acid compositions of cellular and hepatic
lipids. This showed that Hnf4α not only increased elovl5 promoter ac­
tivity and elovl5 mRNA level, but also increased LC-PUFA biosynthesis
both in vitro and in vivo [122]. These data demonstrated that Hnf4α
involved in the regulation of LC-PUFA biosynthesis in rabbitfish by
increasing transcription of Δ4 fads2, Δ6Δ5 fads2 and elovl5, which was
the first demonstration of the role of Hnf4α in vertebrate LC-PUFA
biosynthesis.
3.3.3. Positive roles of Lxrα-Srebp-1 in the transcriptional regulation of
rabbitfish LC-PUFA biosynthesis
To investigate the role of the classical Lxrα-Srebp-1 pathway in the
regulation of LC-PUFA biosynthesis in rabbitfish, lxrα and srebp-1 genes
were first cloned and characterized [132]. In vivo, the expression levels
of srebp-1 and the key LC-PUFA biosynthesis genes, Δ4 fads2, Δ6Δ5
fads2 and elovl5, displayed consistent changes in response to dietary
lipid source and ambient salinity [132]. Additionally, in vitro, the
expression levels of lxrα and the key enzymatic genes were stimulated by
the Lxrα agonist T0901317 in rabbitfish primary hepatocytes. Besides,
the promoter activities of Δ6Δ5 fads2 and elovl5 genes were significantly
increased by over-expression of srebp-1 mRNA in HEK 293T cells, and
the expression of Δ6Δ5 fads2 and elovl5 was decreased by knockdown of
srebp-1 mRNA in SCHL cells [143]. The above results demonstrated that
the Lxrα-Srebp-1 pathway is involved in LC-PUFA biosynthesis in rab­
bitfish, as demonstrated previously in mammals [104,105,107,108],
through stimulating expression of Δ4 fads2, Δ6Δ5 fads2 and elovl5
genes.
3.3.4. Role of Sp1 in determining the transcriptional activity of genes
involved in the rabbitfish LC-PUFA biosynthesis
An Sp1 binding site with high activity was found in rabbitfish Δ6Δ5
fads2 and elovl5 promoters, and overexpression of sp1 mRNA in SCHL
cells significantly increased the expression of Δ6Δ5 fads2 and elovl5, as
well as the LC-PUFA content of the cells [118]. In contrast, the Sp1
binding site was absent from the rabbitfish ∆4 fads2 promoter, but the
activity of this promoter was significantly increased by insertion of the
Sp1 element from the rabbitfish ∆6∆5 fads2 promoter [118]. In addition,
as described above, the activity of orange-spotted grouper fads2 pro­
moter, which lacked an Sp1 binding site, was also significantly increased
after acquiring the Sp1 binding site of rabbitfish Δ6Δ5 fads2 [113].
These results demonstrated that Sp1 is an important transcription factor
involved in the regulation of LC-PUFA biosynthesis by targeting Δ6Δ5
fads2 and elovl5 in rabbitfish [118], which was the first demonstration of
this mechanism in a vertebrate.
3.3.5. Inhibitory role of Pparγ in the transcriptional regulation of rabbitfish
LC-PUFA biosynthesis
In mammals, PPARγ is a key regulator involved in adipocyte differ­
entiation and lipid homeostasis [144] but, to the best of our knowledge,
there has been no report on the regulatory roles of PPARγ in mammalian
LC-PUFA biosynthesis. In rabbitfish primary hepatocytes, the Pparγ
agonists 2-bromopalmitate and fenofibrate increased the expression of
both pparγ and elovl5, but decreased the expression of Δ6Δ5 fads2 [145].
Moreover, the PPRE binding site for Pparγ was identified in the pro­
moter of Δ6Δ5 fads2 gene, and site-directed mutation and EMSA indi­
cated interaction between Pparγ and the promoter of the Δ6Δ5 fads2
gene [121]. Specifically, over-expression of pparγ in SCHL cells inhibited
the expression of Δ6Δ5 fads2 and reduced LC-PUFA biosynthesis ca­
pacity, while pparγ RNAi knockdown increased the expression of Δ6Δ5
D. Xie et al.
fads2. These data demonstrated a negative effect of Pparγ in the regu­
lation of rabbitfish LC-PUFA biosynthesis [121].
3.4. Studies on the transcriptional regulation of LC-PUFA biosynthesis in
other teleosts
Besides rabbitfish, other relevant studies on the transcriptional
regulation of LC-PUFA biosynthesis have been conducted in Atlantic
salmon, Japanese and European seabass, large yellow croaker, golden
pompano, zebrafish and gilthead seabream (Sparus aurata). The main
results are summarized below.
3.4.1. Atlantic salmon
Studies on transcriptional regulation of LC-PUFA biosynthesis in
teleosts was initiated in Atlantic salmon which, second to rabbitfish, is
the species with the most knowledge of the key enzymes of LC-PUFA
biosynthesis. To date, genes including Δ6 fads2 (a/b/c), Δ5 fads2,
elovl5 (a/b), elovl2 and elovl4b have been cloned and functionally
characterized from this species [12,21,22,35,70,73,75,88,117]. In
addition, mechanisms of transcriptional regulation of LC-PUFA biosyn­
thesis have also been investigated in Atlantic salmon. Specifically, the
salmon head kidney cell line was treated with EPA and DHA, and a
positive relationship between the relative expression of the transcription
factors lxr and srebp1 and expression levels of key desaturase and
elongase target genes (Δ6 fads2a, Δ5 fads2, elovl5a and elovl2) was
observed [130], indicating a possible role for the Lxrα-Srebp1 pathway
in the regulation of LC-PUFA biosynthesis in salmon. Later, the regula­
tory role of Lxrα-Srebp1 was confirmed by TF ligand activation and
agonist assays, as well as srebp1 over-expression, which established that
Lxr can activate Srebp1, and Srebp1 was an activator of Δ6 fads2, elovl5a
and elovl5b expression [133]. Moreover, the conserved elements NF-Y
and SRE, as well as the Sp1 binding element, were found in the core
promoter of Atlantic salmon Δ6 fads2 and elovl5 genes through site-
directed mutation and EMSA assay [109,117].
3.4.2. Japanese and European seabass
In Japanese seabass, promoter activity analysis showed that over-
expression of srebp-1 or pparα significantly increased the activity of
fads2 promoter, which confirmed the importance of NF-Y and SRE sites
for fads2 gene expression in this species of seabass [112]. Studies in
seabass were also the first to report on the transcriptional regulation of
LC-PUFA biosynthesis at the level of epigenetic modification in teleosts.
Specifically, the influence of different dietary lipid sources on gene
expression and core promoter methylation of Δ6 fads2 were studied in
European seabass (D. labrax) and Japanese seabass (L. japonicus)
[110,111]. In Japanese seabass, a significant negative correlation was
observed between the CpG methylation of fads2 promoter and hepatic
fads2 transcript levels in response to high dietary n-3 LC-PUFA (pre­
dominantly DHA and EPA, ~2:1). However, the conserved NF-Y, SRE
and Sp1 sites were not methylated, and so the possible inhibition of
fads2 expression by DNA methylation might be related to the inactive
chromatin structure and/or inhibition of another TF [111]. In European
seabass, high dietary n-3 LC-PUFA (predominantly DHA and EPA,
~1.4:1) also reduced expression of Δ6 fads2, but no differences in
methylation at CpG sites were observed in the Δ6 fads2 promoter [110].
Possible reasons for these different results might be the different DHA:
EPA ratios, or n-3 LC-PUFA contents of the diets in the two studies with
the high n-3 LC-PUFA diet (45 % of total fatty acids) being almost 30-
fold greater than that of the low n-3 LC-PUFA diet (1.5 % of total fatty
acids) in the Japanese seabass study, but the high n-3 LC-PUFA diet (1.2
% of diet) was only 4-fold higher than the low diet (0.3 % of diet) in the
European seabass study [110,111].
3.4.3. Large yellow croaker
Evidence collected in large yellow croaker from in vivo feeding trials
and in vitro hepatocyte cell culture studies showed that levels of lxrα,
8
Progress in Lipid Research 82 (2021) 101095
srebp-1, Δ6 fads2, elovl5 and elovl4 mRNA decreased with high dietary
LC-PUFA [81,112,146]. As these data suggested that there might be a
regulatory relationship between Lxrα, Srebp-1 and key LC-PUFA
biosynthesis genes in this species, the Δ6 fads2, elovl5 and elovl4
cDNAs and their corresponding promoters were characterized
[81,112,146]. In HEK 293T cells, the over-expression of lxrα signifi­
cantly increased the activities of the elovl4 and elovl5 promoters, while
over-expression of srebp-1 significantly decreased the promoter activ­
ities of Δ6 fads2 and elovl4 [81,112]. These results indicated that Lxrα
and Srebp-1 were involved in the transcriptional regulation of key genes
in the LC-PUFA biosynthesis pathway. Additionally, in croaker primary
hepatocytes, expression of the elovl4 gene was up-regulated by an Lxrα
agonist and decreased by a Srebp-1 inhibitor, while elovl5 expression
was increased by Lxrα agonist but showed no response to the Srebp-1
inhibitor [112]. These results confirmed that the transcription of
elovl4 can be regulated by both Lxrα and Srebp-1, while suggesting that
the expression of elovl5 might be the target of Lxrα, but not Srebp-1
[112].
3.4.4. Golden pompano
An in vivo feeding trial with golden pompano juveniles suggested that
this species could not synthesize LC-PUFA from C18 PUFA or that such
ability is very limited [147]. However, full-length mRNAs encoding two
putative Elovl elongases and two putative Fads2 desaturases were iso­
lated, and the corresponding enzymatic activities were investigated by
heterologous expression in yeast [62,80,93]. The two putative elongases
were reported to possess Elovl4 and Elovl5 activities, while both Fads2
were reported to display Δ8/Δ5/Δ4 desaturase activities [62,80,93].
Although no Δ6 activity was detected, these data would still suggest that
golden pompano would have the ability to biosynthesize EPA and ARA
from ALA and LA, respectively, via the Δ8 pathway and, furthermore,
also have the ability to produce DHA from EPA via the Δ4 pathway.
These data are unusual and highly unexpected as they are not consistent
with the data from the in vivo feeding trial in golden pompano [147] or
the existing body of knowledge on LC-PUFA biosynthesis and teleost
phylogeny [5,13]. However, if and when the data are confirmed, they
suggest that pompano will represent another highly interesting species
for further study. In this respect, so far the promoters of elovl5 [80],
elovl4 [93], fads2 [62] have been isolated, and promoter activity assays
have shown that Pparαb was the key positive TF in the regulation of
these genes [62,80,93], similar to the regulatory mechanisms in rainbow
trout [112] and mammals [106].
3.4.5. Zebrafish
Zebrafish has all the enzymatic machinery enabling the biosynthesis
of LC-PUFA from the C18 precursors, LA and ALA, via the combined
action of the gene products of Δ6Δ5 fads2 [23], elovl5 [69], elovl2 [94]
and elovl4 [86]. In recent years, the transcriptional regulation of LC-
PUFA biosynthesis has been investigated in zebrafish with NF-Y,
CCAAT boxes, Sp1, SRE and PPRE binding sites recognized as impor­
tant for maintaining and regulating Δ6Δ5 fads2 promoter activity [115],
whereas NF-Y, SRE and Sp1 were vital for elovl5 promoter activity [119].
Moreover, Srebp-1 was shown to interact with SRE in the promoters of
Δ6Δ5 fads2 and elovl5, while NF-Y binds to CCAAT sites in the promoter
of Δ6Δ5 fads2 [115]. Overexpression of Srebp-1 in the zebrafish liver
cell line (ZFL ATCC® CRL-2643™) increased the activities of Δ6Δ5
fads2 and elovl5 promoters, which provided further evidence of the
transcriptional regulation mechanisms in D. rerio [115].
3.4.6. Gilthead seabream
Gilthead seabream is another marine teleost whose LC-PUFA
biosynthesis pathway has attracted attention. The seabream fads2
gene was cloned and characterized with dual ∆6 and ∆8 desaturase ac­
tivity [25,35,148], and PUFA elongation capacity has been also
demonstrated through functional characterization of the Elovl5 and
Elovl4 elongases [70,90]. Like most marine fish, gilthead seabream has a
D. Xie et al.
low capacity to biosynthesize LC-PUFA due to the apparent absence of
Δ5 desaturase activity [35,148] and, thus, an adequate dietary supply of
LC-PUFA is critical for larval growth and development [149]. Although
several studies reported that Ppar, Sp1, Srebp-1, Lxrα, and Np-y may be
involved in the regulation of energy metabolism in gilthead seabream
[150–152], no direct evidence supported the roles of these TF in LC-
PUFA biosynthesis. However, feeding trial studies showed that the
mRNA levels of pparγ, srebp-1, Δ6 fads2, and elovl4 increased in seab­
ream fed low dietary LC-PUFA, which suggested that Pparγ and Srebp-1
may play roles in regulating LC-PUFA biosynthesis [153,154]. Addi­
tionally, two recent studies showed that the promoters of stearoyl-CoA
desaturase 1a and fads2 were responsive to broodstock nutrition, and
that both the parents fed VO diets (low n-3 LC-PUFA) and their offspring
shared increased DNA-methylation [155,156], which revealed epige­
netic mechanisms in the nutritional regulation of LC-PUFA biosynthesis
in seabream.
Overall, concerning transcriptional regulation of LC-PUFA biosyn­
thesis, similar regulatory mechanisms such as the Lxrα-Srebp-1 pathway
are conserved from teleosts to mammals. However, teleosts appear to
possess more complex mechanisms of regulation as a result of the
increased gene repertoire and greater functional diversity of teleost
fads2 and, to some extent, elovl genes [20,34]. For example, Sp1 plays an
important role in determining the promoter activities of Δ6Δ5 fads2 and
elovl5 genes, Hnf4α and Pparα target widely the promoters of Δ6Δ5
fads2, Δ4 fads2, and elovl5 genes as a stimulatory (positive) TF, while
Pparγ has inhibitory (negative) effects on Δ6Δ5 fads2 expression. A
schematic overview of the transcriptional regulation of LC-PUFA
biosynthesis in teleosts based on currently available data is shown in
Fig. 3. However, this is far from a full understanding of transcriptional
regulation of LC-PUFA biosynthesis in teleosts. The influence of fish
species, habitat and feeding habits, as well as impacts of salinity, tem­
perature and dietary nutrients also require to be elucidated. Certainly,
expression of TF and downstream genes, and metabolism were changed
with adaptation to environmental and dietary factors. Teleosts may
respond to changes in these factors via stimuli acting through known cell
signalling cascades, such as calcium ion [Ca2+] and kinase phosphory­
lation among others. However, presently little is known about these
cellular signalling pathways in the regulation of key enzyme genes
involved in LC-PUFA biosynthesis in fish, and this requires to be
regulation of target mRNA expression.
9
Progress in Lipid Research 82 (2021) 101095
investigated in the future.
4. The post-transcriptional regulation of LC-PUFA biosynthesis
in teleosts
It is well known that most eukaryotic genes are also subject to
considerable post-transcriptional regulation, and thus transcribed
mRNA can undergo a variety of modifications including further pro­
cessing, export, localization, turnover, and translation, which are an
integral part of gene expression, and equally as sophisticated and
important as transcriptional control. Recent improvements in high
throughput sequencing and computational prediction methods have
allowed the discovery of several types of non-coding RNA (ncRNA) that,
together with protein effector complexes, can also control the expression
of target mRNA at a post-transcriptional level [157]. Non-coding RNA,
functional RNA molecules that are transcribed from DNA but do not
code for proteins, includes microRNA (or miRNA), small interfering
RNA (siRNA), small nuclear RNA (snRNA) and long non-coding RNA
(lncRNA) [157]. Among them, microRNA (or miRNA) appear as
important post-transcriptional regulators of their mRNA targets via
mRNA degradation and/or translational repression [158]. MiRNA are
highly conserved, small, ncRNA of 18–25 nt length that typically control
the expression of their targets by imperfect base pairing to the 3′ un­
translated regions (3′ UTR) of the target mRNA [159]. Now, miRNA have
emerged as crucial post-transcriptional regulators of lipid metabolism
[160].
While there have been no reports in other vertebrates, the roles of
miRNA in the regulation of LC-PUFA biosynthesis has attracted attention
in teleosts. Recently, several miRNAs have been identified to be involved
in LC-PUFA biosynthesis in the teleost model S. canaliculatus by target­
ing LC-PUFA biosynthesis-related genes as outlined in Fig. 3. These
include miR-17 and miR-146a that directly target key desaturase and
elongase genes involved in LC-PUFA biosynthesis, and miR-24, miR-33,
miR-26a, miR-145 and the miR-15/16 cluster that target TF that, in turn,
regulate the expression of key desaturase and elongase genes involved in
LC-PUFA biosynthesis [143,161–167]. Below, we provide a description
of the most important findings obtained in these studies.
Fig. 3. Schematic overview of transcription factors
(TF) and miRNA involved in the transcriptional and
post-transcriptional regulation of LC-PUFA biosyn­
thesis in teleosts. The main TF binding elements
identified in the core promoters of teleost fads2 and
elovl include GC rich region, nuclear factor Y (NF-Y),
sterol regulatory element (SRE), direct repeat 1 (DR-
1) and peroxisome proliferator response element
(PPRE). The TF include stimulatory protein 1 (Sp1),
sterol regulatory element-binding protein-1 (Srebp-
1), hepatocyte nuclear factor 4 alpha (Hnf4α), liver X
receptor alpha (Lxrα) and peroxisome proliferator-
activated receptors alpha/gamma (Pparα/γ). Several
miRNAs have been shown to be involved in LC-PUFA
biosynthesis in S. canaliculatus, where miR-17 and
miR-146a directly target Δ4 fads2 and elovl5,
respectively. miR-24 and miR-33 directly co-target
insulin-induced gene 1 (insig1), an endoplasmic re­
ticulum (ER) membrane protein that facilitates the
retention of Srebp-1 precursors in the ER and pre­
vents their proteolytic activation in the Golgi appa­
ratus. miR-26a, miR-145 and miR-15/16 cluster
target lxrα, hnf4α and pparγ, respectively. nSrebp-1,
the mature nuclear form of Srebp-1 protein; Scap,
Srebp cleavage-activating protein; TSS, transcrip­
tional start site. Pointed arrows: upregulation of
target mRNA expression. Blunt arrows: down-
D. Xie et al.
4.1. Inhibitory roles of miR-17, miR-146a, miR-26a and miR-145 in the
regulation of LC-PUFA biosynthesis
The first miRNA demonstrated to participate in the regulation of LC-
PUFA biosynthesis in vertebrates was miR-17 in rabbitfish [161]. It is
located in the forepart of the miR-17-92 cluster and its mature sequence
is highly consistent with that of other vertebrate species. In silico analysis
found a conserved complementary site for miR-17 in the 3′ UTR of rab­
bitfish Δ4 fads2 mRNA, and dual luciferase reporter assays demon­
strated that miR-17 targeted the 3′ UTR of Δ4 fads2 directly [161].
Moreover, miR-17 abundance was significantly higher in the liver of
rabbitfish reared at 32 ppt salinity than at 10 ppt salinity, and showed
inverse trends with Δ4 fads2 gene expression and protein quantity. An
inverse expression pattern for miR-17 and Δ4 fads2 was also found in
rabbitfish primary hepatocytes incubated with 100 μM PUFA including
ALA, EPA and DHA. These results demonstrated that miR-17 was
involved in the regulation of LC-PUFA biosynthesis by targeting Δ4
fads2 (Fig. 3).
Similar to miR-17, another miRNA, miR-146a, was found to bind the
3′ UTR of elovl5 that, as noted above, is one of the critical enzymes in the
elongation of C18 and C20 PUFA [5,13,65]. In vitro gain- and loss-of-
function experiments in SCHL cells demonstrated that miR-146a
decreased Elovl5-dependent PUFA elongation product/substrate
indices including 20:3n-6/18:3n-6, 20:4n-3/18:4n-3 and 22:5n-3/
20:5n-3, as well as overall LC-PUFA contents, by inhibiting the expres­
sion of elovl5 [163] (Fig. 3).
Different to the regulatory mechanisms on LC-PUFA biosynthesis
described above for miR-17 and miR-146a by directly targeting Δ4 fads2
and elovl5, respectively, miR-26a was found to exert its regulatory ef­
fects on LC-PUFA biosynthesis indirectly by targeting the 3′ UTR of the
TF lxrα, based on both in silico analysis and dual luciferase reporter as­
says [165]. Overexpression of miR-26a in SCHL cells markedly reduced
the protein levels of Lxrα, Srebp-1 and Δ6Δ5 Fads2 induced by the Lxr
agonist T0901317. On the contrary, knockdown of miR-26a using
antagomirs (i.e. anti-MiR) facilitated Srebp-1 processing and increased
the expression of Δ6Δ5fads2, Δ4 fads2 and elovl5 genes involved in LC-
PUFA biosynthesis, and consequently, promoted LC-PUFA biosynthesis
in both hepatocytes in vitro and rabbitfish in vivo. Combining the results
on miR-26a and those of the role of the Lxrα-Srebp-1 pathway in the
regulation of LC-PUFA biosynthesis as mentioned above (Section 3.3)
[162], it has been suggested that miR-26a plays a critical role in regu­
lating LC-PUFA biosynthesis through targeting the Lxrα-Srebp-1
pathway in rabbitfish [165] (Fig. 3).
Similarly, miR-145 was found to target the 3′ UTR of hnf4α that, as
noted above, is an important TF involved in LC-PUFA biosynthesis in
rabbitfish by regulating expression of ∆4 fads2, Δ6Δ5 fads2 and elovl5
[114,116,120,126,142]. In vitro gain- and loss-of-function experiments
in SCHL cells demonstrated that miR-145 decreased the expression of ∆4
fads2, Δ6Δ5 fads2 and elovl5 genes, and consequently reduced total LC-
PUFA contents by targeting hnf4α [166]. Moreover, knockdown of miR-
145 by intraperitoneal injection with antagomirs increased Hnf4α pro­
tein level and its downstream target genes expression (Δ6Δ5fads2, Δ4
fads2 and elovl5), as well as total LC-PUFA contents in fish tissues,
including liver, brain and eyes, where LC-PUFA biosynthetic activity is
particularly high in fish [83]. These results revealed that miR-145 may
be a key mediator involved in the regulation of LC-PUFA biosynthesis
both in vivo and in vitro through targeting hnf4α in the marine teleost
S. canaliculatus [166] (Fig. 3).
4.2. Stimulatory roles of miR-33 and miR-24 in the regulation of LC-
PUFA biosynthesis
In mammals, miR-33 has been found within an intron of SREBP-1, an
important TF involved in regulating the expression of genes encoding
key enzymes of vertebrate LC-PUFA biosynthesis and, consequently,
miR-33 was reported to modulate lipid metabolism in cooperation with
10
Progress in Lipid Research 82 (2021) 101095
its host, SREBP-1 [168]. Subsequently, miR-33 was investigated in
rabbitfish and confirmed to be an intronic miRNA (intron 16) in the
srebp-1 gene [162]. Moreover, miR-33 was demonstrated to be involved
in the regulation of LC-PUFA biosynthesis by cooperating with Srebp-1
and/or through facilitating Srebp-1 processing by targeting insulin-
induced gene 1 (Insig1) [143,162] (Fig. 3). INSIG1 is an endoplasmic
reticulum (ER) membrane protein that facilitates the retention of
SREBP-1 precursors in the ER and prevents their proteolytic activation
in the Golgi apparatus [169].
Additionally, miR-24 was found to bind the 3′ UTR of insig1 mRNA in
rabbitfish [164]. Overexpression of miR-24 down-regulated Insig1
protein level, and up-regulated mature Srebp-1 protein and gene
expression of Δ4 fads2, Δ6Δ5 fads2 and elovl5 in SCHL cells. Knockdown
of miR-24 increased the expression of insig1 but inhibited the expression
of mature Srebp1 protein and Δ4 fads2, Δ6Δ5 fads2 and elovl5 genes,
and these effects could be attenuated by additional insig1 knockdown
(Fig. 3). Besides, knockdown of miR-24 in SCHL cells not only reduced
MUFA accumulation, but also suppressed LC-PUFA biosynthesis through
inhibiting Insig1-dependent Srebp-1 activation and the expression of
critical enzymes that are required for LC-PUFA biosynthesis [164].
4.3. Role of miRNA clusters in the regulation of LC-PUFA biosynthesis
Compared with individual miRNA, the regulatory model of miRNA
clusters is more complex, and their function is more efficient [170,171].
Recent studies demonstrated the role of the miR-15/16 cluster in the
regulation of LC-PUFA biosynthesis in rabbitfish [167]. First, the
abundance of miR-15/16 cluster was greatly increased in SCHL cells
treated with the LC-PUFA biosynthetic precursor ALA, but was signifi­
cantly depressed by DHA and EPA, which suggested a possible role of the
miR-15/16 cluster in LC-PUFA biosynthesis. Moreover, bioinformatic
analysis showed a potential binding site shared for miR-15 and miR-16
in the 3′ UTR of pparγ, a negative regulator of LC-PUFA biosynthesis in
rabbitfish as mentioned above. In vitro, luciferase reporter assays
revealed that pparγ was a potential target of the miR-15/16 cluster, and
individual or co-overexpression of miR-15 and miR-16 in SCHL cells
significantly inhibited both mRNA and protein levels of Pparγ, but
increased the mRNA levels of Δ6Δ5 fads2, Δ4 fads2 and elovl5. This led
to increased contents of DHA and ARA in the SCHL cells with higher
product/substrate ratios for Δ6Δ5 and Δ4 desaturation activities.
Furthermore, inhibition of Pparγ was more pronounced with co-
overexpression of miR-15 and miR-16 than with individual over­
expression in SCHL cells. Consistently, knockdown of the miR-15/16
cluster gave the opposite results, and increased mRNA levels of LC-
PUFA biosynthesis enzymes were observed after knockdown of pparγ.
These gain- and loss-of-function experiments demonstrated that miR-15
and miR-16 act as a miRNA cluster to enhance LC-PUFA biosynthesis by
targeting Pparγ in rabbitfish, uncovering a novel mechanism that may
also operate in other vertebrates [167].
Overall, studies on the roles of miRNA in the regulation of LC-PUFA
biosynthesis have only been reported in S. canaliculatus, but not yet in
other fish species or other vertebrates, except for miR-193a-5p that was
shown to regulate LC-PUFA biosynthesis by targeting FADS1 in bovine
mammary epithelial cells [172]. As described above, individual miRNA
or clusters regulated rabbitfish LC-PUFA biosynthesis by targeting fads2,
elovl or TF-encoding genes. At present, little is known about how
external factors such as ambient salinity, temperature, or dietary nu­
trients like fatty acids are signaled to cellular cascades to affect miRNA
that can then influence TF with the latter subsequently regulating
enzyme gene transcription or protein translation and, eventually,
enzymic activities. Moreover, it is not known whether feedback and
feed-forward loops are present in teleosts. In these, a TF regulates a
miRNA or a miRNA modulates a TF, with both co-regulating the same
target genes involved in LC-PUFA biosynthesis as reported in mammals
where these regulatory loops are important regulatory motifs in multiple
biological processes [173]. Thus, it is crucial to identify and study
D. Xie et al.
miRNA-TF-gene or TF-miRNA-gene co-regulatory networks in teleosts as
the application of various genome-wide approaches increases in the
future so as to provide a more comprehensive insight into the regulatory
mechanisms of LC-PUFA biosynthesis.
5. Conclusions and perspective
In recent years, the regulatory mechanisms of LC-PUFA biosynthesis
in teleost fish has become a hot research topic in lipid nutrition, as it
provides insight into the metabolic responses that are triggered in fish
when fed diets with high inclusion of VO that dominate the market in
intensive aquaculture. The fundamental biochemical and molecular
studies will facilitate the development of practical strategies that can
enable increased replacement of dietary FO (rich in LC-PUFA) with VO
(rich in C18 PUFA precursors, but devoid of LC-PUFA) in farmed teleost
diets, while ensuring that this does not negatively impact the health of
the fish or its nutritional quality (i.e. content of n-3 LC-PUFA) as human
food [174].
There are at least five TF involved in the transcriptional regulation of
LC-PUFA biosynthesis in teleosts, with Hnf4α, Lxrα-Srebp-1 and Sp1
displaying positive/stimulatory roles, and Pparγ playing negative/
inhibitory effects. Indeed, evidence obtained in S. canaliculatus was the
first demonstration that Hnf4α and Pparγ have major regulatory func­
tions in LC-PUFA biosynthesis and provided clarification of the impor­
tant role of Sp1 in determining the transcriptional control of genes
involved LC-PUFA biosynthesis in vertebrates. With respect to post-
transcriptional regulation of LC-PUFA biosynthesis, studies showed
that specific miRNA or miRNA clusters play important roles by acting
either directly on key fads and elovl genes involved in LC-PUFA
biosynthesis or indirectly by regulating TF that, in turn, themselves
modulate the expression of the fads and elovl genes. While miR-33, miR-
24 and the miR-15/16 cluster have stimulatory roles in LC-PUFA
biosynthesis in teleosts, miR-17, miR-26a, miR-145 and miR-146a
have inhibitory roles. Fig. 3 summarizes the regulatory mechanisms of
LC-PUFA biosynthesis in teleosts, showing several aspects with complex
interactions. Among them, Srebp-1 and Hnf4α might play more central
roles in the regulation of LC-PUFA biosynthesis. For example, Lxrα, miR-
33, miR-24 and Insig1 regulated LC-PUFA biosynthesis via Srebp-1,
while the regulation of Sp1 on key enzymes of LC-PUFA biosynthesis
also depends on Srebp-1. Other factors, such as Pparγ, miR-17, miR-26a,
miR-145, miR-146a and miR-15/16 directly or indirectly target different
nodes to form a complex network to regulate LC-PUFA biosynthesis.
While rabbitfish is a robust model for the investigation of LC-PUFA
biosynthesis in vertebrates, a major challenge that needs to be
addressed in the future is the sequencing of the rabbitfish genome as­
sembly that will enable, among others, the application of gene knockout
methodologies (e.g. CRISPR/Cas9), nowadays regarded as the “gold
standard” in gene function analyses. Further studies are also required to
understand how environmental factors such as salinity, and nutritional
factors such as lipids and fatty acids, affect LC-PUFA biosynthesis.
Current data collected through the studies presented above suggest that
miRNA play pivotal roles in the metabolic responses of teleosts to
environmental challenges affecting lipid homeostasis. Besides, the reg­
ulatory network of LC-PUFA biosynthesis in teleosts needs to be further
investigated and integrated within the overall regulation of various lipid
metabolic pathways. The regulation of both de novo lipogenesis and LC-
PUFA biosynthesis by Srebp-1 and Hnf4α, suggests a close relationship
between the different metabolic pathways, which indicates that we must
consider more aspects when exploring new mechanisms of lipid meta­
bolism, including different pathways and different tissues. This can be
achieved by systematic analysis of the transcriptome, proteome, and
metabolome.
Until now, there has been relatively little research into the post-
transcriptional regulation of LC-PUFA biosynthesis in other fish spe­
cies. The substantial complexity of gene expression after transcription
may be one of the reasons. Lack of commercial antibodies for proteins
11
Progress in Lipid Research 82 (2021) 101095
related to LC-PUFA biosynthesis in different fish species also increases
the difficulties in investigating the multifaceted control of gene
expression at the post-transcriptional level. Studies of post-
transcriptional control by miRNA have provided a glimpse into the
richness and sophistication of mRNA regulation. As the application of
various genome-wide approaches increases, the next decade will bring
an even more comprehensive view of all levels of the cellular regulation
of LC-PUFA biosynthesis in teleosts.
Author contributions
Dizhi Xie, Cuiying Chen, Yewei Dong and Shuqi Wang: Conceptual­
ization, writing original draft; Cuihong You, Óscar Monroig and Douglas
R. Tocher: Writing, review and editing of manuscript; Yuanyou Li:
Writing, design, review and editing, supervision and funding acquisi­
tion. All authors approved the submission and publication of this
manuscript.
Declaration of Competing Interest
None.
Acknowledgements
This work was financially supported by the National Key R&D Pro­
gram of China (2018YFD0900400) and National Natural Science
Foundation of China (31873040, 31110103913, 30972266, 30671629,
31702357), and partly funded through the project IMPROMEGA of the
Ministry of Science, Innovation and Universities, Spanish Government
(grant no. RTI2018-095119-B-I00, MCIU/AEI/FEDER, UE).
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