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

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 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 genome-wide 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, and several fluorescent lipid analogues and probes have been successfully introduced in zebrafish (Table 1). Moreover, non-linear microscopy techniques, in particular CARS (coherent anti-Stokes 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.

Table 1
Fluorescent lipid tracers employed in zebrafish research

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.

Probe name Lipid specificity Method of administration Localization in fish 
BODIPY®-conjugated cholesterol Cholesterol analogue [3Microinjection into the yolk [3]; reconstitution to the food; suitable for live imaging Distributed ubiquitously in the larva; a strong signal (in the yolk, gut and brain) [3
Cholesteryl-BODIPY®-conjugated fatty acid ester 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 Reconstitution to the food [4]; suitable for live imaging When combined with a high-cholesterol diet, gives diffuse fluorescence in the vasculature and bright deposits in blood vessels [4
BODIPY®-conjugated fatty acids Fatty acid analogues 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 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
BODIPY®-conjugated phospholipids Release of a fluorescent BODIPY®-labelled acyl chain upon cleavage by phospholipase A2 [8]; with PED6, the fluorescence is quenched until the cleavage occurs [5Addition to fish water [5]; suitable for live imaging 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
Filipin Binds to sterols with free 3′-OH group Staining of formaldehyde-fixed fish [3Ubiquitous, enriched, e.g. in the gut and brain [3
NBD (nitrobenzoxadiazole)-conjugated cholesterol Cholesterol analogue Addition to fish water (solubilized with bile) [5]; suitable for live imaging Gut and gall bladder [5,9
Nile Red Detects neutral lipids Addition to fish water [10,11]; suitable for live imaging Yolk, gall bladder and fat stores [10,11
Oil Red O Detects neutral lipids Staining of formaldehyde-fixed fish Yolk, head, heart, vasculature, liver and swim bladder [6,1215
Probe name Lipid specificity Method of administration Localization in fish 
BODIPY®-conjugated cholesterol Cholesterol analogue [3Microinjection into the yolk [3]; reconstitution to the food; suitable for live imaging Distributed ubiquitously in the larva; a strong signal (in the yolk, gut and brain) [3
Cholesteryl-BODIPY®-conjugated fatty acid ester 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 Reconstitution to the food [4]; suitable for live imaging When combined with a high-cholesterol diet, gives diffuse fluorescence in the vasculature and bright deposits in blood vessels [4
BODIPY®-conjugated fatty acids Fatty acid analogues 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 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
BODIPY®-conjugated phospholipids Release of a fluorescent BODIPY®-labelled acyl chain upon cleavage by phospholipase A2 [8]; with PED6, the fluorescence is quenched until the cleavage occurs [5Addition to fish water [5]; suitable for live imaging 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
Filipin Binds to sterols with free 3′-OH group Staining of formaldehyde-fixed fish [3Ubiquitous, enriched, e.g. in the gut and brain [3
NBD (nitrobenzoxadiazole)-conjugated cholesterol Cholesterol analogue Addition to fish water (solubilized with bile) [5]; suitable for live imaging Gut and gall bladder [5,9
Nile Red Detects neutral lipids Addition to fish water [10,11]; suitable for live imaging Yolk, gall bladder and fat stores [10,11
Oil Red O Detects neutral lipids Staining of formaldehyde-fixed fish Yolk, head, heart, vasculature, liver and swim bladder [6,1215

Label-free imaging of lipids in the zebrafish yolk using CARS microscopy

Figure 1
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.

Figure 1
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.

ZEBRAFISH LIPID COMPOSITION AND STORAGE

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.

Schematic illustration of the teleost lineage

Figure 2
Schematic illustration of the teleost lineage

Note that medaka (Oryzias latipes), another popular fish model, is evolutionarily quite distant from zebrafish.

Figure 2
Schematic illustration of the teleost lineage

Note that medaka (Oryzias latipes), another popular fish model, is evolutionarily quite distant from zebrafish.

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].

ZEBRAFISH LIPID ABSORPTION

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 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.

Table 2
Proteins involved in intracellular sterol transport conserved in zebrafish

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.

Protein family Orthologues in zebrafish Function in mammals Function in zebrafish 
ABCA1 Two isoforms (ABCA1a and ABCA1b) [26]. Effluxes cholesterol from the cells to apoA-I [27Not characterized 
Caveolins Caveolin-1, -2 and -3, with two isoforms (α and β) for caveolin-1 [28Structural components of caveolae; implicated in signal transduction, transcytosis, cholesterol transport and adipogenesis [29Intestinal cholesterol absorption [9], muscle development [28], tissue patterning [30], lateral line and neuromast development [31
LDL receptor Present in zebrafish [32Uptake of LDL particles into cells [27Not characterized 
NPC proteins NPC1 [33], NPC2 (BC045895) and NPC1L1 (XM_001919973) NPC1 and NPC2 function in late endosomal cholesterol egress and NPC1-L1 in dietary cholesterol absorption [27NPC1 is up-regulated in hypoxia [33]; other functions not characterized 
Oxysterol-binding proteins OSBPL 1A (NM_001045082), OSBPL2 (NM_199872), OSBPL3a (NM_001004646), OSBPL6 (NM_001127405), OSBPL7 (NM_001005927), OSBPL9 (NM_001077805) and OSBPL10 (EH579561) Sterol sensors or transporters that integrate sterol balance to various metabolic processes [34Not characterized 
START-domain proteins Cholesterol-binding members StAR and MLN64 [35StAR shuttles cholesterol to mitochondria for steroidogenesis [27]; MLN64 is a late-endosomal cholesterol-binding protein involved in endosome dynamics [36Not characterized; StAR is expressed in steroidogenic tissues suggesting conserved function [35
Protein family Orthologues in zebrafish Function in mammals Function in zebrafish 
ABCA1 Two isoforms (ABCA1a and ABCA1b) [26]. Effluxes cholesterol from the cells to apoA-I [27Not characterized 
Caveolins Caveolin-1, -2 and -3, with two isoforms (α and β) for caveolin-1 [28Structural components of caveolae; implicated in signal transduction, transcytosis, cholesterol transport and adipogenesis [29Intestinal cholesterol absorption [9], muscle development [28], tissue patterning [30], lateral line and neuromast development [31
LDL receptor Present in zebrafish [32Uptake of LDL particles into cells [27Not characterized 
NPC proteins NPC1 [33], NPC2 (BC045895) and NPC1L1 (XM_001919973) NPC1 and NPC2 function in late endosomal cholesterol egress and NPC1-L1 in dietary cholesterol absorption [27NPC1 is up-regulated in hypoxia [33]; other functions not characterized 
Oxysterol-binding proteins OSBPL 1A (NM_001045082), OSBPL2 (NM_199872), OSBPL3a (NM_001004646), OSBPL6 (NM_001127405), OSBPL7 (NM_001005927), OSBPL9 (NM_001077805) and OSBPL10 (EH579561) Sterol sensors or transporters that integrate sterol balance to various metabolic processes [34Not characterized 
START-domain proteins Cholesterol-binding members StAR and MLN64 [35StAR shuttles cholesterol to mitochondria for steroidogenesis [27]; MLN64 is a late-endosomal cholesterol-binding protein involved in endosome dynamics [36Not characterized; StAR is expressed in steroidogenic tissues suggesting conserved function [35

In fish, a two-step absorption model for dietary lipids has been proposed [22]. It consists of a ‘fast component’ which is a fatty-acid-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.

LIPOPROTEIN METABOLISM

Overall, teleost fish are hyperlipidaemic and hypercholesterolaemic as compared with mammals. Upon feeding on a high-cholesterol diet, the plasma total cholesterol in zebrafish was 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.

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-alcohol-syndrome-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-4-bora-3a,4a-diaza-s-indacene)–cholesterol show that yolk-derived sterol is readily incorporated into the developing zebrafish brain [3].

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 high-cholesterol 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.

Fluorescently labelled sterol added to the diet accumulates in zebrafish tissues

Figure 3
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.

Figure 3
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.

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 Agouti-related 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 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 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 non-alcoholic 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 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.

Abbreviations

     
  • apoA

    apolipoprotein A

  •  
  • apoE

    apolipoprotein E

  •  
  • BODIPY®

    4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

  •  
  • CARS

    coherent anti-Stokes Raman scattering

  •  
  • CNS

    central nervous system

  •  
  • dpf

    days post-fertilization

  •  
  • FIT

    fat-inducing transcript

  •  
  • HDL

    high-density lipoprotein

  •  
  • LDL

    low-density 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-regulatory element-binding protein

  •  
  • SCAP

    SREBP-cleavage-activating protein

  •  
  • TAG

    triacylglycerol

  •  
  • VLDL

    very-low-density lipoprotein

  •  
  • YSL

    yolk syncytial layer

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.).

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