Biochem. J. (2010) 429, 235–242 (Printed in Great Britain)
235
doi:10.1042/BJ20100293
REVIEW ARTICLE
Zebrafish: gaining popularity in lipid research
Maarit HÖLTTÄ-VUORI*, Veijo T. V. SALO*, Lena NYBERG†, Christian BRACKMANN†, Annika ENEJDER†, Pertti PANULA* and
Elina IKONEN*1
*Institute of Biomedicine/Anatomy, Haartmaninkatu 8, 00140 University of Helsinki, Finland, and †Chalmers University of Technology, Department of Chemical and Biological
Engineering, SE-412 96 Göteborg, Sweden
Zebrafish are an increasingly popular vertebrate model organism
in which to study biological phenomena. It has been widely
used, especially in developmental biology and neurobiology,
and many aspects of its development and physiology are
similar to those of mammals. The popularity of zebrafish relies
on its relatively low cost, rapid development and ease of
genetic manipulation. Moreover, the optical transparency of the
developing fish together with novel imaging techniques enable
the direct visualization of complex phenomena at the level of the
entire organism. This potential is now also being increasingly
appreciated by the lipid research community. In the present review
we summarize basic information on the lipid composition and
distribution in zebrafish tissues, including lipoprotein metabolism,
intestinal lipid absorption, the yolk lipids and their mobilization,
as well as lipids in the nervous system. We also discuss studies
in which zebrafish have been employed for the visualization
of whole-body lipid distribution and trafficking. Finally, recent
advances in using zebrafish as a model for lipid-related diseases,
including atherosclerosis, obesity, diabetes and hepatic steatosis
are highlighted. As the insights into zebrafish lipid metabolism
increase, it is likely that zebrafish as a model organism will
become an increasingly powerful tool in lipid research.
INTRODUCTION
has been the lack of convenient methods to perform heritable
targeted mutagenesis. This problem may now be solved by the
recent development of customized zinc-finger nucleases that
introduce targeted frameshift mutations in the genome, thereby
enabling creation of directed zebrafish gene knockouts [1]. In
gene-silencing approaches, it should be taken into account that
zebrafish harbour two copies of many genes owing to a genomewide duplication event. This took place after the bony fishes
diverged from the common ancestor with humans some 350
million years ago. The duplicated genes often exhibit differential
tissue expression patterns with partitioning of ancestral functions,
rather than the evolution of completely new functions [2].
The whole-genome sequencing of zebrafish is almost complete
(http://www.sanger.ac.uk/Projects/D_rerio).
In terms of lipid metabolism, zebrafish as a model system is
still a newcomer. Although considerable amount of information
is available on fish lipid composition in general, there is,
evidently, a bias towards those species that are important as a
food source, such as salmonids. Among different fish species,
there is substantial diversity concerning their living environments
(salinity and temperature) and diet (carnivores, insectivores and
herbivores). Thus results obtained from one fish species are not
necessarily directly applicable to others.
What makes zebrafish particularly attractive as a research tool
is the possibility of applying subcellular resolution methods
to a whole organism. The developing zebrafish embryos are
small and translucent, enabling non-intrusive visualization of
organs and biological processes in vivo with high resolution.
This feature has also been recognized by lipid researchers,
The number of diseases associated with disturbances in
lipid metabolism is rapidly increasing. These include major
health burdens in the Western world, such as atherosclerosis,
Alzheimer’s disease, obesity and Type 2 diabetes. Important work
on the role of lipid imbalance as a contributing factor in these
diseases has been carried out in cell culture models and other in
vitro settings. However, extrapolation of these results to the whole
organism is often challenging. There is a need for models that
allow detailed analysis of lipid metabolism in an intact organism.
All model organisms have their advantages and disadvantages;
the fly (Drosophila melanogaster) and the worm (Caenorhabditis
elegans) have powerful genetics, but their body plans and
metabolic processes differ significantly from those of vertebrates
in many aspects. The mouse bears close resemblance to humans
at the whole-organism level, but the generation of genetically
manipulated mice is slow and expensive. In addition, whole-organ
entities are, for many purposes, difficult to study in situ, limiting
the approaches that can be undertaken.
Zebrafish (Danio rerio) has become a popular model organism
for several lines of biomedical research, including developmental
biology, genetics and neurobiology. As a vertebrate, it resembles
mammals in its development and metabolic processes, but is
more tractable. Zebrafish produce a large number of offspring at
low cost, which makes large-scale screens possible. The genetic
manipulation of zebrafish is relatively feasible, including gene
knockdown by morpholino oligonucleotides and the generation
of fish with transient or stable transgene expression. A drawback
Key words: cholesterol, imaging, lipid, lipidosis, model organism,
zebrafish.
Abbreviations used: apoA, apolipoprotein A; apoE, apolipoprotein E; BODIPY® , 4,4-difluoro-4-bora-3a ,4a -diaza-s -indacene; CARS; coherent antiStokes Raman scattering; CNS, central nervous system; dpf, days post-fertilization; FIT, fat-inducing transcript; HDL, high-density lipoprotein; LDL, lowdensity lipoprotein; LXR, liver X receptor; MTP, microsomal transfer protein; NBD, nitrobenzoxadiazole; NPC, Niemann–Pick type C; NPC1L1, NPC1-like
protein 1; PPAR, peroxisome-proliferator-activated receptor; PUFA, polyunsaturated fatty acid; S1P, sphingosine 1-phosphate; SREBP, sterol-regulatoryelement-binding protein; SCAP, SREBP-cleavage-activating protein; TAG, triacylglycerol; VLDL, very-low-density lipoprotein; YSL, yolk syncytial layer.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
c The Authors Journal compilation c 2010 Biochemical Society
Yolk, gall bladder and fat stores [10,11]
Yolk, head, heart, vasculature, liver and swim bladder
[6,12–15]
Binds to sterols with free 3 -OH group
Cholesterol analogue
Detects neutral lipids
Detects neutral lipids
Filipin
NBD (nitrobenzoxadiazole)-conjugated cholesterol
Nile Red
Oil Red O
Staining of formaldehyde-fixed fish [3]
Addition to fish water (solubilized with bile) [5];
suitable for live imaging
Addition to fish water [10,11]; suitable for live imaging
Staining of formaldehyde-fixed fish
Release of a fluorescent BODIPY® -labelled acyl chain
upon cleavage by phospholipase A2 [8]; with PED6, the
fluorescence is quenched until the cleavage occurs [5]
BODIPY® -conjugated phospholipids
BODIPY® -conjugated fatty acids
Cholesteryl fatty acid ester analogue with fluorophore
in fatty acid. Traces the cholesteryl ester until the
fatty acid is cleaved, thereafter a fatty acid tracer
Fatty acid analogues
Cholesteryl-BODIPY® -conjugated fatty acid ester
Microinjection into the yolk [3]; reconstitution to the food;
suitable for live imaging
Reconstitution to the food [4]; suitable for live imaging
Cholesterol analogue [3]
BODIPY® -conjugated cholesterol
Method of administration
Lipid specificity
Probe name
In mammals, dietary lipids are hydrolysed by lipases and
emulsified by bile acids in the gut, from where they are absorbed.
This process appears to be largely similar in fish; fish bile is
produced in the liver, stored in the gall bladder and delivered
Fluorescent lipid tracers employed in zebrafish research
ZEBRAFISH LIPID ABSORPTION
Table 1
Zebrafish are freshwater fish that belong to the cyprinid family
in the class of Actinopterygii (ray-finned fishes), along with
minnows and carps (Figure 2). They are omnivorous fish that
primarily feed on zoo- and phyto-plankton, as well as insects,
in the wild. In the aquarium, zebrafish are usually fed various
types of dry food (typically with a fat content of 8–15 %) along
with live food, most commonly Artemia (brine shrimp) [18], that
are rich in cholesterol and unsaturated fatty acids. The zebrafish
start to feed at around 5 dpf (days post-fertilization); until that
time, their sole source of energy is the maternally derived yolk.
High levels of PUFAs (polyunsaturated fatty acids) are
characteristic to fish lipids and they need to be obtained from
the diet [19]. Fish are poikilothermic, which results in the need
to modify their lipid composition upon changes in the
temperature: an increase in the level of membrane phospholipid
unsaturation is thought to facilitate the maintenance of optimal
membrane viscosity in low temperatures [19]. Indeed, for
instance in carp and crucian carp brain, it has been shown that
phosphatidylethanolamine and phosphatidylserine incorporate
more PUFAs when the temperature decreases [20,21]. The
tendency towards fatty acid unsaturation during acclimatization
to low temperature may also be reflected in the stored neutral
lipids [19].
Zebrafish, like other teleosts, harbour white fat, but not brown
fat, the latter being associated with homoeothermy. The first
visceral pre-adipocytes appear in association with the pancreas
shortly after the initiation of exogenous nutrition [11]. The
pattern of lipid storage is more diverse than in homoeotherms
[22]. In fish, lipids are mostly stored as TAGs (triacylglycerols)
[19] and the main storage sites in zebrafish include visceral,
intramuscular and subcutaneous adipocyte depots [23]. A recent
study has pinpointed FIT (fat-inducing transcript) 2 as a mediator
of TAG deposition in zebrafish; upon targeted knockdown of
FIT2, the diet-induced accumulation of neutral lipids in the liver
and intestine was inhibited [13]. This is in accordance with
the observation that shRNA (small-hairpin RNA) silencing of
FIT2 in 3T3-LI adipocytes prevented the accumulation of lipid
droplets, and suggests a conserved function for FIT2 in
lipid-droplet formation [13].
BODIPY® , 4,4-difluoro-4-bora-3a,4a-diaza-s -indacene; PED6, N -{[6-(2,4-dinitrophenyl)amino]hexanoyl}-1-palmitoyl-2-BODIPY® -FL-pentanoyl-sn -glycero-3-phosphoethanolamine.
ZEBRAFISH LIPID COMPOSITION AND STORAGE
Short chains are water-soluble and are added to fish water [5];
chains longer than ten carbons which are poorly
water-soluble [6], are induced by microinjection into the
yolk [7]; suitable
for live imaging
Addition to fish water [5]; suitable for live imaging
Localization in fish
and several fluorescent lipid analogues and probes have been
successfully introduced in zebrafish (Table 1). Moreover, nonlinear microscopy techniques, in particular CARS (coherent antiStokes Raman scattering), enables label-free visualization of
lipids [16] (Figure 1). This allows opening of new research
avenues. For example, zebrafish is already utilized as a model
for several neurodegenerative diseases: automated quantitative
behavioural analysis and imaging systems of complete brain
neurotransmitter networks have enabled elaborate studies on
both normal and pathological conditions in the CNS (central
nervous system) [17]. Yet, the precise role of lipid imbalance in
neurodegeneration is poorly understood and difficult to study in an
intact organism. With an increasing assortment of techniques for
lipid imaging in zebrafish, it should be possible to start addressing
the function of lipids in the CNS in a non-invasive manner.
PED6 fluorescence is concentrated in the gut and gall bladder
[5], and also the vasculature when combined with a
high-cholesterol diet [4]; unquenched probe also labels the
pharynx [5], as well as vasculature, even in the
normolipidaemic fish [4]
Ubiquitous, enriched, e.g. in the gut and brain [3]
Gut and gall bladder [5,9]
M. Hölttä-Vuori and others
Distributed ubiquitously in the larva; a strong signal (in the
yolk, gut and brain) [3]
When combined with a high-cholesterol diet, gives diffuse
fluorescence in the vasculature and bright deposits in
blood vessels [4]
Short chains distributed in the gut and gall bladder; long
chains distributed in the yolk, from where they are
metabolized into TAG, cholesteryl esters and phospholipids
[7]
236
Zebrafish in lipid research
Figure 1
237
Label-free imaging of lipids in the zebrafish yolk using CARS microscopy
Microscopy images of 2-day-old zebrafish embryos. (A) CARS microscopy image visualizing lipids via probing of vibrating molecular CH2 groups. The lipid content of the yolk located in the lower
part of the image (yellow colour) generates significantly higher signal intensities compared with the zebrafish body in general (part of the fish eye is visualized with blue colour tones). (B) Brightfield
image of the same field of view. Although structures and shapes can be recognized in both images, the brightfield, naturally, lacks the chemical selectivity.
to the intestine or pyloric caeca via the bile duct [19]. Upon
treatment of zebrafish larvae with atorvastatin, an inhibitor of
HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, which
is involved in cholesterol biosynthesis, the absorption of a
fluorescently quenched phospholipid was prevented, presumably
due to inhibition in the formation of cholesterol-derived bile acids
that are needed for lipid absorption [5].
Studies on zebrafish intestinal lipid absorption have identified
the ffr (fat-free) gene [5,24] and the annexin-2–caveolin-1
complex [9] as important mediators of lipid uptake from the gut.
The drug ezetimibe, which inhibits cholesterol absorption from
the gut by targeting the NPC (Niemann–Pick type C) protein
NPC1L1 (NPC1-like protein 1) [25], was found to inhibit NBD–
cholesterol uptake in zebrafish [9]. This was suggested to be due
to disruption of the annexin-2–caveolin-1 complex. Interestingly,
NPC1L1 is also conserved in the zebrafish genome (Table 2). What
the potential role of NPC1L1 in zebrafish lipid absorption is and
how this relates to the annexin-2–caveolin-1 complex deserves
further investigation.
In fish, a two-step absorption model for dietary lipids has been
proposed [22]. It consists of a ‘fast component’ which is a fattyacid-delivery system; immediately after feeding, a fraction of
dietary fatty acids are found in solution in the plasma, either as
unbound fatty acids (short chain) or bound to carrier proteins
(long chain). The ‘slow component’ represents the synthesis and
transport of TAG-rich chylomicrons in a mechanism similar to
that of mammals [22]. MTP (microsomal transfer protein), which
is responsible for packaging of TAGs into lipoprotein particles, is
conserved in zebrafish. It is expressed in the YSL (yolk syncytial
layer), intestine and liver [37]. MTP depletion resulted in impaired
dietary lipid absorption, indicating that its function in dietary lipid
metabolism is conserved in zebrafish [6].
The inter-organ transport of lipids in fish resembles that in
mammals; the absorbed dietary lipids are delivered from the
intestine to the liver and from there to extrahepatic tissues [22].
In the blood, the lipids are transported by lipoprotein carriers in a
similar manner to that in higher vertebrates, as discussed below.
Figure 2
Schematic illustration of the teleost lineage
Note that medaka (Oryzias latipes ), another popular fish model, is evolutionarily quite distant
from zebrafish.
LIPOPROTEIN METABOLISM
Overall, teleost fish are hyperlipidaemic and hypercholesterolaemic as compared with mammals. Upon feeding on a highcholesterol diet, the plasma total cholesterol in zebrafish was
c The Authors Journal compilation c 2010 Biochemical Society
Not characterized; StAR is expressed in steroidogenic tissues
suggesting conserved function [35]
OSBPL 1A (NM_001045082), OSBPL2 (NM_199872), OSBPL3a
(NM_001004646), OSBPL6 (NM_001127405), OSBPL7
(NM_001005927), OSBPL9 (NM_001077805) and OSBPL10
(EH579561)
Cholesterol-binding members StAR and MLN64 [35]
Oxysterol-binding proteins
c The Authors Journal compilation c 2010 Biochemical Society
START-domain proteins
Present in zebrafish [32]
NPC1 [33], NPC2 (BC045895) and NPC1L1 (XM_001919973)
LDL receptor
NPC proteins
StAR shuttles cholesterol to mitochondria for steroidogenesis [27];
MLN64 is a late-endosomal cholesterol-binding protein involved in
endosome dynamics [36]
Not characterized
Intestinal cholesterol absorption [9], muscle development [28], tissue
patterning [30], lateral line and neuromast development [31]
Not characterized
NPC1 is up-regulated in hypoxia [33]; other functions not characterized
Effluxes cholesterol from the cells to apoA-I [27]
Structural components of caveolae; implicated in signal transduction,
transcytosis, cholesterol transport and adipogenesis [29]
Uptake of LDL particles into cells [27]
NPC1 and NPC2 function in late endosomal cholesterol egress and
NPC1-L1 in dietary cholesterol absorption [27]
Sterol sensors or transporters that integrate sterol balance to various
metabolic processes [34]
Two isoforms (ABCA1a and ABCA1b) [26].
Caveolin-1, -2 and -3, with two isoforms (α and β) for caveolin-1 [28]
ABCA1
Caveolins
Not characterized
Function in zebrafish
Function in mammals
Orthologues in zebrafish
Protein family
Table 2
Accession numbers are from GenBank® . ABCA1, ATP-binding cassette transporter protein A1; MLN, metastatic lymph node; OSBPL, oxysterol-binding protein-like; STAR, steroidogenic acute regulatory protein; StART, StAR-related lipid transfer.
M. Hölttä-Vuori and others
Proteins involved in intracellular sterol transport conserved in zebrafish
238
reported to reach an average of approx. 20.7 mmol/l (800 mg/dl), a
level observed in cholesterol-fed LDL (low-density lipoprotein)receptor-knockout mice [4]. The degree of fatty acid unsaturation
in the fish lipoproteins is high, for instance in the trout more than
60 % of fatty acids are unsaturated [38].
The fish lipoprotein particles and their associated apoproteins
are generally comparable with those of higher vertebrates. The
predominant lipoprotein in the blood of teleost fish, including
zebrafish, is HDL (high-density lipoprotein) [4,19,38]. Fish LDL
particles contain more TAG and less cholesteryl esters than
human LDL [38]. Thus, whereas in humans the majority of
plasma cholesterol is found in the LDL fraction, in fish most
of the circulating cholesterol is in the HDL fraction [38,39]. The
lipoprotein profile is, however, modulated by diet; in zebrafish,
feeding on a high-cholesterol diet resulted in an increase in plasma
VLDL (very-low-density lipoprotein) and LDL amounts [4]. In
addition, in females of oviparous fish species (such as zebrafish),
the level of lipoproteinaemia also depends on the concentration of
circulating vitellogenin, a VHDL (very-high-density lipoprotein)
synthesized by the liver upon oestrogen stimulation [38].
The protein composition of lipoprotein particles has been
characterized most extensively in salmonids; in trout, VLDL
and LDL contain apoB (apolipoprotein B)-like proteins, whereas
in HDL the major apoprotein types are apoA (apolipoprotein
A)-I- and apoA-II-like proteins [38]. In zebrafish, the apoE
(apolipoprotein E) and apoA-I genes have been characterized
and they show 27.5 % and 25.6 % identity with human apoE
and apoA-I sequences respectively [40]. Both of these genes are
highly expressed during embryo development. Zebrafish apoE
transcripts were detected in the brain and eyes, in accordance
with the high expression of apoE in the mammalian CNS [40].
YOLK LIPIDS AND THEIR MOBILIZATION
The yolk is largely depleted during the first week of larval
development, but until then it is a significant factor in lipid
metabolism. This is worth noting, as many of the commonly
employed experimental approaches in zebrafish (morpholino
injections, visualization of transparent embryos and study of organ
development) are carried out while the yolk is still contributing to
the lipid balance.
The major lipid constituents in the yolk are neutral lipids
(TAG, wax and steryl esters) and polar phospholipids, especially
phosphatidylcholine and phosphatidylethanolamine [41]. The
high lipid content in the zebrafish yolk is manifested by the strong
signal it generates in CARS microscopy (Figure 1), similarly
as observed for lipid stores in other model systems [42]. The
ratio of different lipid classes in the yolk varies significantly
from one teleost species to another, but they all can be used
as a fuel during embryonic and larval development. The YSL
is an extra-embryonic tissue important for embryonic and larval
nutrition. It is responsible for the degradation and transfer of yolk
reserves to the embryo. In turbot, it was shown that the yolk lipids
were taken up and packaged into VLDL particles for delivery
to the developing tissues [43]. ApoE expression was detected
at sites of VLDL synthesis and secretion. Interestingly, MTP
also functions in TAG transfer at this site as MTP depletion in
zebrafish impaired yolk consumption [6]. The zebrafish apoE and
apoA-I are both highly expressed in the YSL [40]. In addition,
depletion of apoC2 (apolipoprotein C2) by morpholinos leads to
yolk malabsorption and embryonic growth retardation in zebrafish
[7], further indicating that the yolk lipids are delivered to the
developing organs by lipoprotein carriers.
Zebrafish in lipid research
In addition to being an energy reservoir, yolk lipids
function in signalling and embryonic development. In zebrafish,
Cyp11a1 (P450 cholesterol side-chain-cleavage enzyme) is
expressed in the YSL during epiboly. This enzyme catalyses
the first step in steroid synthesis, namely the conversion
of cholesterol into pregnenolone in mitochondria. The
extra-embryonically produced pregnenolone is responsible
for stabilizing microtubules, either by binding directly to
microtubules or to something that associates with them [44].
This again regulates the movement of cells during early embryo
development.
Another example of yolk lipid signalling is S1P (sphingosine
1-phosphate), a secreted lipid derived from sphingomyelin
metabolism that regulates many cellular functions, including
differentiation, growth and migration. Zebrafish studies have
unravelled a role for S1P in heart development. The S1P
transporters are expressed in the YSL and, in their absence,
myocardial precursors of the developing zebrafish embryo failed
to migrate correctly, leading to cardia bifida (two hearts)
phenotype [45,46].
LIPIDS IN THE ZEBRAFISH NERVOUS SYSTEM
The majority of the central and peripheral nervous system
lipids reside in myelin, a multilayered membrane assembly
that sheaths the axons of central and peripheral nerves. In
zebrafish, the onset of myelination takes place at around 2 dpf, as
judged by the emergence of myelin-associated protein expression,
whereas the first myelinated axons at the ultrastructural level
are apparent at 4 dpf [47]. The major lipids in zebrafish
myelin are phosphatidylethanolamine, phosphatidylcholine and
cholesterol, with small amounts of glycolipids, but no detectable
sphingomyelin [48]. Zebrafish myelin appears to be structurally
more stable than that of mammals or other teleosts, as judged by
challenging the lateral line and optic nerve myelin with various
pH and ionic strengths [48].
In the developing mammalian embryo, impairment of
cholesterol biosynthesis results in severe developmental
abnormalities in the CNS, as the brain is practically exclusively
dependent on de novo cholesterol production (for exceptions,
see [49]). Treatment of zebrafish embryos with atorvastatin
resulted in germ cell migration defects and mild morphological
abnormalities, but this was apparently not due to the lack
of cholesterol. Rather, the defects were attributed to protein
prenylation as the phenotype could be rescued by mevalonate,
but not by squalene [50].
Cholesterol is, however, crucially important for neural
development in zebrafish. If zebrafish embryos are exposed
to alcohol during gastrulation, they develop a fetal-alcoholsyndrome-like neurological defects, and other defects, with an
associated decrease in the cholesterol content of the embryo [51].
Supplementing the embryos with cholesterol prevents them from
developing the fetal-alcohol-syndrome-like defects, presumably
by rescuing the cholesterol modification of the morphogen Sonic
Hedgehog [51]. It is conceivable that, during early embryo
development, the yolk serves as a reservoir for cholesterol
in the developing fish brain. The blood–brain barrier starts
to be functional from 3 dpf onwards [52]; thus, at least up
to this point, yolk-derived circulating lipoproteins should be
able to access the CNS. Accordingly, our observations with
the fluorescent cholesterol derivative BODIPY® (4,4-difluoro-4bora-3a,4a-diaza-s-indacene)–cholesterol show that yolk-derived
sterol is readily incorporated into the developing zebrafish brain
[3].
239
ZEBRAFISH AS A MODEL FOR LIPID-RELATED DISEASES
Atherosclerosis
The development of arteriosclerotic lesions is a widespread
phenomenon in many wildlife species, including poikilothermic
animals [53]. It has been shown that, for instance, Atlantic
salmon develops coronary lesions characterized by muscle cell
proliferation, disruption of the elastica and luminal narrowing
[39]. The number of lesions was shown to increase if the fish were
fed on a cholesterol-enriched diet. Interestingly, whereas a highcholesterol diet was associated with elevated serum cholesterol
in salmons, the coronary lesions were apparently devoid of lipid
deposits [39]. This was suggested to be due, in part, to the high
HDL levels. On the other hand, in tuna, coronary artery lesions
with abundant lipid deposits have been observed [53].
Although the high HDL levels may argue that (zebra)fish are not
a very suitable system to study atherosclerosis, there are factors
that suggest otherwise. The level of lipids in the fish circulation
is high, and the large fraction of PUFAs in lipoproteins may
render them susceptible to oxidation, as has been shown for
rainbow trout [54]. These modified lipoproteins were cleared by
scavenger receptors in a similar manner to mammalian systems.
Moreover, a recent study showed that when fed a high-cholesterol
diet, zebrafish exhibited elevated plasma LDL and VLDL levels,
lipoporotein oxidation and fatty streak formation, in a similar
manner to human atherosclerosis [4]. We have observed that,
upon dietary administration, BODIPY® –cholesterol is distributed
throughout the body in 12-dpf zebrafish (Table 1 and Figure 3A).
The overall labelling intensity was increased when BODIPY® –
cholesterol was fed together with a high amount of unlabelled cholesterol (Figure 3B), probably reflecting a general
increase in the sterol content of the fish.
What makes zebrafish particularly attractive as an atherosclerosis model is that it allows temporal analysis of
atherosclerotic lesion development by imaging lipid deposition
and cellular changes in the vascular wall [4]. Indeed, the zebrafish
model allowed, for the first time, the visualization of macrophage
lipid deposition in vivo [4]. Such approaches are not readily
available in other animal models of atherosclerosis, particularly
mouse, where examination of plaques typically requires that the
animal is killed. Together, these observations suggest that
zebrafish can be used to model various aspects of atherosclerosis
development. Whether the vascular lesions observed in zebrafish
progress to advanced rupture-prone atheromas warrants further
investigation.
Obesity and diabetes
In terms of energy consumption and fasting, fish are able to
survive for much longer periods without food than homoeothermic
animals, and for many species, a period of fasting in the cold
season is part of their life cycle [19]. The metabolic responses
to food deprivation, although apparently similar to those of
mammals, are attenuated in fish [55]. The regulation of zebrafish
energy homoeostasis resembles that of mammals, including a
central melanocortin system that responds to leptin. Zebrafish
that overexpress the endogenous melanocortin antagonist Agoutirelated protein are obese and exhibit increased linear growth
and adipocyte hypertrophy [23]. Moreover, adiponectin and
adiponectin receptor genes have been found to be expressed in
the developing zebrafish embryo and their levels are modulated
by food deprivation similarly as in higher vertebrates [56].
PPARs (peroxisome-proliferator-activated receptors) belong to
a nuclear hormone receptor superfamily that plays a critical role
c The Authors Journal compilation c 2010 Biochemical Society
240
Figure 3
M. Hölttä-Vuori and others
Fluorescently labelled sterol added to the diet accumulates in zebrafish tissues
Zebrafish (12 dpf, ten per group) were fed with 10 μg BODIPY® -cholesterol (BPYchol)/g of fish flakes (Special Diet Services SDS-100, lipid content 14 %) for 7 days. (A) BODIPY® -cholesterol
supplied along with a normal diet (SDS-100). (B) BODIPY® -cholesterol supplied along with a cholesterol-enriched diet {4 % (w/w) cholesterol in SDS-100; prepared as described in [4]}. After
feeding, the fish were killed by immersion in ice-cold water and imaged immediately. The Figures show representative fish for both groups, imaged with equal exposure times. The asterisk indicates
the swim bladder, arrows indicate somites and arrowheads indicate the gut.
as a primary lipid sensor and regulator of lipid metabolism. There
are three PPAR isoforms (α, β and γ ), all of which are conserved
in zebrafish [57]. The distribution of the different isoforms
appears to be similar to that of mammals, with PPARγ serving
as a marker for adipocytes [11]. Furthermore, pharmagolocical
compounds known to modulate the fat content in mammals [PPAR
activators, β-adrenergic agonists, SIRT (sirtuin)-1 activators and
nicotinic acid treatment] were shown to be effective in zebrafish,
highlighting the evolutionary conservation of the key regulators
of lipid metabolism [10].
The zebrafish pancreas has a similar basic structure and cellular
composition to the mammalian pancreas. Therefore it is perhaps
not surprising that zebrafish is emerging as a diabetes research
model [58]. Zebrafish become hyperglycaemic if exposed to
high glucose, and prolonged high blood sugar levels were
associated with retinopathy, reminiscent of that observed in
diabetes patients [59]. In addition, zebrafish blood glucose levels
were shown to decrease upon administration of the anti-diabetic
drug glipizide [60]. Together, these features make zebrafish an
attractive platform to study the links between lipid imbalance and
diabetes.
Cholesterol metabolism and hepatic steatosis
The key transcriptional regulators of cholesterol metabolism are
the SREBP (sterol-regulatory-element-binding protein) and LXR
(liver X receptor) systems. The former acts to increase, and
the latter to decrease, cellular cholesterol levels. The zebrafish
genome encodes only one LXR that by sequence similarity
more closely resembles the mammalian LXRα, but by expression
pattern the LXRβ [61]. The zebrafish LXR responds to known
LXR agonists and is likely to be involved in the regulation of
cholesterol and lipid metabolism, much like in mammals.
In the zebrafish gonzo mutant, the Site-1 protease is
disrupted. This protease activates SREBPs, and its inactivation
in zebrafish gonzo-mutant larvae leads to lipid deficiency
[12]. Essentially the same lipid phenotype was observed when
SCAP (SREBP-cleavage-activating protein) was depleted by
c The Authors Journal compilation c 2010 Biochemical Society
morpholinos, suggesting that the regulation of lipid synthesis
via the SCAP/SREBP pathway is conserved in zebrafish [12].
Interestingly, in addition to the lipid phenotype, gonzo-mutant
fish exhibited defective cartilage development resembling human
chondrodysplasias [12]. This feature was not observed upon
SCAP knockdown, implying that the lipid and cartilage defects
are caused by different mechanisms.
Although exposure of early zebrafish embryos to alcohol leads
to developmental malformations through cholesterol imbalance
[51], later-stage embryos respond to alcohol by developing hepatic
steatosis in a similar manner to that observed in humans [15]. The
development of steatosis required the induction of lipogenesis
through SREBP activation, as it was prevented in gonzo mutants
[15]. In addition to acute alcoholic liver disease, zebrafish can
serve as a model to study the genetics and physiology of nonalcoholic fatty liver. In a genetic screen for mutations causing
hepatomegaly, a novel gene named foie gras was identified
[62]. Foie gras mutants develop large lipid-filled hepatocytes and
steatosis resembling human fatty liver disease. Foie gras is highly
conserved in metazoans, but its function is presently unknown
[62]. It will be of interest to investigate whether this protein plays
a role in the development of mammalian fatty liver disease.
In addition to regulatory proteins, many proteins that bind
cholesterol and participate in its intracellular trafficking and
distribution have orthologues in zebrafish (Table 2). In mammals,
several of these genes have been identified, as their mutations
lead to severe cholesterol transport defects, highlighting the
importance of the proteins in the maintenance of whole-body
cholesterol balance. The loss of function phenotype for most of
these genes has not yet been established in zebrafish, but remains
an attractive research avenue.
CONCLUSIONS
Zebrafish is becoming an increasingly popular vertebrate model
system in several lines of biomedical research because of its
amenability to genetic manipulation and imaging. In addition,
zebrafish produce high numbers of offspring at low cost, which
Zebrafish in lipid research
allows the design of high-throughput analyses in an in vivo system.
In lipid research, zebrafish is still under-utilized relative to its
potential. However, during the last few years, reports on the
use of zebrafish to investigate lipid absorption, atherosclerosis,
obesity and hepatic steatosis have been published. These studies
have revealed remarkable similarities in the lipid metabolism
between zebrafish and mammals. In addition, the techniques for
lipid visualization in zebrafish have already proven to be highly
useful and even applicable to large-scale screens. Together, these
characteristics make zebrafish a powerful tool for lipid research
that allows the dissection of systems that have been difficult to
study in vivo so far, such as the CNS.
FUNDING
The work of our laboratories is supported by the Academy of Finland [grant numbers
123861 (to M.H.-V.), 123743 (to E.I.), 116177 (to P.P.)]; the Swedish Scientific Research
Council (to L.N., C.B. and A.E.); the Biocentrum Helsinki (to E.I.); the Finnish Cultural
Foundation (to M.H.-V.); and by the Sigrid Juselius Foundation (to P.P. and E.I.).
REFERENCES
1 Foley, J. E., Maeder, M. L., Pearlberg, J., Joung, J. K., Peterson, R. T. and Yeh, J. R.
(2009) Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Nat.
Protoc. 4, 1855–1867
2 Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L. and Postlethwait, J. (1999)
Preservation of duplicate genes by complementary, degenerative mutations. Genetics
151, 1531–1545
3 Holtta-Vuori, M., Uronen, R. L., Repakova, J., Salonen, E., Vattulainen, I., Panula, P., Li,
Z., Bittman, R. and Ikonen, E. (2008) BODIPY–cholesterol: a new tool to visualize sterol
trafficking in living cells and organisms. Traffic 9, 1839–1849
4 Stoletov, K., Fang, L., Choi, S.-H., Hartvigsen, K., Hansen, L. F., Hall, C., Pattison, J.,
Juliano, J., Miller, E. R., Almazan, F. et al. (2009) Vascular lipid accumulation, lipoprotein
oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish. Circ. Res.
104, 952–960
5 Farber, S. A., Pack, M., Ho, S. Y., Johnson, I. D., Wagner, D. S., Dosch, R., Mullins, M. C.,
Hendrickson, H. S., Hendrickson, E. K. and Halpern, M. E. (2001) Genetic analysis of
digestive physiology using fluorescent phospholipid reporters. Science 292, 1385–1388
6 Schlegel, A. and Stainier, D. Y. (2006) Microsomal triglyceride transfer protein is required
for yolk lipid utilization and absorption of dietary lipids in zebrafish larvae. Biochemistry
45, 15179–15187
7 Pickart, M. A., Klee, E. W., Nielsen, A. L., Sivasubbu, S., Mendenhall, E. M., Bill, B. R.,
Chen, E., Eckfeldt, C. E., Knowlton, M., Robu, M. E. et al. (2006) Genome-wide reverse
genetics framework to identify novel functions of the vertebrate secretome. PLoS ONE 1,
e104
8 Farber, S. A., Olson, E. S., Clark, J. D. and Halpern, M. E. (1999) Characterization of
Ca2+ -dependent phospholipase A2 activity during zebrafish embryogenesis. J. Biol.
Chem. 274, 19338–19346
9 Smart, E. J., De Rose, R. A. and Farber, S. A. (2004) Annexin 2–caveolin 1 complex is a
target of ezetimibe and regulates intestinal cholesterol transport. Proc. Natl. Acad. Sci.
U.S.A. 101, 3450–3455
10 Jones, K., Alimov, A., Rilo, H., Jandacek, R., Woollett, L. and Penberthy, W. T. (2008) A
high throughput live transparent animal bioassay to identify non-toxic small molecules or
genes that regulate vertebrate fat metabolism for obesity drug development. Nutr. Metab.
(London) 5, 23
11 Flynn, 3rd, E. J., Trent, C. M. and Rawls, J. F. (2009) Ontogeny and nutritional control of
adipogenesis in zebrafish (Danio rerio ). J. Lipid Res. 50, 1641–1652
12 Schlombs, K., Wagner, T. and Scheel, J. (2003) Site-1 protease is required for cartilage
development in zebrafish. Proc. Natl. Acad. Sci. U.S.A. 100, 14024–14029
13 Kadereit, B., Kumar, P., Wang, W. J., Miranda, D., Snapp, E. L., Severina, N., Torregroza,
I., Evans, T. and Silver, D. L. (2008) Evolutionarily conserved gene family important for fat
storage. Proc. Natl. Acad. Sci. U.S.A. 105, 94–99
14 Raldua, D., Andre, M. and Babin, P. J. (2008) Clofibrate and gemfibrozil induce an
embryonic malabsorption syndrome in zebrafish. Toxicol. Appl. Pharmacol. 228,
301–314
15 Passeri, M. J., Cinaroglu, A., Gao, C. and Sadler, K. C. (2009) Hepatic steatosis in
response to acute alcohol exposure in zebrafish requires sterol regulatory element
binding protein activation. Hepatology 49, 443–452
241
16 Enejder, A., Brackmann, C. and Svedberg, F. (2010) Coherent anti-Stokes Raman
scattering microscopy of cellular lipid storage. IEEE J. Sel. Topics Quantum Electron.,
doi:10.1109/JSTQE.2009.2032512
17 Panula, P., Sallinen, V., Sundvik, M., Kolehmainen, J., Torkko, V., Tiittula, A.,
Moshnyakov, M. and Podlasz, P. (2006) Modulatory neurotransmitter systems and
behavior: towards zebrafish models of neurodegenerative diseases. Zebrafish 3, 235–247
18 Brand, M., Granato, M. and Nüsslein-Volhard, C. (2002) Keeping and raising zebrafish. In
Zebrafish, A Practical Approach. (Nüsslein-Volhard, C. and Dahm, R., eds), pp. 7–37,
Oxford University Press, Oxford
19 Henderson, R. J. and Tocher, D. R. (1987) The lipid composition and biochemistry of
freshwater fish. Prog. Lipid Res. 26, 281–347
20 Buda, C., Dey, I., Balogh, N., Horvath, L. I., Maderspach, K., Juhasz, M., Yeo, Y. K. and
Farkas, T. (1994) Structural order of membranes and composition of phospholipids in fish
brain cells during thermal acclimatization. Proc. Natl. Acad. Sci. U.S.A. 91, 8234–8238
21 Kakela, R., Mattila, M., Hermansson, M., Haimi, P., Uphoff, A., Paajanen, V., Somerharju,
P. and Vornanen, M. (2008) Seasonal acclimatization of brain lipidome in a eurythermal
fish (Carassius carassius ) is mainly determined by temperature. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 294, R1716–R1728
22 Sheridan, M. A. (1988) Lipid dynamics in fish: aspects of absorption, transportation,
deposition and mobilization. Comp. Biochem. Physiol. B 90, 679–690
23 Song, Y. and Cone, R. D. (2007) Creation of a genetic model of obesity in a teleost. FASEB
J. 21, 2042–2049
24 Ho, S. Y., Lorent, K., Pack, M. and Farber, S. A. (2006) Zebrafish fat-free is required for
intestinal lipid absorption and Golgi apparatus structure. Cell Metab. 3, 289–300
25 Garcia-Calvo, M., Lisnock, J., Bull, H. G., Hawes, B. E., Burnett, D. A., Braun, M. P., Crona,
J. H., Davis, Jr, H. R., Dean, D. C., Detmers, P. A. et al. (2005) The target of ezetimibe is
Niemann-Pick C1-Like 1 (NPC1L1). Proc. Natl. Acad. Sci. U.S.A. 102, 8132–8137
26 Annilo, T., Chen, Z. Q., Shulenin, S., Costantino, J., Thomas, L., Lou, H., Stefanov, S. and
Dean, M. (2006) Evolution of the vertebrate ABC gene family: analysis of gene birth and
death. Genomics 88, 1–11
27 Ikonen, E. (2008) Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol.
Cell Biol. 9, 125–138
28 Nixon, S. J., Wegner, J., Ferguson, C., Mery, P. F., Hancock, J. F., Currie, P. D., Key, B.,
Westerfield, M. and Parton, R. G. (2005) Zebrafish as a model for caveolin-associated
muscle disease: caveolin-3 is required for myofibril organization and muscle cell
patterning. Hum. Mol. Genet. 14, 1727–1743
29 Cohen, A. W., Hnasko, R., Schubert, W. and Lisanti, M. P. (2004) Role of caveolae and
caveolins in health and disease. Physiol. Rev. 84, 1341–1379
30 Fang, P. K., Solomon, K. R., Zhuang, L., Qi, M., McKee, M., Freeman, M. R. and Yelick,
P. C. (2006) Caveolin-1α and -1β perform nonredundant roles in early vertebrate
development. Am. J. Pathol. 169, 2209–2222
31 Nixon, S. J., Carter, A., Wegner, J., Ferguson, C., Floetenmeyer, M., Riches, J., Key, B.,
Westerfield, M. and Parton, R. G. (2007) Caveolin-1 is required for lateral line neuromast
and notochord development. J. Cell Sci. 120, 2151–2161
32 Gregg, R. G., Willer, G. B., Fadool, J. M., Dowling, J. E. and Link, B. A. (2003) Positional
cloning of the young mutation identifies an essential role for the Brahma chromatin
remodeling complex in mediating retinal cell differentiation. Proc. Natl. Acad. Sci. U.S.A.
100, 6535–6540
33 van der Meer, D. L., van den Thillart, G. E., Witte, F., de Bakker, M. A., Besser, J.,
Richardson, M. K., Spaink, H. P., Leito, J. T. and Bagowski, C. P. (2005) Gene expression
profiling of the long-term adaptive response to hypoxia in the gills of adult zebrafish. Am.
J. Physiol. Regul. Integr. Comp. Physiol. 289, R1512–R1519
34 Yan, D. and Olkkonen, V. M. (2008) Characteristics of oxysterol binding proteins. Int. Rev.
Cytol. 265, 253–285
35 Bauer, M. P., Bridgham, J. T., Langenau, D. M., Johnson, A. L. and Goetz, F. W. (2000)
Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in
vertebrates. Mol. Cell. Endocrinol. 168, 119–125
36 Holtta-Vuori, M., Alpy, F., Tanhuanpaa, K., Jokitalo, E., Mutka, A. L. and Ikonen, E. (2005)
MLN64 is involved in actin-mediated dynamics of late endocytic organelles. Mol. Biol.
Cell 16, 3873–3886
37 Marza, E., Barthe, C., Andre, M., Villeneuve, L., Helou, C. and Babin, P. J. (2005)
Developmental expression and nutritional regulation of a zebrafish gene homologous to
mammalian microsomal triglyceride transfer protein large subunit. Dev. Dyn. 232,
506–518
38 Babin, P. J. and Vernier, J. M. (1989) Plasma lipoproteins in fish. J. Lipid. Res. 30,
467–489
39 Farrell, A. P., Saunders, R. L., Freeman, H. C. and Mommsen, T. P. (1986) Arteriosclerosis
in Atlantic salmon: effects of dietary cholesterol and maturation. Arteriosclerosis 6,
453–461
40 Babin, P. J., Thisse, C., Durliat, M., Andre, M., Akimenko, M. A. and Thisse, B. (1997)
Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are
highly expressed during embryonic development. Proc. Natl. Acad. Sci. U.S.A. 94,
8622–8627
c The Authors Journal compilation c 2010 Biochemical Society
242
M. Hölttä-Vuori and others
41 Wiegand, M. D. (1996) Composition, accumulation and utilization of yolk lipids in teleost
fish. Rev. Fish Biol. Fish. 6, 259–286
42 Hellerer, T., Axang, C., Brackmann, C., Hillertz, P., Pilon, M. and Enejder, A. (2007)
Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes
Raman scattering (CARS) microscopy. Proc. Natl. Acad. Sci. U.S.A. 104, 14658–14663
43 Poupard, G., Andre, M., Durliat, M., Ballagny, C., Boeuf, G. and Babin, P. J. (2000)
Apolipoprotein E gene expression correlates with endogenous lipid nutrition and yolk
syncytial layer lipoprotein synthesis during fish development. Cell Tissue Res. 300,
251–261
44 Hsu, H. J., Liang, M. R., Chen, C. T. and Chung, B. C. (2006) Pregnenolone stabilizes
microtubules and promotes zebrafish embryonic cell movement. Nature 439,
480–483
45 Osborne, N., Brand-Arzamendi, K., Ober, E. A., Jin, S. W., Verkade, H., Holtzman, N. G.,
Yelon, D. and Stainier, D. Y. (2008) The spinster homolog, two of hearts, is required for
sphingosine 1-phosphate signaling in zebrafish. Curr. Biol. 18, 1882–1888
46 Kawahara, A., Nishi, T., Hisano, Y., Fukui, H., Yamaguchi, A. and Mochizuki, N. (2009)
The sphingolipid transporter spns2 functions in migration of zebrafish myocardial
precursors. Science 323, 524–527
47 Brosamle, C. and Halpern, M. E. (2002) Characterization of myelination in the developing
zebrafish. Glia 39, 47–57
48 Avila, R. L., Tevlin, B. R., Lees, J. P., Inouye, H. and Kirschner, D. A. (2007) Myelin
structure and composition in zebrafish. Neurochem. Res. 32, 197–209
49 Mutka, A. L. and Ikonen, E. (2010) Genetics and molecular biology: brain cholesterol
balance – not such a closed circuit after all. Curr. Opin. Lipidol. 21, 93–94
50 Thorpe, J. L., Doitsidou, M., Ho, S. Y., Raz, E. and Farber, S. A. (2004) Germ cell
migration in zebrafish is dependent on HMGCoA reductase activity and prenylation. Dev.
Cell 6, 295–302
51 Li, Y. X., Yang, H. T., Zdanowicz, M., Sicklick, J. K., Qi, Y., Camp, T. J. and Diehl, A. M.
(2007) Fetal alcohol exposure impairs Hedgehog cholesterol modification and signaling.
Lab. Invest. 87, 231–240
Received 23 February 2010/14 April 2010; accepted 6 May 2010
Published on the Internet 28 June 2010, doi:10.1042/BJ20100293
c The Authors Journal compilation c 2010 Biochemical Society
52 Jeong, J. Y., Kwon, H. B., Ahn, J. C., Kang, D., Kwon, S. H., Park, J. A. and Kim, K. W.
(2008) Functional and developmental analysis of the blood–brain barrier in zebrafish.
Brain Res. Bull. 75, 619–628
53 Vastesaeger, M. M. and Delcourt, R. (1962) The natural history of atherosclerosis.
Circulation 26, 841–855
54 Froystad, M. K., Volden, V., Berg, T. and Gjoen, T. (2002) Metabolism of oxidized and
chemically modified low density lipoproteins in rainbow trout: clearance via scavenger
receptors. Dev. Comp. Immunol. 26, 723–733
55 Wang, T., Hung, C. C. and Randall, D. J. (2006) The comparative physiology of food
deprivation: from feast to famine. Annu. Rev. Physiol. 68, 223–251
56 Nishio, S., Gibert, Y., Bernard, L., Brunet, F., Triqueneaux, G. and Laudet, V. (2008)
Adiponectin and adiponectin receptor genes are coexpressed during zebrafish
embryogenesis and regulated by food deprivation. Dev. Dyn. 237, 1682–1690
57 Ibabe, A., Grabenbauer, M., Baumgart, E., Fahimi, H. D. and Cajaraville, M. P. (2002)
Expression of peroxisome proliferator-activated receptors in zebrafish (Danio rerio ).
Histochem. Cell Biol. 118, 231–239
58 Kinkel, M. D. and Prince, V. E. (2009) On the diabetic menu: zebrafish as a model for
pancreas development and function. BioEssays 31, 139–152
59 Gleeson, M., Connaughton, V. and Arneson, L. S. (2007) Induction of hyperglycaemia in
zebrafish (Danio rerio ) leads to morphological changes in the retina. Acta Diabetol. 44,
157–163
60 Elo, B., Villano, C. M., Govorko, D. and White, L. A. (2007) Larval zebrafish as a model for
glucose metabolism: expression of phosphoenolpyruvate carboxykinase as a marker for
exposure to anti-diabetic compounds. J. Mol. Endocrinol. 38, 433–440
61 Archer, A., Lauter, G., Hauptmann, G., Mode, A. and Gustafsson, J. A. (2008)
Transcriptional activity and developmental expression of liver X receptor (LXR) in
zebrafish. Dev. Dyn. 237, 1090–1098
62 Sadler, K. C., Amsterdam, A., Soroka, C., Boyer, J. and Hopkins, N. (2005) A genetic
screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver
disease. Development 132, 3561–3572