review article zebrafish as a model to study the role of...

Review ArticleZebrafish as a Model to Study the Role of PeroxisomeProliferating-Activated Receptors in Adipogenesis and Obesity

Marjo J Den Broeder1 Victoria A Kopylova1 Leonie M Kamminga23 and Juliette Legler1

1 Institute for Environmental Studies Faculty of Earth and Life Sciences VU University 1081 HV Amsterdam Netherlands2Department of Molecular Biology Faculty of Sciences Radboud Institute for Molecular Life Science Radboud UniversityNijmegen Netherlands3Radboud University Nijmegen Medical Center Nijmegen Netherlands

Correspondence should be addressed to Juliette Legler julietteleglervunl

Received 17 August 2015 Revised 29 October 2015 Accepted 5 November 2015

Academic Editor Stephane Mandard

Copyright copy 2015 Marjo J Den Broeder et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The Peroxisome Proliferator-Activated Receptors (PPARs) PPARA and PPARD are regulators of lipid metabolism with importantroles in energy release through lipid breakdown while PPARG plays a key role in lipid storage and adipogenesis The aim of thisreview is to describe the role of PPARs in lipid metabolism adipogenesis and obesity and evaluate the zebrafish as an emergingvertebratemodel to study the function of PPARs Zebrafish are an appropriatemodel to study human diseases including obesity andrelated metabolic diseases as pathways important for adipogenesis and lipid metabolism which are conserved between mammalsand fish This review synthesizes knowledge on the role of PPARs in zebrafish and focuses on the putative function of PPARs inzebrafish adipogenesis Using in silico analysis we confirm the presence of five PPARs (pparaa pparab pparda ppardb and pparg)in the zebrafish genomewith 67ndash74 identity to human andmouse PPARsDuring development ppardab paralogs and pparg showmRNA expression around the swim bladder and pancreas the region where adipocytes first develop whereas pparg is detectable inadipocytes at 15 days post fertilization (dpf)This review indicates that the zebrafish is a promising model to investigate the specificfunctions of PPARs in adipogenesis and obesity

1 Introduction

In the last 30 years obesity has become a worldwide epi-demic and according to the World Health Organization(WHO) over 700 million people can be characterized asobese [1] Obesity is a risk factor for developing type IIdiabetes mellitus cardiovascular diseases and hypertensionas well as cancer Over the last decade many studies havedescribed the important biological functions of PeroxisomeProliferator-Activated Receptors (PPARs) and their role inobesity Genetic variation in PPARs results in altered fatdeposition and body weight as studies on the develop-ment of obesity T2DM dyslipidemia and cardiovasculardiseases have identified single nucleotide polymorphisms(SNPs) in PPAR genes [2] PPARs are important factors foradipocyte differentiation and energy homeostasis and arehighly expressed in tissue with active lipid metabolism In

addition PPARs are involved in embryonic development celldifferentiation and inflammation [3ndash5]

The aimof this review is to describe the function of PPARsin lipid metabolism and adipogenesis in particular their rolein obesity and related disorders As numerous animal modelshave been used to study the origin of obesity and to gainbetter knowledge of PPAR-related molecular mechanismswe evaluate the zebrafish as an emerging model to studyadipogenesis We hypothesize that PPARs in zebrafish havesimilar functions as in mammals

(1) Peroxisome Proliferator-Activated Receptors PPARs arenuclear hormone receptors which belong to the NR1C sub-family of steroid receptor superfamily The classificationsof PPARs in the nuclear receptor family are describedin a nomenclature system according to Nuclear Recep-tors Nomenclature Committee [6] Three PPARs have been

Hindawi Publishing CorporationPPAR ResearchVolume 2015 Article ID 358029 11 pageshttpdxdoiorg1011552015358029

2 PPAR Research

identified in vertebrates namely PPARA (hPPAR PPAR120572and NR1C1) PPARD (NR1C2 PPAR120573120575 FAAR NUCI andNUCII) and PPARG (NR1C3 PPAR120574) which are encodedby different genes [4] The zebrafish orthologs of PPARAPPARD and PPARG are referred to as Pparaa PparabPparda Ppardb and Pparg according to the ZFIN nomen-clature (httpzfinorg)

Like other nuclear receptors PPARs have a proteinstructure that generally consists of four parts namely an N-terminal domain (NTD) a DNA-binding domain (DBD) aligand binding domain (LBD) and a connective structure(hinge) [7 8] The NTD contains a ligand-independentactivation factor-1 (AF-1) The DBD consists of two zincfingers that specifically bind to the peroxisome proliferatorresponse element (PPRE) in the promotor regions of PPARtarget genes [9] The C-terminal part of the protein theLBD consists of 13 120572-helices and 4 120573-sheets which formthe ligand binding pocket The LBD contains the liganddependent activation factor-2 (AF-2) and ligand binding toAF-2 results in activation of the LBD [3 10] Expressionof PPARs is predominantly in the nucleus though PPARAand PPARG are also found in lower concentrations in thecytoplasm as well [11 12] PPARs can be shuttled between thecytoplasm and the nucleus by export receptors that recognizetwo different nuclear localization signals (NLSs) on the PPARprotein [12]

PPARs regulate transcriptional gene activation throughheterodimerization with retinoid X receptors (RXR) [13 14]All PPARs can form a complex with RXR and heterodimer-ization between PPAR and RXR is ligand independent [1516] In the absence of ligands PPARRXR heterodimersbound to PPREs will act as a transcriptional repressor dueto binding of corepressor proteins such as nuclear receptorcorepressor 1 (NCoR) and silencing mediator of retinoicacid and thyroid hormone receptor (SMRT) Binding ofa specific PPAR agonist to LBD leads to the release ofcorepressor complex and recruitment of the coactivationfactors Coactivators of PPARs include the steroid receptorcoactivator-1 (SRC-1) CREB-binding protein (CBP) PPAR-binding protein (PBP) P300 cyclin G2 PPAR-interactingprotein (PRIP) and PPAR120574 coactivator-1 (PGC-1) [17ndash19]As a consequence of a conformational change of the LBDthe PPARRXR heterodimer binds to the PPRE presentin promoter regions in order to regulate transcription oftarget genes by facilitating RNA polymerase II functionThe PPRE consists of direct repeats (DRs) containing twohexanucleotide sequences AGGTCA which are separatedby one nucleotide [20] The 51015840 flanking site of the PPREis important for PPAR isoformRXR heterodimer bindingspecificity and consists of a 7-nucleotide sequence consensus(C[AG][AG]A[AT]CT) [21]

The LBDof the PPARs is activated by specific endogenousagonists such as fatty acids fatty acids derivatives phos-pholipids eicosanoids and prostaglandins (Table 1) Manysynthetic ligands of PPARS have been developed for thetreatment of diabetes mellitus and high levels of triglyceridesand cholesterol such as the thiazolidine drugs (TZDs) andfibrates (Table 1) Recently chemicals present in the environ-ment throughhuman activity such as pesticides and phthalate

ester plasticizers have also been identified as PPAR ligands[22 23]

(2) Biological Function of PPARs in Lipid Metabolism andAdipocyte Differentiation PPARA is mainly involved infatty acid oxidation and is highly expressed in tissue withmitochondrial and peroxisomal 120573-oxidation such as brownadipose tissue (BAT) liver and to a lower extent also in heartkidney and muscles [24] PPARA is activated by long chainunsaturated fatty acids eicosanoids and synthetic fibrateswhich have been developed to treat dyslipidemia by reducingtriglyceride levels [25 26]

PPARD is ubiquitously expressed and has a commonfunction in fatty acid metabolism comparable to PPARAPPARD also seems to be involved in embryo implantationkeratinocyte differentiation and wound healing [27 28]PPARD is a promising drug target for heart defects causedby diabetes A recent study has shown that PPARD inducesglucose transport to the heart muscle cells and that diabetespatients have decreased PPARD expression in cardiac musclewhen glycemic levels are high [29] Synthetic agonists forPPARD have been developed for the treatment of obesity andrelated conditions as overexpression increases glucose influxreduces damage improves insulin sensitivity and reduceslipid accumulation [30]

In humans and mice the PPARG gene has three splicevariants PPARG1 PPARG2 and PPARG3 PPARG1 andPPARG3 translate into identical proteins whereas PPARG2contains an extra 28 amino acid regions in the NTD dueto alternative splicing [31] PPARG2 is an essential regulatorof adipocyte differentiation and lipid storage in WAT whilePPARG13 has a more general function in lipid metabolismand is expressed in the colon andmacrophages [32 33]Manysynthetic agonists have also been designed for PPARG2 withthe most well known being the TZDs and fibrates both usedfor the treatment of metabolic disorders like type 2 diabetesmellitus (T2DM) TZDs that target PPARG are used toincrease insulin sensitivity and reduce glycemia in the treat-ment of diabetes and additionally PPARG activation hasshown protective effects on the vasculature [34] Activationof PPARG by TZDs has also been linked to increased weightgain in patients based on PPARG function in adipocytedifferentiation and its involvement in lipid homeostasis [35]As PPARG has also been shown to regulate certain processesin cancer development it might be a possible additionaltarget in the treatment of cancer New generation drugs thattarget both PPARA and PPARG are very promising and havealready been used in treatment as they show hypolipidemichypotensive anti-inflammatory and antiatherogenic action[29 36 37]

2 Zebrafish as Model for Obesity

Numerous animal models have been used to study theetiology of obesity in order to gain better understanding ofmolecular mechanisms and possible treatments In the lastdecade zebrafish (Danio rerio) shown in adult and larvalstages in Figure 1 have emerged as an excellent model tostudy human diseases [38] due to a number of advantages

PPAR Research 3

Table 1 Endogenous and synthetic ligands of vertebrate PPARs

PPAR Ligand type Potential agonists

PPARA

Endogenous

Fatty acids (lauric acid linoleic acid linolenic acid arachidonic acid eicosapentaenoic acid (Xenopus)docosahexaenoic acid (Xenopus) petroselinic acid (Xenopus) oleic acid (Xenopus) and elaidic acid(Xenopus)) [66 67]Eicosanoids (fatty acid-derived) (eg leukotriene B

4) [68 69]

Prostaglandin J2 (fatty acid-derived) [70]Endocannabinoids [71]

SyntheticNonsteroidal anti-inflammatory drugs (NSAIDs) [72]Fibrates (eg gemfibrozil Bezafibrate clofibrate fenofibrate ciprofibrate pirinixic acid (Wy 14643) andGW2331) [3 67 73 74]ETYA (581114-eicosatetraynoic acid) (Xenopus) [66]

PPARDEndogenous Fatty acids (eg docosahexaenoic acid linoleic acid) [69]

Very Low Density Lipoproteins (VLDL) components [75]

Synthetic Fibrates (Wy-14643 Bezafibrate) [69]GW501516 GW800644 [76 77]

PPARG

Endogenous Fatty acids (docosahexaenoic acid linoleic acid (mouse)) [14 69]Prostaglandin J2 metabolite 15-deoxy-delta 1214-PGJ2 [70 78]

Synthetic

NSAIDs [72]Thiazolidinediones [79]ETYA (581114-eicosatetraynoic acid) (mouse) [14]Prostaglandin J2 derivatives [70 78]Fibrates (Bezafibrate clofibrate and GW2331) [14 67]

of this system Zebrafish develop rapidly and have a shortlife cycle and detailed genome sequence information isavailable The availability of new genome editing techniqueslike TALENs [39] and CRISPR-Cas9 [40] as well as severaltransgenesis tools (MultiSiteGateway Tol2 BAC transgenesisGal4UAS and Q system) enables the study of the functionof genes Zebrafish can also be used for high-throughputforward genetic and chemical screens [41ndash43] facilitating theidentification of molecules that regulate biological functions

Zebrafish are a promising model for obesity research aslipid metabolism pathways are conserved between mammalsand fish [44ndash46] Zebrafish have the key organs that areimportant for energy homeostasis and metabolism in mam-mals aswell as other key functions such as appetite regulationin the brain [47] insulin regulation [48] endocrine signalingthrough leptin [49] and lipid storage in white adipocytes [5051] Like humans zebrafish kept on a high caloric diet showincreased plasma triglyceride levels and hepatic steatosis aswell as comparable expression patterns of genes involved inlipid metabolism such as LEP SREBP PPARA PPARG andNR3H1 [45] It has also been shown that obesity in zebrafishcoincides with an increased plasma fibrinogen concentrationthat is induced via IL-6 and IL-1Bl secreted from visceralwhite adipose tissue Zebrafish as well as mice rats andhumans also produce a higher amount of IL-6 and IL-1Bupon high caloric feeding adding to the evidence that majormetabolic pathways between fish and mammals are verysimilar [45] It is important to note however that zebrafishmay not be an appropriate model for studying thermogenesisin higher vertebrates since brown adipose tissue has notbeen identified in poikilothermic animals Zebrafish appearto not make thermogenic brown adipocytes relying insteadon thyroid hormone-mediated processes to generate heat in

their muscles [52] Although uncoupling proteins (UCPs 1ndash5) have been identified in zebrafish that are expressed inbrain liver and muscle tissue but not in adipose tissue [53]differences in thermogenesis between mammals and fish areimportant to consider

One major advantage of the zebrafish as a model ofobesity is its optical transparence which allows temporalmonitoring of adipocyte formation and fatty acid uptake invivo [54] adipocytes can be visualized in developing larvaewith various dyes including the sudanophilic dye Oil RedO (ORO) or Sudan dyes (Sudan III Sudan IV and SudanBlack B) and fluorescent dyes like Nile Red or LipidGreen(Figure 1(c)) [50 55] ORO and Sudan dyes are lipophilic(fat-soluble) dyes that can be applied as a soluble colorantfor neutral lipids and cholesteryl esters Nile Red binds toboth neutral lipids and phospholipids while LipidGreen onlybinds to neutral lipids [55 56] Dyes such as Sudan BlackB and ORO are fixative based dyes and fixation techniquesare time consuming and may also cause deformation of lipiddroplets in tissue [57] while Nile Red and LipidGreen canbe applied in vivo The potential of using Nile Red stainingin zebrafish larvae as a whole-organism test for screeningpharmaceuticals and toxicological agents has recently beendemonstrated [58]

3 Adipogenesis and LipidMetabolism in Zebrafish

Adipose tissue develops from pluripotent mesenchymal stemcells (MSCs) and commitment to this cell lineage givesrise to preadipocytes (determination phase) and subsequentterminal differentiation adipocytes (differentiation phase)(Figure 1(d)) [59 60] In the transcriptional cascade leading

4 PPAR Research

(a)

(d)

(b)

(c) (c998400)

Mesenchymal stem cells Adipoblast Preadipocyte

AdipocyteTerminal differentiationDetermination

Antiadipogenic factors

Adipogenic factors

RXR

LXRGATA

23

Krox20

Early adipogenic factors

Ligands

Target gene activation

Fabp11a adiponectin glut 4 LPL and leptin

SREBP1c

Wnt genes

catenin

EZH2

Sirt1 PGC1aFOXO1

Klf5

120573-

CEBP120572

CEBP120573

CEBP120575

PPARD

PPARG

Figure 1 (a) An adult female and male zebrafish (b) Developing zebrafish larvae (15 dpf) (c + c1015840) A transmission light image of adipocytesin developing larvae (15 dpf) (left) and a fluorescent image after staining lipids with LipidGreen (right) (d) Transcriptional network of factorsimportant for adipocyte differentiation in zebrafish and mammals

PPAR Research 5

Table 2 Location and characteristics of the PPAR genes in zebrafish genome (GRCz10 Havana and NCBI)

PPAR Gene Chromosome location GRCz10 NCBI Nucleotide cds (bp) Protein (aa) Exonspparaa ENDARG00000031777 4 rev CM0028881 NM 001161333 1413 470 6pparab ENDARG00000054323 25 forw CM0029091 NM 001102567 1380 459 7pparda ENDARG00000044525 22 forw CM0029061 XM 694808 XM005168286 1491 496 7ppardb ENDARG00000009473 8 forw CM0028921 NM 131468 1554 517 7pparg ENDARG00000031848 11 rev CM0028951 NM 131467 1584 527 7

to the differentiation of WAT (Figure 1(d)) the nuclearhormone receptor PPARG and CCAATenhancer-bindingprotein (CEBP120572) are key players Two other members ofthe CEBP family CEBP120573 and CEBP120575 are also importantfactors during differentiation into adipocytes [59 61 62]CEBP120572 is a transcription factor that regulates PPARGexpression but also autoregulates its own expression Inzebrafish orthologs of these genes are expressed in adipocytesand liver [51]The terminal differentiationmarkers like leptinand fabp11a are also expressed Adiponectin has also beenfound in adult zebrafish adipose tissue [63]

During the first 4-5 days of development zebrafishembryos are dependent on nutrients provided by the yolk sacwhich contains essential fat-soluble vitamins and triacylglyc-erol (TAG) as well as cholesterol At days 5-6 the yolk sacis depleted and it is essential that larvae are provided withfood to start to eat The first signs of adipogenesis becomevisible at 8 days post fertilization (dpf) in the visceral cavityclose to the pancreas but only in the minority of larvae Inmost larvae the first adipocytes translocate asymmetricallyto the right visceral cavity [50] Most adipocytes are observedfrom 12 dpf onwards (standard length (SL) of gt 5mm) inthe pancreatic area [51] In our laboratory using LipidGreenstaining adipocytes are clearly visible at 15 dpf in the visceralregion in a subset of larvae (Figure 1(c)) The amount ofadipocytes is correlatedwith size of the larvae rather than agesuggesting body-length dependent lipid storage in adipocytes[50 51] At 17 dpf all larvae have WAT in the pancreatic andvisceral area indicating that visceral WAT development isnot dependent on size but regulated by age Subcutaneous(20 dpf SL gt 82mm) and cranial (22 dpf SL gt 94mm)adipocytes develop in a size-dependent manner [51] Adultfish have the largest adipocyte deposits in the visceral regionsbut smaller deposits are found subcutaneously in the tailand jaw and in the periorbital regions Zebrafish adipocytesare metabolically active and are able to communicate viagap junctions [64] The adipocyte lipid droplet size can varybetween 1 and 100 120583m and while most contain one largedroplet some may contain multiple smaller droplets in theearly stage of development These characteristics are con-formant to mammalian adipocyte development and provideevidence that zebrafish contain WAT similar to mammals[50 65]

4 PPARs in Zebrafish

The presence of five ppar genes in the zebrafish genome hasbeen described previously [80ndash82] We performed an in silicostudy to examine the homology of zebrafish PPARs with

orthologs in different species Using the TBLASTX searchtool in Ensembl database (Ensembl GRCz10 httpwwwensemblorgDanio rerioInfoIndex) we checked for allfive ppar genes in the newest version of zebrafish genomesequence available (GRC10) Based on the HavanaEnsemblmerged sequences and the mammalianzebrafish proteinsequences cDNA and Nucleotide Database (NCBI) tran-scripts could be identified (Table 2) The zebrafish genomehas undergone complete genome duplication in the teleostlineage after the divergence of fish and mammal ancestors[83 84] Because of this zebrafish have two PPARA (pparaapparab) and PPARD (pparda ppardb) genes that are locatedon separate chromosomes (ohnologs) Only one PPARG(pparg) gene has been identified pparaa is located on thereverse strand of chromosome 4 while pparab is located atforward strand of chromosome 25 The orthologs of PPARDare located on chromosome 22 (pparda) and chromosome8 (ppardb) and are both positioned on the forward strandIn zebrafish pparg gene is positioned on chromosome 11 onthe reverse strand (Table 2) Comparing genomic regions ofPPARs in human and zebrafish we found conserved syntenyregions using The Synteny Database [85] pparaa and pparabshowed both synteny to the same region at human Chr22 and showed synteny between their location on zebrafishgenome Also pparda and ppardb have synteny to the samelocation at human Chr 6 and as their PPARA orthologspparda and ppardb showed synteny between their locationon zebrafish genome The zebrafish genome surroundingpparg is highly conserved compared to human Chr 3 (Sup-plemental data 3AndashC in Supplementary Material availableonline at httpdxdoiorg1011552015358029) The con-served regions contain the same genes in both organismswhich indicates a conserved functional relationship betweensyntenic genes

Pparaa and Pparab encode for proteins that are 470 and459 amino acids (aa) long respectively Pparda consists of496 aa and Ppardb is 517 aa long Pparg is the longest PPARin zebrafish and is 527 aa in size Protein alignment andthe phylogenetic tree are based on 49 amino acid sequencesfrom 15 different species (Supplemental Data 1) The pro-tein sequences were subjected to homology analyses usingPRALINE multiple sequence alignment (httpwwwibivunlprogramspralinewww) (Supplemental Data 1 and Sup-plemental Data 2) The phylogenetic tree was constructedusing the softwareGeneious 90 (BLOSUM62matrix GeneticDistancemodel Jukes-Cantor Biomatters httpwwwgene-iouscom) (Figure 2) All zebrafish PPARs show proteinidentity to human and mice orthologs with similarity ofhuman PPARA to zebrafish Pparab and Pparaa of 74 and

6 PPAR Research

006

PPARD

Birds

M musculus

Birds

Birds

Amphibians

Amphibians

Amphibians

Fish

Mam

mal

s

Mam

mal

s

Mam

mal

s

Fish

Fish

PPARG

PPARA

R norvegicusH sapiensM fascicularisM mulatta

B taurusC lupus

G gallusX laevis

S salar

D rerio gene bD rerio gene a

M musculusR norvegicus

H sapiensM fascicularisM mulatta

B taurusC lupus

G gallus

G gallus

S salar

S salar


M musculus iso 1M musculus iso 2R norvegicus iso 1

R norvegicus iso 2H sapiens iso 1H sapiens iso 2

M fascicularisM mulatta

B taurus iso 1B taurus iso 2

C lupus

X laevis

X laevis

D rerio

O cuniculus (rabbit)

S scrofa (boar)

S scrofa (boar)

S scrofa (boar)

O latipes (medaka)

O latipes (medaka)

O latipes (medaka)

O mykiss (trout)

O mykiss (trout)

O mykiss (trout)

Figure 2 Phylogenetic tree of the PPAR orthologs in various organisms based on amino acid sequence difference (BLOSUM62 matrix) Thephylogenetic tree is generated using the Geneious 90 software Protein accession numbers information is provided in Supplemental Data 1

67 respectively Zebrafish Pparda and Ppardb showed 71and 73 similarity to human PPARD Besides that zebrafishPparg is 67 similar to human PPARG (Table 3)

Amino acid sequence analysis of different homologuesrevealed that PPARs consist of a highly conserved pro-tein family The phylogenetic tree shows also that PPARAPPARD and PPARG proteins form distinct clusters of pro-tein although PPARA and PPARD were clustered togetherand the PPARG branch stands alone The first gene duplica-tion event of the PPARgene family probably occurred in bonyfish before being separated from birds and mammals Thesecond gene duplication is vertebrate specific and resulted inthe different PPAR isotypes (A D and G) [86 87] After thegene duplication events the PPARs acquired ligand bindingcapacities in an independent manner [88] and started toevolve by mutations in each PPARS resulting in refinedspecificity for ligands [87] Zhao and colleagues [82] alignedthe sequences of the DBD and LBD between human and11 different species The DBD and LBD for all PPAR arehighly conserved between the different species except for

the DBD and LDB in Xenopus PPARD which are far muchless conserved (83 and 75 resp) One explanation canbe that PPARD is less evolved in Xenopus compared to theother species and therefore has a separated position in thephylogenetic tree

The zebrafish PPAR proteins contain several large regionsof amino acids that are highly conserved (SupplementalData 2) The N-terminal region of zebrafish PPARs is lesssimilar as this region is highly variable between organismsThe N-terminal region contains the conserved AF-1 thatcan be activated by endogenous factors like hormones andcytokines In contrast the AF-2 site located in the C-terminalregion remains well conserved Like human PPARs zebrafishPparaa Pparab and Pparg contain a threonineserine-richNLS2 site that is involved in cytoplasm-nucleus shuttlingof the PPARs This site was not found in Ppard ohnologshowever which have a threonineserine-rich part earlier inthe protein which could possibly result in a similar actionThe overall conservation of PPARG is fairly high betweenorganisms except for three fish species including the Japanese

PPAR Research 7

Table 3 Comparison of PPARs between human mouse and zebrafish

PPAR Human Mouse ZebrafishPPARA

Genes 1 1 2Splice variants 1 1 1 per geneIsoforms 1 1 1 per gene

Size (cds bp) 1407 1407 pparaa 1413pparab 1380

Protein similarity to zebrafish 67 for pparaa74 for pparab

65 for pparaa71 for pparab

PPARDGenes 1 1 2Splice variants 1 1 1 per geneIsoforms 1 1 1 per gene

Size (cds bp) 1326 1323 pparda 1491ppardb 1554

Protein similarity to zebrafish 71 for pparda73 for ppardb

70 for pparda73 for ppardb

PPARGGenes 1 1 1Splice variants 4 1 1Isoforms 2 2 1

Size (cds bp) PPARG1 = 1434PPARG2 = 1518

PPARG1 = 1428PPARG2 = 1518 pparg 1584

Protein similarity to zebrafish 67 67

medaka (Oryzias latipes) salmon (Salmo salar) and rainbowtrout (Oncorhynchus mykiss) which have several (up to 5)additional amino acid regions throughout the protein In allfish species the LDB of PPARG is less conserved to human(74ndash78) and this can be explained by the rapid evolutionaryrate of PPARG in teleosts [82] When comparing human andzebrafish PPARG two major differences between zebrafishPparg and human PPARG were found Firstly the first 30amino acids from the N-terminus differ highly betweenzebrafish Pparg and both hPPARG isoforms In this regionof the protein hPPARG2 contains extra 28 amino acidscompared to the hPPARG1 isoform Conversely zebrafishPparg contains only 16 amino acids in this region that differin all except two amino acids from hPPARG2 Secondlyzebrafish Pparg has an extra 27 amino acid regions startingfrom the 156th amino acid Other fish species also have extraamino acid sequence so it is possible that during evolutionteleost fish gained additional coding sequences which canhave implications for protein folding and functionality Wealso observed that all fish species analysed have an extraprotein sequence in the ligand binding domain visible atamino acid position 401 in the protein alignment This extrasequence is not a part of the ligand binding pocket andpresumably should not interfere with binding to specificligands However a previous study has shown differencesin ligand binding specificity as sequence comparison ofzebrafish PPARg to human and mice PPARG revealed resid-ual differences within the LDB [89]

The phylogenetic tree shows that the zebrafish PPARAohnologs have evolved further from each other comparedto the zebrafish PPARD ohnologs (Figure 2) The zebrafishPPARDohnologs havemost likely retained the same function(subfunctionalization) while the zebrafish PPARA ohnologsmight have been subject to neofunctionalization [80] pparabhas more similarities with salmon and rainbow trout whilepparaa is on a separate branch in the phylogenetic tree andhas a higher homology with Japanese medaka Additionallyzebrafish PPARA and PPARD ohnologs seem to be morerelated to each other than to zebrafish PPARG

In fish PPARs have been identified and characterizedbut not much is known about their expression duringdevelopmental stages and adipogenesis Zebrafish PPARs areexpressed during development and so far little is knownof the expression profiles of the paralogs pparaa pparabpparda and ppardb The limited expression studies that havebeen performed show that ppardb and pparg mRNA arealready expressed early in development (5ndash10 hours postfertilization (hpf)) in whole embryo From 20 hpf on theexpression is more intensive in the head region and continuesto be specifically expressed in the head branchial archesand pectoral fin at 36 hpf From this time point expressiondata for pparda is also available and comparable to thatof ppardb and pparg At 5 dpf gene expression is detectedaround the swim bladder (pparda ppardb and pparg) in theliver (pparda pparg) in the intestinal bulb (pparda) and alsoin the complete intestinal tract (pparg) [80] The expression

8 PPAR Research

patterns of ppard paralogs and pparg can be an indicationthat they play a role in adipogenesis since it is known thatfirst adipocytes arise in the pancreatic region underneaththe swim bladder [50 51] During the larval stage (15 dpf)pparg mRNA is localized in developing adipocytes withinthe pancreas and the intestinal epithelium in the same cellswhere Nile Red staining was observed [50] Expression of thepparg gene is specifically detected in visceral adipose tissueand pancreas as opposed to liver [51]

5 Conclusions

PPARs are important factors in energy homeostasis and obe-sity development While PPARA and PPARD are necessaryfor lipid breakdown PPARG plays a role in lipid accumu-lation and adipogenesis Single nucleotide polymorphismsin PPARs have been implicated in the development of obe-sity T2DM lipodystrophy dyslipidemias and cardiovascularrisk The zebrafish is an emerging model for research in obe-sity and related metabolic diseases and could be very usefulfor providing insight into the role of PPARs in the origins ofobesity All five PPAR genes (pparaa pparab pparda ppardband pparg) showprotein similarity to human andmice PPARsvarying from 67 to 74 Three of the five zebrafish PPARs(pparda ppardb and pparg) show expression of mRNA inregions around swim bladder and pancreas that correlate tothe sites where first adipocytes will develop and at 15 dpfpparg expression is detected in developing adipocytes Thiscolocalization suggests a role for PPARs in adipogenesisHowever detailed expression studies of PPARs during earlystages of adipocyte development are not available and todetermine the subcellular expression of the different PPARsin zebrafish specific antibody staining needs to be performedor transgenic fish lines should be generated In additionmoreconclusive evidence of the function of PPAR in zebrafishadipogenesis can be obtained by the generation of mutantlines Taken together this review shows that the zebrafish isa promising model for elucidating the specific functions ofPPARs in adipogenesis and obesity

Conflict of Interests

The authors declare that they do not have any conflict ofinterests

Acknowledgment

This research is financially supported by Netherlands Organ-isation for Scientific Research (NWO) VIDI86409005 andASPASIA015006018

References

[1] WHO ldquoWorld health statistics 2012rdquo World Health Organiza-tion vol 27 no 1 pp 179ndash187 1949

[2] E L Yong J Li and M H Liu ldquoSingle gene contributionsgenetic variants of peroxisome proliferator-activated receptor(isoforms 120572 120573Δ and 120574) and mechanisms of dyslipidemiasrdquoCurrent Opinion in Lipidology vol 19 no 2 pp 106ndash112 2008

[3] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 no 1 pp 409ndash4352002

[4] B Desvergne andW Wahli ldquoPeroxisome proliferator-activatedreceptors nuclear control of metabolismrdquo Endocrine Reviewsvol 20 no 5 pp 649ndash688 1999

[5] K Tachibana D Yamasaki K Ishimoto and T Doi ldquoThe role ofPPARs in cancerrdquo PPAR Research vol 2008 Article ID 10273715 pages 2008

[6] Nuclear Receptors Nomenclature C ldquoA unified nomenclaturesystem for the nuclear receptor superfamilyrdquo Cell vol 97 no 2pp 161ndash163 1999

[7] M M Aagaard R Siersbaeligk and S Mandrup ldquoMolecularbasis for gene-specific transactivation by nuclear receptorsrdquoBiochimica et Biophysica ActamdashMolecular Basis of Disease vol1812 no 8 pp 824ndash835 2011

[8] D J Mangelsdorf C Thummel M Beato et al ldquoThe nuclearreceptor superfamily the second decaderdquoCell vol 83 no 6 pp835ndash839 1995

[9] J D Tugwood I Issemann R G Anderson K R Bundell WL McPheat and S Green ldquoThe mouse peroxisome proliferatoractivated receptor recognizes a response element in the 51015840flanking sequence of the rat acyl CoA oxidase generdquoThe EMBOJournal vol 11 no 2 pp 433ndash439 1992

[10] V Zoete A Grosdidier and O Michielin ldquoPeroxisomeproliferator-activated receptor structures ligand specificitymolecular switch and interactions with regulatorsrdquo Biochimicaet Biophysica Acta vol 1771 no 8 pp 915ndash925 2007

[11] T Helledie M Antonius R V Soslashrensen et al ldquoLipid-bindingproteins modulate ligand-dependent trans-activation by per-oxisome proliferator-activated receptors and localize to thenucleus as well as the cytoplasmrdquo Journal of Lipid Research vol41 no 11 pp 1740ndash1751 2000

[12] T Umemoto and Y Fujiki ldquoLigand-dependent nucleo-cytoplasmic shuttling of peroxisome proliferator-activatedreceptors PPAR120572 and PPAR120574rdquo Genes to Cells vol 17 no 7 pp576ndash596 2012

[13] P Tontonoz R A Graves A I Budavari et al ldquoAdipocyte-specific transcription factorARF6 is a heterodimeric complex oftwo nuclear hormone receptors PPAR gamma and RXR alphardquoNucleic Acids Research vol 22 no 25 pp 5628ndash5634 1994

[14] P Tontonoz E Hu and B M Spiegelman ldquoStimulation ofadipogenesis in fibroblasts by PPAR1205742 a lipid-activated tran-scription factorrdquo Cell vol 79 no 7 pp 1147ndash1156 1994

[15] V Chandra P Huang Y Hamuro et al ldquoStructure of the intactPPAR-120574-RXR-120572 nuclear receptor complex on DNArdquo Naturevol 456 no 7220 pp 350ndash356 2008

[16] N Rochel F Ciesielski J Godet et al ldquoCommon architecture ofnuclear receptor heterodimers on DNA direct repeat elementswith different spacingsrdquo Nature Structural and Molecular Biol-ogy vol 18 no 5 pp 564ndash570 2011

[17] V Aguilar J-S Annicotte X Escote J Vendrell D Langinand L Fajas ldquoCyclin G2 regulates adipogenesis through PPARgamma coactivationrdquo Endocrinology vol 151 no 11 pp 5247ndash5254 2010

[18] Y Zhu C Qi C Calandra M S Rao and J K Reddy ldquoCloningand identification of mouse steroid receptor coactivator-1(mSRC-1) as a coactivator of peroxisome proliferator-activatedreceptor gammardquo Gene Expression vol 6 no 3 pp 185ndash1951996

PPAR Research 9

[19] Y Zhu C Qi S Jain M S Rao and J K Reddy ldquoIsolation andcharacterization of PBP a protein that interacts with peroxi-some proliferator-activated receptorrdquo The Journal of BiologicalChemistry vol 272 no 41 pp 25500ndash25506 1997

[20] S A Kliewer K Umesono D J Mangelsdorf and R M EvansldquoRetinoid X receptor interacts with nuclear receptors in retinoicacid thyroid hormone and vitamin D3 signallingrdquo Nature vol355 no 6359 pp 446ndash449 1992

[21] G Venkatachalam M K Sakharkar A P Kumar and MV Clement ldquoPPRESearch peroxisome proliferator activatorelement search databaserdquo International Journal of IntegrativeBiology vol 8 no 1 pp 37ndash42 2009

[22] A Janesick and B Blumberg ldquoMinireview PPAR120574 as the targetof obesogensrdquo Journal of Steroid Biochemistry and MolecularBiology vol 127 no 1-2 pp 4ndash8 2011

[23] A Janesick and B Blumberg ldquoObesogens stem cells and thedevelopmental programming of obesityrdquo International Journalof Andrology vol 35 no 3 pp 437ndash448 2012

[24] P Escher O Braissant S Basu-Modak L Michalik W Wahliand B Desvergne ldquoRat PPARs quantitative analysis in adult rattissues and regulation in fasting and refeedingrdquo Endocrinologyvol 142 no 10 pp 4195ndash4202 2001

[25] BM Forman J Chen and RM Evans ldquoHypolipidemic drugspolyunsaturated fatty acids and eicosanoids are ligands forperoxisome proliferator-activated receptors alpha and deltardquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 9 pp 4312ndash4317 1997

[26] K Schoonjans B Staels and J Auwerx ldquoRole of the peroxisomeproliferator-activated receptor (PPAR) in mediating the effectsof fibrates and fatty acids on gene expressionrdquo Journal of LipidResearch vol 37 no 5 pp 907ndash925 1996

[27] Y Barak D Liao W He et al ldquoEffects of peroxisomeproliferator-activated receptor 120575 on placentation adiposityand colorectal cancerrdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 1 pp 303ndash308 2002

[28] N S Tan L Michalik N Noy et al ldquoCritical roles of PPAR120573120575in keratinocyte response to inflammationrdquo Genes and Develop-ment vol 15 no 24 pp 3263ndash3277 2001

[29] E M Burkart N Sambandam X Han et al ldquoNuclear receptorsPPAR120573120575 and PPAR120572 direct distinct metabolic regulatory pro-grams in the mouse heartrdquoThe Journal of Clinical Investigationvol 117 no 12 pp 3930ndash3939 2007

[30] Y-X Wang C-H Lee S Tiep et al ldquoPeroxisome-proliferator-activated receptor 120574 activates fatmetabolism to prevent obesityrdquoCell vol 113 no 2 pp 159ndash170 2003

[31] L Fajas D Auboeuf E Raspe et al ldquoThe organizationpromoter analysis and expression of the human PPAR120574 generdquoThe Journal of Biological Chemistry vol 272 no 30 pp 18779ndash18789 1997

[32] D Ren T N Collingwood E J Rebar A P Wolffe and H SCamp ldquoPPAR120574 knockdownby engineered transcription factorsexogenous PPAR1205742 but not PPAR1205741 reactivates adipogenesisrdquoGenes and Development vol 16 no 1 pp 27ndash32 2002

[33] GMedina-Gomez S LGray L Yetukuri et al ldquoPPARgamma2prevents lipotoxicity by controlling adipose tissue expandabilityand peripheral lipid metabolismrdquo PLoS Genetics vol 3 no 4article e64 pp 634ndash647 2007

[34] A E Caballero R Saouaf S C Lim et al ldquoThe effects oftroglitazone an insulin-sensitizing agent on the endothelialfunction in early and late type 2 diabetes a placebo-controlled

randomized clinical trialrdquoMetabolism Clinical and Experimen-tal vol 52 no 2 pp 173ndash180 2003

[35] T M Larsen S Toubro and A Astrup ldquoPPARgamma agonistsin the treatment of type II diabetes is increased fatnesscommensurate with long-term efficacyrdquo International Journalof Obesity vol 27 no 2 pp 147ndash161 2003

[36] N Marx T Bourcier G K Sukhova P Libby and J PlutzkyldquoPPAR120574 activation in human endothelial cells increases plas-minogen activator inhibitor type-1 expression PPAR120574 as apotential mediator in vascular diseaserdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 19 no 3 pp 546ndash551 1999

[37] A Raji E W Seely S A Bekins G H Williams and DC Simonson ldquoRosiglitazone improves insulin sensitivity andlowers blood pressure in hypertensive patientsrdquo Diabetes Carevol 26 no 1 pp 172ndash178 2003

[38] J Ablain and L I Zon ldquoOf fish andmen using zebrafish to fighthuman diseasesrdquo Trends in Cell Biology vol 23 no 12 pp 584ndash586 2013

[39] T Cermak E L Doyle M Christian et al ldquoEfficient designand assembly of custom TALEN and other TAL effector-basedconstructs for DNA targetingrdquo Nucleic Acids Research vol 39no 12 article e82 2011

[40] W Y Hwang Y Fu D Reyon et al ldquoEfficient genome editingin zebrafish using a CRISPR-Cas systemrdquoNature Biotechnologyvol 31 no 3 pp 227ndash229 2013

[41] G N Wheeler and A W Brandli ldquoSimple vertebrate modelsfor chemical genetics and drug discovery screens lessons fromzebrafish and Xenopusrdquo Developmental Dynamics vol 238 no6 pp 1287ndash1308 2009

[42] M Tsang ldquoZebrafish a tool for chemical screensrdquo Birth DefectsResearch Part CmdashEmbryo Today Reviews vol 90 no 3 pp 185ndash192 2010

[43] R T Peterson and M C Fishman ldquoDesigning Zebrafishchemical screensrdquo in Methods in Cell Biology vol 105 chapter23 pp 525ndash541 Elsevier 2011

[44] A Schlegel and D Y R Stainier ldquoLessons from lsquolowerrsquo organ-isms what worms flies and zebrafish can teach us about humanenergy metabolismrdquo PLoS Genetics vol 3 no 11 article e1992007

[45] T Oka Y Nishimura L Zang et al ldquoDiet-induced obesityin zebrafish shares common pathophysiological pathways withmammalian obesityrdquo BMC Physiology vol 10 article 21 2010

[46] J L Anderson J D Carten and S A Farber ldquoZebrafish lipidmetabolism frommediating early patterning to themetabolismof dietary fat and cholesterolrdquoMethods in Cell Biology vol 101pp 111ndash141 2011

[47] S-INishio YGibert L Berekelya et al ldquoFasting inducesCARTdown-regulation in the zebrafish nervous system in a cannabi-noid receptor 1-dependent mannerrdquo Molecular Endocrinologyvol 26 no 8 pp 1316ndash1326 2012

[48] V Jimenez-Amilburu S Jong-Raadsen J Bakkers H P Spainkand R Marın-Juez ldquoGLUT12 deficiency during early devel-opment results in heart failure and a diabetic phenotype inzebrafishrdquo Journal of Endocrinology vol 224 no 1 pp 1ndash152015

[49] M R Dalman Q Liu M D King B Bagatto and R L Lon-draville ldquoLeptin expression affects metabolic rate in zebrafishembryos (D Rerio)rdquo Frontiers in Physiology vol 4 article 1602013

[50] E J Flynn C M Trent and J F Rawls ldquoOntogeny andnutritional control of adipogenesis in zebrafish (Danio rerio)rdquoJournal of Lipid Research vol 50 no 8 pp 1641ndash1652 2009

10 PPAR Research

[51] D Imrie andK C Sadler ldquoWhite adipose tissue development inzebrafish is regulated by both developmental time and fish sizerdquoDevelopmental Dynamics vol 239 no 11 pp 3013ndash3023 2010

[52] A G Little and F Seebacher ldquoThe evolution of endothermy isexplained by thyroid hormone-mediated responses to cold inearly vertebratesrdquo Journal of Experimental Biology vol 217 no10 pp 1642ndash1648 2014

[53] Y-C Tseng R-D Chen M Lucassen et al ldquoExploring uncou-pling proteins and antioxidant mechanisms under acute coldexposure in brains of fishrdquo PLoS ONE vol 6 no 3 Article IDe18180 2011

[54] M Holtta-Vuori V T V Salo L Nyberg et al ldquoZebrafishgaining popularity in lipid researchrdquo Biochemical Journal vol429 no 2 pp 235ndash242 2010

[55] J H Lee J-H So J H Jeon et al ldquoSynthesis of a newfluorescentsmall molecule probe and its use for in vivo lipid imagingrdquoChemical Communications vol 47 no 26 pp 7500ndash7502 2011

[56] H S Chun J H Jeon H S Pagire et al ldquoSynthesis ofLipidGreen2 and its application in lipid and fatty liver imagingrdquoMolecular Biosystems vol 9 no 4 pp 630ndash633 2013

[57] S Fukumoto and T Fujimoto ldquoDeformation of lipid droplets infixed samplesrdquo Histochemistry and Cell Biology vol 118 no 5pp 423ndash428 2002

[58] A Tingaud-Sequeira N Ouadah and P J Babin ldquoZebrafishobesogenic test a tool for screening molecules that targetadiposityrdquo Journal of Lipid Research vol 52 no 9 pp 1765ndash17722011

[59] K W Park D S Halperin and P Tontonoz ldquoBefore they werefat adipocyte progenitorsrdquo Cell Metabolism vol 8 no 6 pp454ndash457 2008

[60] Q Q Tang and M D Lane ldquoAdipogenesis from stem cell toadipocyterdquoAnnual Review of Biochemistry vol 81 no 1 pp 715ndash736 2012

[61] S R Farmer ldquoTranscriptional control of adipocyte formationrdquoCell Metabolism vol 4 no 4 pp 263ndash273 2006

[62] D P Ramji and P Foka ldquoCCAATenhancer-binding proteinsstructure function and regulationrdquo Biochemical Journal vol365 no 3 pp 561ndash575 2002

[63] S-I Nishio Y Gibert L Bernard F Brunet G TriqueneauxandV Laudet ldquoAdiponectin and adiponectin receptor genes arecoexpressed during zebrafish embryogenesis and regulated byfood deprivationrdquo Developmental Dynamics vol 237 no 6 pp1682ndash1690 2008

[64] T Yanagiya A Tanabe and K Hotta ldquoGap-junctional com-munication is required for mitotic clonal expansion duringadipogenesisrdquo Obesity vol 15 no 3 pp 572ndash582 2007

[65] A Tingaud-Sequeira A Knoll-Gellida M Andre and P JBabin ldquoVitellogenin expression in white adipose tissue infemale teleost fishrdquoBiology of Reproduction vol 86 no 2 article38 2012

[66] H Keller C Dreyer J Medin A Mahfoudi K Ozatoand W Wahli ldquoFatty acids and retinoids control lipidmetabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimersrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 90 no 6 pp 2160ndash2164 1993

[67] S A Kliewer S S Sundseth S A Jones et al ldquoFatty acidsand eicosanoids regulate gene expression through direct inter-actions with peroxisome proliferator-activated receptors 120572 and120574rdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 9 pp 4318ndash4323 1997

[68] P R Devchand H Keller J M Peters M Vazquez F JGonzalez and W Wahli ldquoThe PPAR120572-leukotriene B4 pathwayto inflammation controlrdquo Nature vol 384 no 6604 pp 39ndash431996

[69] K Yu W Bayona C B Kallen et al ldquoDifferential activationof peroxisome proliferator-activated receptors by eicosanoidsrdquoJournal of Biological Chemistry vol 270 no 41 pp 23975ndash23983 1995

[70] S A Kliewer J M Lenhard T M Willson I Patel D CMorris and J M Lehmann ldquoA prostaglandin J2 metabolitebinds peroxisome proliferator-activated receptor gamma andpromotes adipocyte differentiationrdquo Cell vol 83 no 5 pp 813ndash819 1995

[71] S E OrsquoSullivan ldquoCannabinoids go nuclear evidence for acti-vation of peroxisome proliferator-activated receptorsrdquo BritishJournal of Pharmacology vol 152 no 5 pp 576ndash582 2007

[72] JM Lehmann JM Lenhard B B Oliver GM Ringold and SA Kliewer ldquoPeroxisome proliferator-activated receptors 120572 and120574 are activated by indomethacin and other non-steroidal anti-inflammatory drugsrdquo The Journal of Biological Chemistry vol272 no 6 pp 3406ndash3410 1997

[73] S Kersten B Desvergne and W Wahli ldquoRoles of PPARS inhealth and diseaserdquo Nature vol 405 no 6785 pp 421ndash4242000

[74] T M Willson P J Brown D D Sternbach and B R HenkeldquoThe PPARs from orphan receptors to drug discoveryrdquo Journalof Medicinal Chemistry vol 43 no 4 pp 527ndash550 2000

[75] A Chawla C-H Lee Y Barak et al ldquoPPARdelta is a very low-density lipoprotein sensor in macrophagesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 3 pp 1268ndash1273 2003

[76] RANgala C J Stocker AG Roy et al ldquoAnew highly selectivemurine peroxisome proliferator-activated receptor 120575 agonistincreases responsiveness to thermogenic stimuli and glucoseuptake in skeletal muscle in obese micerdquo Diabetes Obesity andMetabolism vol 13 no 5 pp 455ndash464 2011

[77] W R Oliver Jr J L Shenk M R Snaith et al ldquoA selectiveperoxisome proliferator-activated receptor 120575 agonist promotesreverse cholesterol transportrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no9 pp 5306ndash5311 2001

[78] B M Forman P Tontonoz J Chen R P Brun B MSpiegelman and R M Evans ldquo15-deoxy-Δ1214-prostaglandinJ2is a ligand for the adipocyte determination factor PPAR120574rdquo

Cell vol 83 no 5 pp 803ndash812 1995[79] JM Lehmann L BMoore T A Smith-OliverWOWilkison

T M Willson and S A Kliewer ldquoAn antidiabetic thiazolidine-dione is a high affinity ligand for peroxisome proliferator-activated receptor 120574 (PPAR120574)rdquo The Journal of Biological Chem-istry vol 270 no 22 pp 12953ndash12956 1995

[80] S Bertrand B Thisse R Tavares et al ldquoUnexpected novelrelational links uncovered by extensive developmental profilingof nuclear receptor expressionrdquo PLoS Genetics vol 3 no 11article e188 2007

[81] J M McPartland M Glass I Matias R W Norris and C WKilpatrick ldquoA shifted repertoire of endocannabinoid genes inthe zebrafish (Danio rerio)rdquo Molecular Genetics and Genomicsvol 277 no 5 pp 555ndash570 2007

[82] Y Zhao K Zhang J P Giesy and J Hu ldquoFamilies of nuclearreceptors in vertebrate models characteristic and comparativetoxicological perspectiverdquo Scientific Reports vol 5 article 85542015

PPAR Research 11

[83] M A Gates L Kim E S Egan et al ldquoA genetic linkage mapfor zebrafish comparative analysis and localization of genes andexpressed sequencesrdquo Genome Research vol 9 no 4 pp 334ndash347 1999

[84] O Jaillon J-M Aury F Brunet et al ldquoGenome duplicationin the teleost fish Tetraodon nigroviridis reveals the earlyvertebrate proto-karyotyperdquo Nature vol 431 no 7011 pp 946ndash957 2004

[85] J M Catchen J S Conery and J H Postlethwait ldquoAutomatedidentification of conserved synteny after whole-genome dupli-cationrdquo Genome Research vol 19 no 8 pp 1497ndash1505 2009

[86] L Michalik B Desvergne C Dreyer M Gavillet R N LauriniandWWahli ldquoPPAR expression and function during vertebratedevelopmentrdquo International Journal of Developmental Biologyvol 46 no 1 pp 105ndash114 2002

[87] T Zhou X Yan GWang et al ldquoEvolutionary pattern and regu-lation analysis to support why diversity functions existed withinPPAR gene family membersrdquo BioMed Research Internationalvol 2015 Article ID 613910 11 pages 2015

[88] H Escriva F Delaunay and V Laudet ldquoLigand binding andnuclear receptor evolutionrdquo BioEssays vol 22 no 8 pp 717ndash727 2000

[89] A RiuMGrimaldi A leMaire et al ldquoPeroxisome proliferator-activated receptor 120574 is a target for halogenated analogs ofbisphenol Ardquo Environmental Health Perspectives vol 119 no 9pp 1227ndash1232 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 PPAR Research

identified in vertebrates namely PPARA (hPPAR PPAR120572and NR1C1) PPARD (NR1C2 PPAR120573120575 FAAR NUCI andNUCII) and PPARG (NR1C3 PPAR120574) which are encodedby different genes [4] The zebrafish orthologs of PPARAPPARD and PPARG are referred to as Pparaa PparabPparda Ppardb and Pparg according to the ZFIN nomen-clature (httpzfinorg)

Like other nuclear receptors PPARs have a proteinstructure that generally consists of four parts namely an N-terminal domain (NTD) a DNA-binding domain (DBD) aligand binding domain (LBD) and a connective structure(hinge) [7 8] The NTD contains a ligand-independentactivation factor-1 (AF-1) The DBD consists of two zincfingers that specifically bind to the peroxisome proliferatorresponse element (PPRE) in the promotor regions of PPARtarget genes [9] The C-terminal part of the protein theLBD consists of 13 120572-helices and 4 120573-sheets which formthe ligand binding pocket The LBD contains the liganddependent activation factor-2 (AF-2) and ligand binding toAF-2 results in activation of the LBD [3 10] Expressionof PPARs is predominantly in the nucleus though PPARAand PPARG are also found in lower concentrations in thecytoplasm as well [11 12] PPARs can be shuttled between thecytoplasm and the nucleus by export receptors that recognizetwo different nuclear localization signals (NLSs) on the PPARprotein [12]

PPARs regulate transcriptional gene activation throughheterodimerization with retinoid X receptors (RXR) [13 14]All PPARs can form a complex with RXR and heterodimer-ization between PPAR and RXR is ligand independent [1516] In the absence of ligands PPARRXR heterodimersbound to PPREs will act as a transcriptional repressor dueto binding of corepressor proteins such as nuclear receptorcorepressor 1 (NCoR) and silencing mediator of retinoicacid and thyroid hormone receptor (SMRT) Binding ofa specific PPAR agonist to LBD leads to the release ofcorepressor complex and recruitment of the coactivationfactors Coactivators of PPARs include the steroid receptorcoactivator-1 (SRC-1) CREB-binding protein (CBP) PPAR-binding protein (PBP) P300 cyclin G2 PPAR-interactingprotein (PRIP) and PPAR120574 coactivator-1 (PGC-1) [17ndash19]As a consequence of a conformational change of the LBDthe PPARRXR heterodimer binds to the PPRE presentin promoter regions in order to regulate transcription oftarget genes by facilitating RNA polymerase II functionThe PPRE consists of direct repeats (DRs) containing twohexanucleotide sequences AGGTCA which are separatedby one nucleotide [20] The 51015840 flanking site of the PPREis important for PPAR isoformRXR heterodimer bindingspecificity and consists of a 7-nucleotide sequence consensus(C[AG][AG]A[AT]CT) [21]

The LBDof the PPARs is activated by specific endogenousagonists such as fatty acids fatty acids derivatives phos-pholipids eicosanoids and prostaglandins (Table 1) Manysynthetic ligands of PPARS have been developed for thetreatment of diabetes mellitus and high levels of triglyceridesand cholesterol such as the thiazolidine drugs (TZDs) andfibrates (Table 1) Recently chemicals present in the environ-ment throughhuman activity such as pesticides and phthalate

ester plasticizers have also been identified as PPAR ligands[22 23]

(2) Biological Function of PPARs in Lipid Metabolism andAdipocyte Differentiation PPARA is mainly involved infatty acid oxidation and is highly expressed in tissue withmitochondrial and peroxisomal 120573-oxidation such as brownadipose tissue (BAT) liver and to a lower extent also in heartkidney and muscles [24] PPARA is activated by long chainunsaturated fatty acids eicosanoids and synthetic fibrateswhich have been developed to treat dyslipidemia by reducingtriglyceride levels [25 26]

PPARD is ubiquitously expressed and has a commonfunction in fatty acid metabolism comparable to PPARAPPARD also seems to be involved in embryo implantationkeratinocyte differentiation and wound healing [27 28]PPARD is a promising drug target for heart defects causedby diabetes A recent study has shown that PPARD inducesglucose transport to the heart muscle cells and that diabetespatients have decreased PPARD expression in cardiac musclewhen glycemic levels are high [29] Synthetic agonists forPPARD have been developed for the treatment of obesity andrelated conditions as overexpression increases glucose influxreduces damage improves insulin sensitivity and reduceslipid accumulation [30]

In humans and mice the PPARG gene has three splicevariants PPARG1 PPARG2 and PPARG3 PPARG1 andPPARG3 translate into identical proteins whereas PPARG2contains an extra 28 amino acid regions in the NTD dueto alternative splicing [31] PPARG2 is an essential regulatorof adipocyte differentiation and lipid storage in WAT whilePPARG13 has a more general function in lipid metabolismand is expressed in the colon andmacrophages [32 33]Manysynthetic agonists have also been designed for PPARG2 withthe most well known being the TZDs and fibrates both usedfor the treatment of metabolic disorders like type 2 diabetesmellitus (T2DM) TZDs that target PPARG are used toincrease insulin sensitivity and reduce glycemia in the treat-ment of diabetes and additionally PPARG activation hasshown protective effects on the vasculature [34] Activationof PPARG by TZDs has also been linked to increased weightgain in patients based on PPARG function in adipocytedifferentiation and its involvement in lipid homeostasis [35]As PPARG has also been shown to regulate certain processesin cancer development it might be a possible additionaltarget in the treatment of cancer New generation drugs thattarget both PPARA and PPARG are very promising and havealready been used in treatment as they show hypolipidemichypotensive anti-inflammatory and antiatherogenic action[29 36 37]

2 Zebrafish as Model for Obesity

Numerous animal models have been used to study theetiology of obesity in order to gain better understanding ofmolecular mechanisms and possible treatments In the lastdecade zebrafish (Danio rerio) shown in adult and larvalstages in Figure 1 have emerged as an excellent model tostudy human diseases [38] due to a number of advantages

PPAR Research 3



PPARA

Endogenous


4) [68 69]






PPARG


Synthetic








4 PPAR Research

(a)

(d)

(b)

(c) (c998400)




Adipogenic factors

RXR

LXRGATA

23

Krox20


Ligands



SREBP1c

Wnt genes

catenin

EZH2

Sirt1 PGC1aFOXO1

Klf5

120573-

CEBP120572

CEBP120573

CEBP120575

PPARD

PPARG


PPAR Research 5









6 PPAR Research

006

PPARD

Birds

M musculus

Birds

Birds

Amphibians

Amphibians

Amphibians

Fish

Mam

mal

s

Mam

mal

s

Mam

mal

s

Fish

Fish

PPARG

PPARA


B taurusC lupus

G gallusX laevis

S salar




B taurusC lupus

G gallus

G gallus

S salar

S salar






C lupus

X laevis

X laevis

D rerio


S scrofa (boar)

S scrofa (boar)

S scrofa (boar)

O latipes (medaka)

O latipes (medaka)

O latipes (medaka)

O mykiss (trout)

O mykiss (trout)

O mykiss (trout)






PPAR Research 7


















8 PPAR Research


5 Conclusions




Acknowledgment


References



















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 3



PPARA

Endogenous


4) [68 69]






PPARG


Synthetic








4 PPAR Research

(a)

(d)

(b)

(c) (c998400)




Adipogenic factors

RXR

LXRGATA

23

Krox20


Ligands



SREBP1c

Wnt genes

catenin

EZH2

Sirt1 PGC1aFOXO1

Klf5

120573-

CEBP120572

CEBP120573

CEBP120575

PPARD

PPARG


PPAR Research 5









6 PPAR Research

006

PPARD

Birds

M musculus

Birds

Birds

Amphibians

Amphibians

Amphibians

Fish

Mam

mal

s

Mam

mal

s

Mam

mal

s

Fish

Fish

PPARG

PPARA


B taurusC lupus

G gallusX laevis

S salar




B taurusC lupus

G gallus

G gallus

S salar

S salar






C lupus

X laevis

X laevis

D rerio


S scrofa (boar)

S scrofa (boar)

S scrofa (boar)

O latipes (medaka)

O latipes (medaka)

O latipes (medaka)

O mykiss (trout)

O mykiss (trout)

O mykiss (trout)






PPAR Research 7


















8 PPAR Research


5 Conclusions




Acknowledgment


References



















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 PPAR Research

(a)

(d)

(b)

(c) (c998400)




Adipogenic factors

RXR

LXRGATA

23

Krox20


Ligands



SREBP1c

Wnt genes

catenin

EZH2

Sirt1 PGC1aFOXO1

Klf5

120573-

CEBP120572

CEBP120573

CEBP120575

PPARD

PPARG


PPAR Research 5









6 PPAR Research

006

PPARD

Birds

M musculus

Birds

Birds

Amphibians

Amphibians

Amphibians

Fish

Mam

mal

s

Mam

mal

s

Mam

mal

s

Fish

Fish

PPARG

PPARA


B taurusC lupus

G gallusX laevis

S salar




B taurusC lupus

G gallus

G gallus

S salar

S salar






C lupus

X laevis

X laevis

D rerio


S scrofa (boar)

S scrofa (boar)

S scrofa (boar)

O latipes (medaka)

O latipes (medaka)

O latipes (medaka)

O mykiss (trout)

O mykiss (trout)

O mykiss (trout)






PPAR Research 7


















8 PPAR Research


5 Conclusions




Acknowledgment


References



















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 5









6 PPAR Research

006

PPARD

Birds

M musculus

Birds

Birds

Amphibians

Amphibians

Amphibians

Fish

Mam

mal

s

Mam

mal

s

Mam

mal

s

Fish

Fish

PPARG

PPARA


B taurusC lupus

G gallusX laevis

S salar




B taurusC lupus

G gallus

G gallus

S salar

S salar






C lupus

X laevis

X laevis

D rerio


S scrofa (boar)

S scrofa (boar)

S scrofa (boar)

O latipes (medaka)

O latipes (medaka)

O latipes (medaka)

O mykiss (trout)

O mykiss (trout)

O mykiss (trout)






PPAR Research 7


















8 PPAR Research


5 Conclusions




Acknowledgment


References



















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 PPAR Research

006

PPARD

Birds

M musculus

Birds

Birds

Amphibians

Amphibians

Amphibians

Fish

Mam

mal

s

Mam

mal

s

Mam

mal

s

Fish

Fish

PPARG

PPARA


B taurusC lupus

G gallusX laevis

S salar




B taurusC lupus

G gallus

G gallus

S salar

S salar






C lupus

X laevis

X laevis

D rerio


S scrofa (boar)

S scrofa (boar)

S scrofa (boar)

O latipes (medaka)

O latipes (medaka)

O latipes (medaka)

O mykiss (trout)

O mykiss (trout)

O mykiss (trout)






PPAR Research 7


















8 PPAR Research


5 Conclusions




Acknowledgment


References



















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 7


















8 PPAR Research


5 Conclusions




Acknowledgment


References



















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 PPAR Research


5 Conclusions




Acknowledgment


References



















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 9


































10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















10 PPAR Research


































PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 11













of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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