review article fishing for nature s hits: establishment of...

Review ArticleFishing for Naturersquos Hits Establishment of the Zebrafish asa Model for Screening Antidiabetic Natural Products

Nadia Tabassum1 Hongmei Tai2 Da-Woon Jung1 and Darren R Williams1

1New Drug Targets Laboratory School of Life Sciences Gwangju Institute of Science and Technology Gwangju 500-712Republic of Korea2Department of Endocrinology Yanji Hospital Jilin 133000 China

Correspondence should be addressed to Da-Woon Jung junggistackr and Darren R Williams darrengistackr

Received 31 August 2015 Accepted 28 October 2015

Academic Editor Attila Hunyadi

Copyright copy 2015 Nadia Tabassum et alThis is an open access article distributed under theCreativeCommonsAttributionLicensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Diabetes mellitus affects millions of people worldwide and significantly impacts their quality of life Moreover life threateningdiseases such asmyocardial infarction blindness and renal disorders increase themorbidity rate associated with diabetes Variousnatural products frommedicinal plants have shown potential as antidiabetes agents in cell-based screening systems However manyof these potential ldquohitsrdquo fail in mammalian tests due to issues such as poor pharmacokinetics andor toxic side effects To addressthis problem the zebrafish (Danio rerio) model has been developed as a ldquobridgerdquo to provide an experimentally convenient animal-based screening system to identify drug candidates that are active in vivo In this review we discuss the application of zebrafishto drug screening technologies for diabetes research Specifically the discovery of natural product-based antidiabetes compoundsusing zebrafish will be described For example it has recently been demonstrated that antidiabetic natural compounds can beidentified in zebrafish using activity guided fractionation of crude plant extracts Moreover the development of fluorescent-taggedglucose bioprobes has allowed the screening of natural product-based modulators of glucose homeostasis in zebrafish We hopethat the discussion of these advances will illustrate the value and simplicity of establishing zebrafish-based assays for antidiabeticcompounds in natural products-based laboratories

1 Introduction

Over the past three decades the prevalence of diabetesmellitus (DM) has dramatically increased in both developingand developed countries affectingmillions of adults and thisnumber is expected to increase to approximately 439 millionadults by 2030 [1] DM is a heterogeneous group of metabolicdisorders that occurs either due to resistance of the body torespond to insulin or due to the insufficient production ofinsulin in the pancreas both of which cause elevated levelsof blood sugar (hyperglycemia) [2] Prolonged hyperglycemiacauses severe and potentially fatal complications such ascardiovascular disease nephropathy and retinopathy [3]Numerous antidiabetes drugs have been developed butthey cause side effects of varying severity such as nauseaweight gain or cardiovascular issues [4] Since ancient timesherbal medicines have also been used to treat diabetes indifferent cultures (example reviews are [5 6]) The bioactive

natural products isolated fromherbalmedicines have been animportant source of novel drugs for various diseases [7] Forexample between 1981 and 2002 almost half of the 877 smallmolecule New Chemical Entity (NCE) therapeutics werenatural products or their synthetic derivatives [8] Moreoverapproximately 50 of all new commercially available drugsare developed from natural products [7] Natural prod-ucts are an attractive starting point for the drug discoveryprocess because many possess structural complexity thatcannot be achieved using chemical synthesis In additionsynthetic analogues of natural products can be developedwhich possess superior safety and efficacy profiles [9 10]However over the past two decades natural products-baseddrug discovery by pharmaceutical companies has largelywaned This is because natural products are generally notsuitable for large scale drug screening protocols comparedto synthetic compounds Costs and technical difficultiesassociated with the isolation purification and quality control

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 287847 16 pageshttpdxdoiorg1011552015287847

2 Evidence-Based Complementary and Alternative Medicine

of novel natural products also preclude their use in the drugdiscovery industry However natural products-based drugdiscovery continues to flourish in academia A recent exam-ple is the development of the anticancer drug ecteinascidin743 (from the sea squirt Ecteinascidia turbinate) (Figure 2)which obtained clinical approval to treat metastatic softtissue sarcoma [10] Therefore the development of powerfulhigh-content screening assays for natural products wouldcomplement their established advantages as candidates fordrug discovery

Over the past two decades major progress has beenachieved in the development of screening technologies suchas high throughput screening (HTS) [11] and chemical syn-thesis strategies such as diversity orientated synthesis [12]However the number of licensed compounds obtainedagainst novel drug targets is considerably low (just two tothree compounds per year) Moreover a single pharmaceu-tical company can spend m100ndash500million annually for inves-tigating 30ndash50 targets [13] and a new compound typicallyrequires 12 years to enter the drug market [14] There arenumerous reasons to account for this low number of newdrugs such as increasingly rigorous safety evaluations andthe reduced efficiency of target-based discovery (known asEroomrsquos Law) [12] Another major bottleneck in the drugdiscovery process is the failure of promising candidate com-pounds in animal tests due to issues related to absorptionsolubility metabolic stability distribution andor toxicityThese failures also arise because cell-based screening systemscannot recapitulate the physiological interactions that arecrucial in the evolution of some disorders such as metabolicdiseases [15] Another significant reason for the rejection ofcandidate drugs is that discovery methods rely mainly on theexistence of identifiable and ldquoscreenablerdquo targets [16] Thislate-stage attrition of drugs in development is highly costlyand time consuming for pharmaceutical companies Thesedrawbacks of target driven drug discovery have inducedresearchers to screen compounds including natural prod-ucts in whole animal systems to identify new targets andultimately successful drugs In the next section of this reviewwe compare conventional screening systems with animal-based approaches and introduce the zebrafish as an idealmodel system for in vivo drug discovery applications

2 Development of Animal-Based ScreeningSystems for Drug Discovery

The mouse (Mus musculus) is the most widely used animalmodel in biomedical research and can be considered as theldquowork horserdquo model organism [28] It possesses numerousadvantages for research purposes such as sharing 90genome homologywith humans in addition tomany geneticphysiological and organ anatomical similarities Anotherimportant advantage is that genetic manipulation in themouse can be used to model the action of potential drugs viathe knockdown of candidate target genes Thus the mouse isan invaluable model for the discovery of new targets for ther-apeutic interventions [29] However from the perspectiveof drug screening the mouse has significant disadvantages

restricting its use in the pharmaceutical field The majorproblem is the expense involved in maintaining large mousecolonies for compound screening There are also biologicalissues For example transgenic animals that are used tomodel disease may not be suitable for screening becauseof the potential initiation of compensatory gene expressionmechanisms during development This could be problematicfor discovering compounds that should be active in humans[16] Consequently alternative animal-based platforms forscreening have been investigated Two prominent examplesare invertebratemodels the fruit flyDrosophilamelanogasterand the roundworm Caenorhabditis elegans [30 31] Despiteoffering certain advantages such as applicability to HTSand the preservation of some physiological contexts thatare similar to humans these invertebrate models have notbecome widely used for drug screeningThis is because theseinvertebrates have markedly reduced genetic homology withhumans compared to the mouse In addition some of themajor organ systems present in humans such as respiratoryand intravascular circulatory systems are not present ininvertebrates [32] Another issue is the thick cuticle whichaffects drug penetration and complicates the interpretationof screening data (this is especially relevant for C elegans)[33 34] Thus the clinical potential of novel compoundsdiscovered in invertebrates may not be sufficient to justifyscreening in these animal systems

3 Zebrafish Swimming into Placein the Drug Discovery Field

These above-described limitations on mammalian and inver-tebrate models for compound screening provided impetusto develop alternative animal model systems for studyingdrug responses Consequently the zebrafish (Danio rerio) hasrisen to prominence The zebrafish is a small fresh waterteleost that was developed as an animal model in the 1980sfor the study of developmental biology and embryogenesis[36 37] A set of landmark studies published together ina single issue of the journal Development (1996) helpedto establish this animal model for characterizing vertebratedevelopmental pathways Further studies and the productionof gene targeting techniques such as anitisense morpholinosand the generation of transgenic fish enabled zebrafish tobecome an attractive model for studying human disease [3839] Critically the logistical advantages of using zebrafishhave also convinced the research community to adopt thisanimal model These advantages are listed as follows

(1) In contrast to rodents zebrafish embryos are opticallytransparent and zebrafish development is externalwhich permits direct observation of embryonic organsystems under a wide variety of laboratory conditions[40] For its size zebrafish embryos are relativelylarge which assists their handling in the laboratory

(2) Embryonic development is very rapid compared tomammals The entire body plan is established by24 hours after fertilization (hpf) and most majororgan systems such as the digestive tract and thecardiovascular system are developed 2 days after

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Comparison of different animal models for screening antidiabetic compounds

Invertebrates Zebrafish Rodents Large mammals (eg dogsand rabbits)

Silkworms can developdiabetes mellitus when fed aglucose-rich diet [17]

They can induce DM bysimple immersion in highglucose water [18]

Developed diabetes mellituswithin a few days by chemicalinjection or after a few weeksvia feeding a high-fat diet [19]

Developed diabetes mellitusby removing their pancreas[20]

Primary screening ofantidiabetic drugs is possible[21]

High throughput screening forantidiabetic compounds ispossible (eg [22])

High throughput screening forantidiabetic compounds is notpossible

High throughput screeningfor antidiabetic compoundsis not possible

Requiring less time to developdiabetes mellitus compared tosome mammalian models

They require less time forscreening and less amount oftest compound relative tomammalian models

Time consuming as it may takeseveral days to develop diabetesmellitus via feeding [23]

They take several days todevelop diabetes mellitus[23]

Inexpensive to useinvertebrate models and lesslogistical requirementscompared to mammals

Inexpensive and easier tohandle compared tomammalian models

Expensive and harder to handledue to relatively large size(compared to fish orinvertebrates)

Expensive andlogisticalhandling problemsdue to large size

Reduced ethical issuescompared to mammalianmodels

Both adult and larval zebrafishare suitable for screeningstudies

Some ethical issues dependingon country of use (eg securedhousing required in UK orUSA) [24]

Ethical issues as anexperimental model

Amenable tofluorescence-based imagingand quantification of glucoseuptake [25]

Fluorescence imaging ofwhole organism is possible forglucose uptake analysisdiabetes-related reporter genebased screening is alsopossible [26]

Mouse are amenable forfluorescence tracer-basedimaging of whole-body insulinsensitivity and hepatic glucoseproduction [27]

Fluorescent-based imagingof glucose homeostasis notpossible

fertilization (dpf) Complete embryogenesis (hatch-ing) occurs by 72 hpf Therefore several differentbiological processes andor disease mechanisms canbe analyzed during these early developmental stages(Figure 1) [41]

(3) The small size of the zebrafish embryo (5mm at7 dpf) relatively low cost for maintenance comparedto rodents and high fecundity (a single female can layup to 200 eggs per week) make screening possible in96- or 384-well microtiter plates [42 43]

(4) It is now established that drugs against certain organsor tissues that differ or are absent in humans can alsobe discovered or tested in zebrafish For example thedrug rosuvastatin (Figure 2) used to treat prostatecancer was identified by zebrafish chemical screeningeven though male zebrafish do not possess a devel-oped prostate gland only-prostate-like cells [44]

(5) In contrast to insect models for drug screeningzebrafish are vertebrates and possess higher genetichomology with humans (approximately 80 com-pared to the fruit flyDrosophilamelanogaster (sim60)and the roundworm Caenorhabditis elegans (sim36))[13 45 46]

All of the characteristics listed above make zebrafish anideal model system for drug discovery efforts (Figure 1)A comparison of the advantages of zebrafish-based drugtesting approaches in relation to invertebrates and mammals

in the context of diabetes research is shown in Table 1It should also be noted that another prominent small teleostfish model has been developed the medaka (Japanese ricefish Oryzias latipes) [47 48] Medaka possesses certainadvantages for laboratory use compared to zebrafish such as asmaller genome andmore rapid development time Howeverzebrafish is the more established fish model and have beenshown to possess some significant physiological differencescompared to medaka such as greater cardiac regenerationafter injury [49] The potential of zebrafish for diabetes-related drug screening approaches have been realized overthe past decade with multiple strategies developed such asvisual assessment of glucose uptake or quantitative analysisof glucose homeostasis using the microplate format Thesedevelopments and their application for natural products-based screening of antidiabetic compounds are described inthe next section of this review

31TheDevelopment of Zebrafish-Based Screening for Antidia-betes Natural Products Interestingly the first use of zebrafishlarvae to screen natural products and synthetic compoundsformodulators of cell physiology was reported over fifty yearsago [50] Surprisingly this research was largely ignored untilthe 1990s In this decade combinatorial synthesis of smallmolecule libraries was developed allowing the production ofcompound libraries comprising thousands of smallmolecules[51] This progress necessitated the requirement for rapidand efficient screening systems for bioactive compounddiscovery As described in the previous section of this review


3 cm

(a)

EyeOtolith (ear)

HeartLiver

Intestinaltract

Ventralfin

Cloaca

Caudalhematopoieticregion

Muscle

Tailfin

(b)

(1) Primaryscreening to

identify candidateldquohitrdquo compounds

(2) Drug targetidentification

and mechanisticstudies

(3) Toxicologicalanalysis

(4) Assessing

effects

(5) Preclinicalinvestigation in

diseasesmodels

Noveldrugldquooff-targetrdquo

(c)

Figure 1 (a) An adult zebrafish (b) Embryonic development of zebrafish is rapid with the major organ systems such as nervouscardiovascular and digestive tissues being formed within 36 hours of fertilization [35] (image adapted from Wikimedia and used underthe Creative Commons Attribution-Share Alike 40 International license) (c) The zebrafish model can facilitate multiple steps of the drugdiscovery process

the pharmaceutical research community realized that ananimal-based screening system with the potential for highthroughput screeningvalidation could greatly assist drugdiscovery Consequently the zebrafish model was developedfor compound screening based on numerous experimentalldquoreadoutsrdquo such as developmental phenotypes [24] pigmen-tation modulation [52ndash54] tissue regeneration modulation[55] and radiation sensitization [56] The development ofzebrafish-based antidiabetes compound screening is basedupon the discovery of marked similarities in glucose home-ostasis with mammals These similarities which justify theuse of zebrafish to screen for antidiabetic compounds areoutlined below

32 Zebrafish Can Be Used to Model Pancreatic Beta Cell Neo-genesis Diabetes type I is characterized by the destruction ofinsulin producing beta cells in the pancreatic islets produc-ing insulin deficiency and resultant hyperglycemia Insulinsecretion from beta cells is induced by increases in bloodglucose level after meal times (postprandial) Dysregulatedhyperglycemia causes deleterious effects onmany organs andtissues such as the heart kidney and retina [2] Interestinglyunder certain physiological conditions such as increasedmetabolic demand 120573 cell mass can increase via three mech-anisms (i) differentiation of resident precursor cells (ii)transdifferentiation of other pancreatic cell types and (iii)120573 cell proliferation [57] Thus inducing 120573 cell neogenesis


NHO

HO

S

NO

O

OOO

N

OH

HOO

Ecteinascidin 743

NN

F

NSO

O

HO

OHOHO

Rosuvastatin

N NN

O

NN

Cl

Trazodone

OH HNHO

HO

Isoprenaline

NN

O

Cl

PK 11195

N

N

Cl

O

ClRo5-4864

HO

HOH

H

HO

OH

Maslinic acid

OHO O

N

O

OO

Ampkinone

OF

HHO

H

OOH

OH

Dexamethasone

HNNH

O SO O

O

N

N

Glipizide

NH

OOHO

O

O

Fraxidin

OHOHO

OH

OH O N NO

N

GB2-Cy3

N+

O

Trigonelline

N+

Ominus

OHO

Retinoic acid

H3CCH3CH3

CH3

CH3OHN

NO

N

HO

HO

OHOH

2-NBDG

NO2

N

NH NH

Metformin

NH

NH2

PP-II-A03

Cl

HN NNN

NH

NH

HN

OO

ONH2

N SS

S N

S

Disulfiram

H3CH3C

OH O

O

OH

OH

Emodin

CH3

NNN

N

OHO

HO

HN O

midoadenosine5998400-N-ethylcarboxa-

NH2

F

F

O

NH

HN

OO

O

Ph

DAPT

O

O

OHOHOH

HO

HO

Chlorogenic acid

CO2H

O

O

HO

HO

OH H

H

OHH

H

HO

cpp532

CH2OHNN N

NO

O

Caffeine

H3CCH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

Figure 2 Chemical structures of compounds discussed in this review


in type 1 diabetic patients could be a promising therapeuticstrategy Moreover inducing 120573 cell transdifferentiation fromother cell types could provide a cell source for transplantationinto diabetic patients [58 59]

Traditionally the mouse has been used as a model tostudy the restoration of120573 cellmassHowever recent studies inzebrafish have shown its potential as a model of mammalian120573 cell regeneration In addition pancreas development andregeneration can be studied in transparent zebrafish embryos[60]

Despite the small size of this tropical fish its pancreaticstructure is highly similar to mammals and is also comprisedof two types of glandular tissues exocrine and endocrineMany pancreatic developmental genes and hormones thatregulate glucose metabolism in zebrafish such as insulinand glucagon resemble those of mammals [61 62] Forexample the well-characterized early marker of pancreaticdevelopment pdx-1 (pancreatic and duodenal homeobox-1)is conserved between mammals and zebrafish [63] Bothzebrafish and mammalian embryos share the positive regu-latory relationship between hedgehog and pdx-1 in pancreasprecursor cell specification [64] In a related study it wasshown that perturbation of pancreatic genes in the zebrafishproduced phenotypes that closely resembled the equivalenthuman disease For examplemutations of the nuclear proteinvhnf1 (tcf2) are associated with maturity-onset diabetes of theyoung type V (MODY5) Vhnf1 mutant zebrafish embryosdisplayed developmental defects in the pancreas and the liveralong with the formation of kidney cysts which are alsofound in MODY5 patients [65 66] Interestingly the bloodglucose concentration of adult zebrafish is close to the humanphysiological level (50ndash75mgdL and 100mgdL resp) [367]

These similarities between mammalian and zebrafishbeta cells provided research impetus to develop compoundscreening systems for 120573 cell neogenesis For example trans-genic fish expressing fluorescent-tagged nfsB (dihydropteri-dine reductase expressed in the embryonic pancreas) weregenerated to allow visualization of drug-dependent pancre-atic cell ablation The potential of this system for image-based compound screening was validated using the prodrugmetronidazole which is known to be cytotoxic for beta cells[68] Recently Rovira et al utilized a transgenic zebrafishmodel to carry out screening of the Johns Hopkins DrugLibrary (consisting of around 1500 FDAand foreign approveddrugs) for compounds that promote 120573 cell differentiationTheir moderate-throughput screening system used multiwellplates to permit visualization of pancreatic cells in livinglarvae and led to identification of three FDA approveddrugs that induce significant 120573 cell differentiation (1) disul-firam (2) DAPT (N-[N-(35-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) and (3) the fungal-derivednatural product mycophenolic acid (Figure 2) [69] In arelated study Andersson et al used the zebrafish screeningplatform to discover compounds that induce 120573 cell regen-eration [70] A library of approximately 7000 compounds(including FDA approved drugs and natural compounds)was screened in a transgenic zebrafish model showing 120573cell specific expression of nitroreductase [Tg(insCFP-NTR)]

which converts metronidazole (MTZ) into a cytotoxic prod-uct that specifically kills beta cells After the removal oftested compounds larvae showed reconstitution of the betacell mass which could be quantified by microscopy Themost promising enhancer of 120573 cell regeneration was found tobe NECA (51015840-N-ethylcarboxamidoadenosine an adenosineanalogue) NECA was further tested in a mouse model ofdiabetes which validated its ability to induce 120573 cell regen-eration in mammals

Until recently drug discovery for compounds that induce120573 cell proliferation did not attract significant attention frompharmaceutical companies due to the poorly understood sig-naling pathways regulating this process Recently a zebrafish-based screeningmodel has demonstrated its utility for screen-ing to identify inducers of beta cell proliferation Tsuji etal developed an in vivo imaging approach that utilized afluorescent ubiquitylation-based cell cycle indicator 20 smallmolecules among 883 were identified as having the abilityto induce beta cell proliferation in zebrafish Among theseretinoic acid (the metabolite of vitamin A) and the antide-pressant trazodone (Figure 2) have already been shown toincrease mammalian beta cell proliferation indicating thatzebrafish screening can detect bioactive molecules that alsofunction in theirmammalian counterparts (Figure 3) [71 72]Of note it has also been shown that various active com-ponents from coffee (caffeine trigonelline and chlorogenicacid) can also induce beta cell regeneration in alloxan-treatedzebrafish [73] In this study beta cells were visualized usingInsGFP transgenic fish or by simply treating fish with afluorescent-tagged glucose tracer

Interestingly it is now possible to model the effects ofinsulin resistance on beta cell numbers using the zebrafishsystem Transgenic fish were generated that overexpressedadominant-negative version of the insulin-like growth factor-1 receptor [6] Young fish show normal glucose tolerancebecause they respond to insulin resistance by inducingbeta cell proliferation However in older fish this responsebecame less effective and beta cell numbers decreased whichproduced insulin resistance Thus it can be envisaged thatantidiabetics drug candidates that target insulin resistancecan be assayed using these transgenic zebrafish

33 Zebrafish as a Model Animal for the Quantitative Analysisof Glucose Homeostasis For effective diabetes drug discoveryresearch using zebrafish it would be desirable to study bloodglucose regulation in this model and correlate its relationshipwith the humanmetabolic system Although the small size ofzebrafish embryos precludes the collection of blood samplesfor measuring glucose level this is possible in adult zebrafishFor example methods for the microsampling of whole bloodand plasmahave been developed formeasuring blood glucosein fasting and refed zebrafish [67] To measure glucose levelsin zebrafish embryos which are becoming suitable for highthroughput screening [74 75] Jurczyk et al developed afluorescent dual enzyme assay to detect free glucose in theembryos [76] This technology demonstrated that zebrafishpancreatic islets produce a regulatory glucose system at anearly developmental stage (48 hpf) In addition targeting


Tg(insH2BGFP) Tg(insCFP-NTR)

MTZ(ablation)

Drug treatment(regeneration) Analysis

50 80 128 (hpf)

(a)

(b)

Num

ber o

f bet

a cel

ls

DMSO Retinoic acid Trazodone Prednisolone

lowast

lowast16

14

12

10

8

6

4

2

0

(c)

Figure 3 (a) Schematic diagram for zebrafish-based screeningof compounds that promote beta cell regeneration Transgenicfish expressing GFP in the beta cells (insH2BGFP) were bredwith transgenic fish that express nitroreductase specifically intheir beta cells Tg(insCFP-NTR) Beta cells were ablated usingMTZ treatment from 50 to 80 hpf At 80 hpf Tg(insH2BGFP)Tg(insCFP-NTR) larvae were treated with the compounds for 48 hThe numbers of Tg(insH2BGFP) + beta cells were counted at128 hpf (b) Microscopic images of Tg(insH2BGFP) + beta cells in128 hpf larvae treated with 1 120583M retinoic acid 10 120583M trazodone or10120583M prednisolone dissolved in 1 DMSO (c) Quantification ofbeta cell regeneration per larva at 128 hpf following treatment withhit compounds from 80 to 128 hpf Error bars represent SEM lowast119875 lt005 compared to DMSO treated controls Image reproduced from[71] under the Creative Commons Attribution (CC BY) license

zebrafish pdx-1 gene expression during embryogenesis pro-duced islet hypoplasia and persistent hyperglycemia Thisresult correlateswith previous studies reporting that impairedpdx-1 activity causes defective pancreas development inhumans [77 78]

The regulatory mechanisms controlling blood glucoselevels in zebrafish also sharemany similaritieswithmammalsElo et al tested three FDA approved antidiabetic drugsglipizidemetformin and rosiglitazone (Figure 2) in zebrafish

[79] 96 hpf larvae were exposed to cAMP and dexametha-sone to activate zebrafish phosphoenolpyruvate carboxyki-nase (zfPEPCK) which regulates blood glucose levels viagluconeogenesis in the liver and kidneys ofmammals [80 81]Well-known antidiabetes drugs such as metformin down-regulate PEPCK expression and this enzyme is used as a read-out in mammalian cell culture models to check the efficacy ofantidiabetic compounds [26 82] Glipizide metformin androsiglitazone all successfully inhibited cAMPdexamethasoneactivation of zfPEPCK expression even after treatment withrelatively small doses such as 1 120583M for rosiglitazone Thesefindings were significant for zebrafish-based antidiabetesdrug discovery because it indicated that hypoglycemia-inducing drugs which function via PEPCK inhibition couldbe detected using zebrafish larvae which are amenablefor 96-well plate format screening [79] Subsequent to thisstudy that was based on measuring zfPEPCK expressionvia quantitative RT-PCR Gut et al engineered transgeniczebrafish with a luciferase luminescent PEPCK reporter toutilize larvae in aHTS platform [22] To validate their screen-ing system two known modulators of blood glucose levelin humans were tested metformin which induces hypo-glycemia and isoprenaline which induces hyperglycemia(Figure 2) [83ndash85] Isoprenaline and metformin stronglyinduced or reduced PEPCK reporter expression in zebrafishlarvae respectively Glucose levels in the larvae were alsoreduced by metformin and this reduction could be overcomeby cotreatment with isoprenaline 2400 compounds werescreened (a collection of natural compounds FDA approveddrugs and other bioactive chemicals) 60 compounds wereidentified as modulators of PEPCK expression Interestinglytwo of the most prominent hit compounds the translocatorprotein (TSPO) ligands PK 11195 and Ro5-4864 decreasedglucose levels in the larvaewhile producing increased PEPCKexpression In a mammalian model of diet-induced obesitythese compounds reduced blood glucose level intoleranceand inhibited the development of hepatosteatosis (fatty liverdisease)Thus compounds that modulate PEPCK expressionin larval zebrafish are drug candidates for treating metabolicdiseases such as diabetes in mammals

An ideal approach for detecting compounds that affectglucose homeostasis in zebrafish would be to directly visual-ize glucose flux in vivo Fluorescent-tagged glucose bioprobeshave been used to visualize glucose uptake in cells (reviewedin [86]) Lee et al utilized these probes to develop a screeningsystem for measuring glucose flux in zebrafish larvae [26]This screening system is relatively simple because it is appli-cable for wild type zebrafish (transgenic fish may be subjectto quarantine controls) and glucose flux is measured usingthe commercially available probe 2-(N-(7-nitrobenz-2-oxa-13-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG Figure 2)Glucose flux in 72 hpf zebrafish larvae could be assessedby fluorescent microscope analysis of the zebrafish eyewhich expresses relatively high levels of glucose transporter(GLUT) proteins [87] Their screening system was validatedusing the natural product purgative resin emodin (6-methyl-138-trihydroxyanthraquinone Figure 2) which is a knowninducer of glucose uptake [88] Antidiabetic compounds thatinduce glucose uptake could also be quantified by lysing


UP423 UP521 UP32 UP33 UP523

UntreatedUP methanol

fraction

UP ethylacetatefraction

UP hexanefraction

0

20

40

60

80

100

Untreated Methanol Ethylacetate

Hexane

Fluo

resc

ence

inte

nsity

Fraction tested

EmodinUntreated UP22 UP3111 UP353

020406080

100120140

Untreated Emodin UP22 UP353 UP423 UP521 UP33 UP523

Fluo

resc

ence

inte

nsity

UP32(fraxidin)

UP3111(maslinic acid)

Compound treatment

Activity guided fractionation for antidiabetic compounds using zebrafish

(a)

(b)

Figure 4 Activity guided fractionation of the inner shell of the Japanese chestnut tree Castanea crenata using the fluorescent probe 2-NBDG in zebrafish larvae (a)Themethanol fraction produced significant glucose uptake in zebrafish compared to the hexane or ethyl acetatefraction The red line on the graph indicates the threshold for selecting a ldquohitrdquo drug (ie 2-NBDG uptake value for the zebrafish eye shouldshow a ge100 increase compared to that of the untreated larvae) (b) The methanol fraction was purified to isolate eight compounds UP22(scopoletin 4) UP3111 (maslinic acid) UP353 (fragransin) UP423 (4-ketopentanoic acid) UP521 (4-hydroxy-5-methoxycinnamic acid)UP32 (fraxidin) UP33 (678-trimethoxycoumarin) andUP523 (345-trimethoxycinnamic acid)These compoundswere tested for glucoseuptake in the zebrafish (10120583gmL dose for 1 h) and compared with emodin (a known inducer of glucose uptake) Fraxidin and maslinic acidwere identified as hit compounds for inducing glucose uptake (figure modified from [26])

the larvae and measuring 2-NBDG fluorescence in a micro-plate reader Interestingly the applicability of this screeningsystem for natural products-based research was demon-strated using activity guided fractionation of compoundsfrom the inner shell from the Japanese chestnut tree (Cas-tanea crenata) (shown in Figure 4)The strongest performingcompound was identified as fraxidin which had no pre-viously reported antidiabetic activity This compound was

confirmed as novel insulinmimetic with activity inmammalsvia testing in a mammalian adipocyte system The knownantidiabetic compound maslinic acid (Figure 2) [89] wasalso identified in this study Additionally screening of acollection of saponin based natural products isolated fromKorean ginseng (Panax ginseng) allowed identification ofa novel antidiabetic compound termed cpp532 (Figure 2)Visualizing glucose homeostasis via fluorescent probe uptake


GB2 alone

Ampkinone + GB2

Rosiglitazone + GB2

Zebrafish-based screening for antidiabetic compounds using the probe GB2-Cy3

(a)

(b)

(c)

lowast

lowast

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Control1 DMSO

Ampkinone10120583M

Rosiglitazone10120583gmL

Drug treatment

90

80

70

60

50

40

30

20

10

0

20

19

18

17

16

15

14

13

12

11

10Control

1 DMSOAmpkinone

10120583MRosiglitazone10120583gmL

Drug treatment

lowastlowast

Figure 5 The fluorescent probe GB2-Cy3 can be used to test candidate antidiabetic compounds in zebrafish (a) The known antidiabeticcompounds ampkinone (10120583M) and rosiglitazone (10 120583gmL) increased probe uptake in larval zebrafish From a screening perspective probeuptake can be readily quantified by fluorescentmicroplate reader analysis of lysed larvae forGB2-Cy3 uptake (b) or image-based quantificationof fluorescent signal from the zebrafish eye (lowast=119901 lt 005 compared to the control group) (figure reproduced with permission of the RoyalSociety of Chemistry from [92])

in the eye is the first demonstration that glucose flux in azebrafish-based system can bemonitored directly and offers anovel approach for antidiabetic drug discovery [26] Interest-ingly the 2-NBDGglucose probe has also shown applicabilityfor visualizing glucose uptake by insulin sensitive tissues inmice [27 86]

Unfortunately the widely available 2-NBDG glucoseprobe does have certain disadvantages that may restrict itsuse for visualizing glucose homeostasis in vivo For example2-NBDG requires a high treatment concentration (600120583Mfor zebrafish) suffers from rapid photobleaching and hasrelatively low sensitivity compared to recently developed glu-cose probes which possess stronger fluorophores such as Cy3[90] To further optimize the zebrafish larvae-based screen-ing system for antidiabetes drug discovery the 2-NBDGprobe was replaced with GB2-Cy3 (Figure 2) [91 92] Cell-based analyses had previously shown the superior imaging

properties of GB2-Cy3 compared to 2-NBDG [93] Inzebrafish larvae it was shown that GB2-Cy3 is approximatelytenfold more sensitive for monitoring glucose flux and couldbe used at a treatment concentration as low as 5120583M(120 timeslower than 2-NBDG) [92] The sensitivity of GB2-Cy3 fluo-rescence to known modulators of glucose homeostasis wasdemonstrated using the natural product emodin (Figure 2)Additionally two other known antidiabetic drugs were testedto validate the GB2-Cy3 probe in this zebrafish systemampkinone and rosiglitazone (Figure 5) [94] These resultsindicate that zebrafish larvae-based in vivo screening forantidiabetic natural products using the probeGB2-Cy3wouldbe more experimentally robust compared to screening basedon the 2-NBDG probe However it should be noted that testscreening such as the activity guided fractionation approachdescribed for 2-NBDG (Figure 4) was not attempted forthe GB2-Cy3 probe In addition unlike 2-NBDG GB2-Cy3


is not commercially available at this time which restrictsthe suitability of this probe for use by the natural productsresearch community

34 Candidate Antidiabetic Compound Validation inZebrafish-Based Models of Diabetic Complications As men-tioned in the introduction of this review the zebrafish offersnumerous logistical and technical advantages for the assess-ment of new drug candidates prior to preclinical analysisin rodent-based models In the context of antidiabetes drugdiscovery zebrafish that manifest the secondary compli-cations of diabetes such as kidney disease or retinopathywould be a valuable resource for simple initial validationof ldquohitrdquo compounds identified by screening In humansdiabetes produces numerous debilitating complications suchas neuropathy nephropathy retinopathy cardiovascular dis-eases peripheral artery disease stroke periodontal diseaseand increased susceptibility to opportunist infections[95 96] A number of mammalian models of type 2 diabetespresent secondary complications that are observed in humans(reviewed in [97 98]) Interestingly zebrafish models ofdiabetic complications have been developed which allowexperimentally convenient assessment of novel antidiabeticcompounds in a vertebrate system

A significant advantage of using zebrafish for diabetesresearch is that hyperglycemia can be induced by simplyadding glucose to the fish water (eg [99]) In contrastrodent models of diabetes typically require the injectionof the toxic glucose analogues streptozotocin or alloxanwhich preferentially kill pancreatic 120573 cells However theseanalogues also produce significant side effects For examplestreptozotocin is tumorigenic in the kidney lung and liverand alloxan produces liver and kidney necrosis as a byproductof its metabolism [100]

It has been shown that diabetic retinopathy can be mod-elled in zebrafish [18] Over a 4-week period the fish wereimmersed in a 2 or 0 glucose solution alternatingbetween the two solutions every 24 h which was shownto induced hyperglycemia spikes (0 versus 2 glucose)After 4 weeks the eyes were dissected and assessed bymicroscopic examination Fish exposed to high glucosewater presented decreased thickness of the retinal innerplexiform layer (IPL) and inner nuclear layer (INL) whichis also observed in diabetes patients By comparison theseretinal layers in diabetic rats rendered by streptozotocintreatment showed less thinning compared to hyperglycemiazebrafish [3] A subsequent study based on the same methodto induce hyperglycemia reported that thickened dilatedblood vessels were present in the central region of theretina which resembles the pathophysiology observed inhuman patients [101 102] During the proliferative stageof diabetic retinopathy the cytokine vascular endothelialgrowth factor (VEGF) is upregulated and induces bloodvessel cell proliferation (angiogenesis) Upregulated VEGFexpression was also observed in the diabetic zebrafish Thisprovides a relative straightforward model system to testnovel antidiabetic compounds for preventative effects on theprogression of retinopathy becauseVEGFhas been identifiedas a promising drug target for this complication [103]

Cardiovascular issues such as coronary heart disease andstroke are major complications of diabetes [104] Exposureof 6 hpf zebrafish embryos to 05 glucose water until 24 hpfproduced defective cardiac development and altered expres-sion of major cardiac markers Therefore the potential fornovel antidiabetic compounds to protect against cardiovas-cular complications could be tested in this zebrafish system[105] Kidney nephropathy is another major complication ofdiabetes and is a major cause of dialysis in developed coun-tries [106] Prolonged hyperglycemia results in diffuse scar-ring and thickening of the glomerular basement membrane(GBM) in the kidney This diabetic complication can alsobe modelled in zebrafish [3] Microscopic analysis revealeda significant increase in zebrafish kidney GBM thickness 3weeks after the onset of diabetes Interestingly in this studydiabetes was induced in the fish by intraperitoneal injectionof streptozotocin (as an alternative route streptozotocin wasalso be injected into the caudal fin) In addition disruptionof the zebrafish ortholog of solute carrier family 12 member3 (a sodiumchloride transporter in kidney that is linkedto diabetic nephropathy (DN) in humans [107]) produceshistopathological changes in the kidney that resemble humanDN [108] Thus the potential protective effects of novelcompounds on DN can also be readily assessed in thezebrafish system

Diabetes produces defects in wound healing which islinked to hyperglycemia-induced negative regulation of insu-lin-responsive growth factors such as insulin-like growthfactor-1 (IGF-1) [109 110]This leads to complications such asdiabetic foot ulcers which are a leading cause of amputationsaffecting 15 of all diabetes patients [111] Of note this com-plication can also be modeled in zebrafish and provides anopportunity for compound screening to identify enhancers ofdiabetic wound healing [3] Healthy zebrafish readily regen-erate different tissue types after amputationresection such asfins spinal cord and even portions of the ventricle brain orretina [112]The caudal fin of zebrafish is a particularly attrac-tivemodel for studying themolecularmechanisms regulatingregeneration due to the finrsquos relatively simple structure whichis unnecessary for survival and undergoes rapid regeneration[113] Amputation of the caudal fin in diabetic zebrafishresulted in reduced healingregeneration compared to fishwith normoglycemia which could be imaged readily usinglight microscopy Thus the zebrafish fin amputation diabeticmodel can be used to determine the signals and mechanismsregulating regeneration in the context of diabetes [3]

Overall the zebrafish has been used successfully tomodelnumerous diabetic complications observed in humans Thisprovides an experimentally attractive model system for therapidly confirming the therapeutic effect(s) of novel antidia-betic compounds identified by zebrafish-based screening

4 Conclusion

Diabetes is a serious threat to human health and numerousdrug treatments have been developed for this disease How-ever none of the currently approved drugs can completelycure diabetes and they are associated with side effects such asgastrointestinal problems for the commonly prescribed drug


metformin [114] Late stage type II diabetes can be effectivelytreated by a combination therapy of oral hypoglycemic drugsplus insulin [115] However insulin requires subcutaneousinjection for delivery (inhalable forms of insulin have beendeveloped but they are unlikely to be cost-effective [116])Therefore there is an urgent research need to develop newmore effective antidiabetic drugs that produce fewer sideeffects In this review we described the development of dia-betes relevant assays using the experimentally convenientzebrafish model system and discuss the advantages of thismodel for natural products researchWithin the past 25 yearsthe popularity of natural products research has diminishedin the drug discovery field because of major advances in themolecular biology field and the establishment of combina-torial chemistry These advances provided the technology todesign compounds that target specific drug targets Howeverthere is renewed interest in natural products-based drugdiscovery and development which is due in part to theestablishment of the ldquo-omicsrdquo sciences such as proteom-ics genomics and metabolomics which allow detailed char-acterization of the effects of natural compounds on globalgene expression patterns and complete signaling pathwayanalysis (these advances are discussed inmore detail in [117])As described in this review the zebrafish is now establishedas a powerful validated screening system for drug discov-ery prior to preclinical testing in mammals Although theapplication of this system to antidiabetes drug discovery isrelatively recent compared to other research fields (such ascancer therapeutics) it is now known that zebrafish andhumans share significant overlapping biologicalmechanismsto regulate glucose homeostasis These mechanisms canbe analyzed in fish larvae in a 96-well plate format thatfacilitates drug discovery screening [79] Remarkably thezebrafish system can be used to test compounds that showhigh potential for antidiabetes drug development such asnovel modulators of pancreatic 120573 cell regeneration which aredestroyed in type 1 diabetes The great advantage of usingzebrafish-based screening for antidiabetic drug candidates isthat it only requires wild-type fish which can be purchasedfrom a pet store [26] Just two or three fish tanks are neededto set up a small scale zebrafish facility Additional equipmentsuch as breeding chambers and a culturing cylinder forArtemia (a preferred food source for zebrafish) is also readilyavailable at low cost or can even be ldquohome-builtrdquo [118] Thusa basic zebrafish setup can be housed on a single benchin the laboratory From the standpoint of natural productsresearch a zebrafish based assay system can be incorporatedinto the laboratory using a similar amount of space as a cellculture facility but with reduced set-up and running costsTherefore a natural products research laboratory with aninterest in drug screening and validation could employ thezebrafish system to provide vertebrate-based assays for theircompounds Such animal-based analysis could potentiallyenhance the scope and impact of their research From theperspective of diabetes drug discovery the regulatory mech-anisms of glucose homeostasis in zebrafish have been shownto possess significant homology with humans (eg [79])Over the past ten years much research progress has beenachieved in establishing zebrafish as a diabetes animal model

This can be achieved relatively simply by a single injectionof streptozotocin or immersion in high glucose water [119]Of significance pathophysiologic aspects of diabetes can alsobe modelled such as beta cell loss and epigenetic modifi-cations that produce ldquometabolic memoryrdquo after the onset ofhyperglycemia [68 120 121] These discoveries have laid thefoundation for antidiabetes drug screening using zebrafishand provide further validation that compounds discoveredin zebrafish can also be effective in mammals Thereforezebrafish can be used to circumvent themajor ldquobottleneckrdquo indrug discovery which is the failure of primary hits from cell-based screens to be effective in mammalian model systems

Two major types of screening protocol have been devel-oped for antidiabetic compounds in zebrafish and are val-idated in mammalian systems [22 26] One protocol isbased on transgenic zebrafish larvae expressing a fluorescentPEPCK reporter gene [22] and the second is based on mon-itoring of glucose homeostasis using a fluorescent bioprobe[26 92] The advantage of the second protocol is that itemploys wild-type fish and does not require prior knowledgeof the drug target for screening The advantage of the firstprotocol is that modulators of the well-known antidiabetesdrug target PEPCK can be identified

From the viewpoint of natural products research it hasbeen demonstrated that activity guided fractionation ofantidiabetic compounds from plant extracts can be carriedout in zebrafish In addition a library of natural productshas been screened in this zebrafish system which producedthe discovery of a novel antidiabetic drug candidate thatis effective in a mammalian system [26] The developmentof larval zebrafish-based screening for antidiabetic agentsis a recent research advance that allows natural productsscientistsrsquo access to a simple convenient and cheap assay fortesting their novel compounds in vivo

The zebrafish is gaining more interest as a tool for drugdiscovery because it has been demonstrated that pharmacoki-netic analyses can be undertaken in this model [122 123]Moreover a recent study has shown that the effect of com-pound glycosylation on biological activity can be assessed inthe transparent zebrafish larvae [124] This is especially rele-vant for natural products research because plants generallystore chemicals as glycosides which are then activated byenzyme hydrolysis [125] Thus the zebrafish model can beconsidered as a ldquostand-alonerdquo system in which antidiabetesdrug screening validation effects on secondary diabeticcomplications and pharmacokinetics can be investigatedOverall we hope that this review has raised awareness ofthe attributes of zebrafish-based screening andwill encouragenatural products researchers to use this model to screen orvalidate their novel compounds for antidiabetic activity

Abbreviations

2-NBDG 2-(N-(7-Nitrobenz-2-oxa-13-diazol-4-yl)amino)-2-deoxyglucose

cAMP Cyclic adenosine monophosphateDEX DexamethasoneDM Diabetes mellitusDN Diabetic nephropathy


dpf Days after fertilizationFDA Food and Drug AdministrationGBM Glomerular basement membraneGlipizide GLIPGLUT Glucose transporterHh Hedgehog signaling pathwayhpf Hours after fertilizationHTS High throughput screeningINL Inner nuclear layerIPL Inner plexiform layerMet MetronidazoleNTR Nitroreductasepdx-1 Pancreatic and duodenal homeobox-1PEPCK Phosphoenolpyruvate carboxykinasePSL Photoreceptor layerPTU 1-Phenyl-2-thioureaROSI RosiglitazoneSTZ StreptozotocinTriCA Tricyclic antidepressantsVS Virtual screening

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Nadia Tabassum wrote the paper Hongmei Tai edited thepaper and contributed important discussions about relevantstudies Da-Woon Jung and Darren R Williams planned theoutline of the paper and designedselected the figures Allauthors have read and approved the final version of the paper

Acknowledgments

Our research is supported by the following grants (1) BasicScience Research Program through the NRF funded by theKorean government MSIP (NRF-2015R1A2A2A11001597)(2) a grant from the Integrative Aging Research Center ofGwangju Institute of Science and Technology The authorsapologize to investigators whose work could not be discusseddue to space limitations

References

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[2] M Stumvoll B J Goldstein and T W Van Haeften ldquoType 2diabetes principles of pathogenesis and therapyrdquo The Lancetvol 365 no 9467 pp 1333ndash1346 2005

[3] A S OlsenM P Sarras Jr and R V Intine ldquoLimb regenerationis impaired in an adult zebrafish model of diabetes mellitusrdquoWoundRepair andRegeneration vol 18 no 5 pp 532ndash542 2010

[4] R Mukesh and P Namita ldquoMedicinal plants with antidiabeticpotentialmdasha reviewrdquoAmerican-Eurasian Journal of Agriculturalamp Environmental Sciences vol 13 pp 81ndash94 2013

[5] H Zhang F Liang and R Chen ldquoAncient records and modernresearch on themechanisms of Chinese herbal medicines in thetreatment of diabetes mellitusrdquo Evidence-Based Complementary

and Alternative Medicine vol 2015 Article ID 747982 14 pages2015

[6] M Zarshenas S Khademian and M Moein ldquoDiabetes andrelated remedies in medieval Persian medicinerdquo Indian Journalof Endocrinology and Metabolism vol 18 no 2 pp 142ndash1492014

[7] N Mandrekar and N L Thakur ldquoSignificance of the zebrafishmodel in the discovery of bioactive molecules from naturerdquoBiotechnology Letters vol 31 no 2 pp 171ndash179 2009

[8] D J Newman G M Cragg and K M Snader ldquoNaturalproducts as sources of new drugs over the period 1981ndash2002rdquoJournal of Natural Products vol 66 no 7 pp 1022ndash1037 2003

[9] Y Luo R E Cobb and H Zhao ldquoRecent advances in naturalproduct discoveryrdquo Current Opinion in Biotechnology vol 30pp 230ndash237 2014

[10] A L Harvey R Edrada-Ebel and R J Quinn ldquoThe re-emer-gence of natural products for drug discovery in the genomicserardquo Nature Reviews Drug Discovery vol 14 no 2 pp 111ndash1292015

[11] A D Rodrigues ldquoPreclinical drug metabolism in the age ofhigh-throughput screening an industrial perspectiverdquo Pharma-ceutical Research vol 14 no 11 pp 1504ndash1510 1997

[12] Y Zhang J Gong and Z Yang ldquoEfficient total synthesis ofbioactive natural products a personal recordrdquo The ChemicalRecord vol 14 no 4 pp 606ndash622 2014

[13] J Knowles and G Gromo ldquoA guide to drug discovery targetselection in drug discoveryrdquo Nature Reviews Drug Discoveryvol 2 no 1 pp 63ndash69 2003

[14] C H Williams and C C Hong ldquoMulti-step usage of in vivomodels during rational drug design and discoveryrdquo Interna-tional Journal ofMolecular Sciences vol 12 no 4 pp 2262ndash22742011

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[16] M A Lindsay ldquoTarget discoveryrdquoNature Reviews Drug Discov-ery vol 2 no 10 pp 831ndash838 2003

[17] YMatsumoto E Sumiya T Sugita and K Sekimizu ldquoAn inver-tebrate hyperglycemic model for the identification of anti-diabetic drugsrdquo PLoS ONE vol 6 no 3 Article ID e18292 2011

[18] M Gleeson V Connaughton and L S Arneson ldquoInduction ofhyperglycaemia in zebrafish (Danio rerio) leads to morpholog-ical changes in the retinardquo Acta Diabetologica vol 44 no 3 pp157ndash163 2007

[19] MWei L Ong M T Smith et al ldquoThe streptozotocin-diabeticrat as a model of the chronic complications of human diabetesrdquoHeart Lung and Circulation vol 12 no 1 pp 44ndash50 2003

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[21] Y Matsumoto M Ishii Y Hayashi et al ldquoDiabetic silkwormsfor evaluation of therapeutically effective drugs against type IIdiabetesrdquo Scientific Reports vol 5 Article ID 10722 2015

[22] P Gut B Baeza-Raja O Andersson et al ldquoWhole-organismscreening for gluconeogenesis identifies activators of fastingmetabolismrdquo Nature Chemical Biology vol 9 no 2 pp 97ndash1042013

[23] E U Etuk ldquoAnimals models for studying diabetes mellitusrdquoAgriculture and Biology Journal of North America vol 1 no 2pp 130ndash134 2010

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[38] J S Eisen ldquoZebrafish make a big splashrdquo Cell vol 87 no 6 pp969ndash977 1996

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[40] A E Melby R M Warga and C B Kimmel ldquoSpecification ofcell fates at the dorsal margin of the zebrafish gastrulardquo Devel-opment vol 122 no 7 pp 2225ndash2237 1996

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[42] B Kar and S Subbiah ldquoZebrafish an in vivo model for thestudy of human diseasesrdquo International Journal of Genetics andGenomics vol 1 no 1 pp 6ndash11 2013

[43] T P Barros W K Alderton H M Reynolds A G Roach andS Berghmans ldquoZebrafish an emerging technology for in vivopharmacological assessment to identify potential safety liabil-ities in early drug discoveryrdquo British Journal of Pharmacologyvol 154 no 7 pp 1400ndash1413 2008

[44] C Wang W Tao Y Wang et al ldquoRosuvastatin identified froma zebrafish chemical genetic screen for antiangiogenic com-pounds suppresses the growth of prostate cancerrdquo EuropeanUrology vol 58 no 3 pp 418ndash426 2010

[45] J PHughes S S Rees S B Kalindjian andK L Philpott ldquoPrin-ciples of early drug discoveryrdquo British Journal of Pharmacologyvol 162 no 6 pp 1239ndash1249 2011

[46] A L Hopkins and C R Groom ldquoThe druggable genomerdquoNature Reviews Drug Discovery vol 1 no 9 pp 727ndash730 2002

[47] W Lee C-W Kang C-K Su K Okubo and Y NagahamaldquoScreening estrogenic activity of environmental contaminantsandwater samples using a transgenicmedaka embryo bioassayrdquoChemosphere vol 88 no 8 pp 945ndash952 2012

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[49] K Ito M Morioka S Kimura M Tasaki K Inohaya and AKudo ldquoDifferential reparative phenotypes between zebrafishandmedaka after cardiac injuryrdquoDevelopmental Dynamics vol243 no 9 pp 1106ndash1115 2014

[50] R W Jones and M N Huffman ldquoFish embryos as bio-assaymaterial in testing chemicals for effects on cell division anddifferentiationrdquo Transactions of the American MicroscopicalSociety vol 76 no 2 pp 177ndash183 1957

[51] S L Schreiber ldquoChemical genetics resulting from a passion forsynthetic organic chemistryrdquo Bioorganic amp Medicinal Chem-istry vol 6 no 8 pp 1127ndash1152 1998

[52] D-W Jung DWilliams S M Khersonsky et al ldquoIdentificationof the F1F0 mitochondrial ATPase as a target for modulatingskin pigmentation by screening a tagged triazine library inzebrafishrdquoMolecular BioSystems vol 1 no 1 pp 85ndash92 2005

[53] C-T Yang and S L Johnson ldquoSmall molecule-induced ablationand subsequent regeneration of larval zebrafish melanocytesrdquoDevelopment vol 133 no 18 pp 3563ndash3573 2006

[54] T-Y Choi J-H Kim D H Ko et al ldquoZebrafish as a newmodelfor phenotype-based screening ofmelanogenic regulatory com-poundsrdquo Pigment Cell Research vol 20 no 2 pp 120ndash127 2007

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[62] N S Yee K Lorent and M Pack ldquoExocrine pancreas develop-ment in zebrafishrdquo Developmental Biology vol 284 no 1 pp84ndash101 2005

[63] W M Milewski S J Duguay S J Chan and D F SteinerldquoConservation of PDX-1 structure function and expression inzebrafishrdquo Endocrinology vol 139 no 3 pp 1440ndash1449 1998

[64] S Roy TQiao CWolff andPW Ingham ldquoHedgehog signalingpathway is essential for pancreas specification in the zebrafishembryordquo Current Biology vol 11 no 17 pp 1358ndash1363 2001

[65] Z Sun and N Hopkins ldquoVhnf1 the MODY5 and familialGCKD-associated gene regulates regional specification of thezebrafish gut pronephros and hindbrainrdquo Genes and Develop-ment vol 15 no 23 pp 3217ndash3229 2001

[66] A Seth D L Stemple and I Barroso ldquoThe emerging useof zebrafish to model metabolic diseaserdquo Disease Models andMechanisms vol 6 no 5 pp 1080ndash1088 2013

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[68] H Pisharath J M Rhee M A Swanson S D Leach and MJ Parsons ldquoTargeted ablation of beta cells in the embryoniczebrafish pancreas using E coli nitroreductaserdquo Mechanisms ofDevelopment vol 124 no 3 pp 218ndash229 2007

[69] M Rovira W Huang S Yusuff et al ldquoChemical screen iden-tifies FDA-approved drugs and target pathways that induceprecocious pancreatic endocrine differentiationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 108 no 48 pp 19264ndash19269 2011

[70] O Andersson B A Adams D Yoo et al ldquoAdenosine signalingpromotes regeneration of pancreatic 120573 cells in vivordquoCell Metab-olism vol 15 no 6 pp 885ndash894 2012

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[72] Y Ohta Y Kosaka N Kishimoto et al ldquoConvergence of theinsulin and serotonin programs in the pancreatic 120573-cellrdquo Dia-betes vol 60 no 12 pp 3208ndash3216 2011

[73] Y H Nam B N Hong I Rodriguez et al ldquoSynergistic poten-tials of coffee on injured pancreatic islets and insulin action viaKATP channel blocking in zebrafishrdquo Journal of Agricultural andFood Chemistry vol 63 no 23 pp 5612ndash5621 2015

[74] D M Reif L Truong D Mandrell S Marvel G Zhang andR L Tanguay ldquoHigh-throughput characterization of chemical-associated embryonic behavioral changes predicts teratogenicoutcomesrdquo Archives of Toxicology 2015

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[78] E H Hani D A Stoffers J-C Chevre et al ldquoDefective muta-tions in the insulin promoter factor-1 (IPF-1) gene in late-onsettype 2 diabetes mellitusrdquo The Journal of Clinical Investigationvol 104 no 9 pp R41ndashR48 1999

[79] B Elo C M Villano D Govorko and L A White ldquoLarvalzebrafish as a model for glucose metabolism expression ofphosphoenolpyruvate carboxykinase as a marker for exposureto anti-diabetic compoundsrdquo Journal of Molecular Endocrinol-ogy vol 38 no 3-4 pp 433ndash440 2007

[80] A Mitrakou ldquoKidney its impact on glucose homeostasis andhormonal regulationrdquo Diabetes Research and Clinical Practicevol 93 no 1 pp S66ndashS72 2011

[81] G Rosella J D Zajac S J Kaczmarczyk S Andrikopoulosand J Proietto ldquoImpaired suppression of gluconeogenesisinduced by overexpression of a noninsulin-responsive phos-phoenolpyruvate carboxykinase generdquo Molecular Endocrinol-ogy vol 7 no 11 pp 1456ndash1462 1993

[82] L Yuan R Ziegler and A Hamann ldquoInhibition of phospho-enolpyruvate carboxykinase gene expression by metformin incultured hepatocytesrdquo Chinese Medical Journal vol 115 no 12pp 1843ndash1848 2002

[83] A B Eisenstein ldquoCurrent concepts of gluconeogenesisrdquo TheAmerican Journal of Clinical Nutrition vol 20 no 3 pp 282ndash289 1967

[84] C A Millward J D Heaney D S Sinasac et al ldquoMice with adeletion in the gene forCCAATenhancer-binding protein120573 areprotected against diet-induced obesityrdquo Diabetes vol 56 no 1pp 161ndash167 2007

[85] M Foretz S Hebrard J Leclerc et al ldquoMetformin inhibits hep-atic gluconeogenesis inmice independently of the LKB1AMPKpathway via a decrease in hepatic energy staterdquo Journal ofClinical Investigation vol 120 no 7 pp 2355ndash2369 2010

[86] J Um J Lee D Jung and D Williams ldquoSugars that glow in thedark fluorescent tagged glucose bioprobes and their facilitationof the drug discovery processrdquo Current Medicinal Chemistryvol 22 no 15 pp 1793ndash1807 2015

[87] Y-C Tseng R-D Chen J-R Lee S-T Liu S-J Lee and P-P Hwang ldquoSpecific expression and regulation of glucose trans-porters in zebrafish ionocytesrdquoAmerican Journal of Physiology -Regulatory Integrative and Comparative Physiology vol 297 no2 pp R275ndashR290 2009

[88] J Xue W Ding and Y Liu ldquoAnti-diabetic effects of emodininvolved in the activation of PPAR120574 on high-fat diet-fed andlow dose of streptozotocin-induced diabetic micerdquo Fitoterapiavol 81 no 3 pp 173ndash177 2010

[89] J Liu H Sun W Duan D Mu and L Zhang ldquoMaslinicacid reduces blood glucose in KK-Ay micerdquo Biological ampPharmaceutical Bulletin vol 30 no 11 pp 2075ndash2078 2007

[90] W H Kim J Lee D-W Jung and D R Williams ldquoVisualizingsweetness increasingly diverse applications for fluorescent-tagged glucose bioprobes and their recent structural modifica-tionsrdquo Sensors vol 12 no 4 pp 5005ndash5027 2012

[91] J Park Y L Hyang M-H Cho and B P Seung ldquoDevelop-ment of a Cy3-labeled glucose bioprobe and its application in


bioimaging and screening for anticancer agentsrdquo AngewandteChemiemdashInternational Edition vol 46 no 12 pp 2018ndash20222007

[92] J Park J I Um A Jo et al ldquoImpact of molecular charge onGLUT-specific cellular uptake of glucose bioprobes and in vivoapplication of the glucose bioprobe GB2-Cy3rdquo Chemical Com-munications vol 50 no 66 pp 9251ndash9254 2014

[93] H Y Lee J J Lee J Park and S B Park ldquoDevelopment offluorescent glucose bioprobes and their application on real-timeand quantitative monitoring of glucose uptake in living cellsrdquoChemistrymdashA European Journal vol 17 no 1 pp 143ndash150 2011

[94] S Oh S J Kim J H Hwang et al ldquoAntidiabetic and antiobesityeffects of ampkinone (6f) a novel small molecule activator ofAMP-activated protein kinaserdquo Journal of Medicinal Chemistryvol 53 no 20 pp 7405ndash7413 2010

[95] M P Gilbert ldquoScreening and treatment by the primary careprovider of common diabetes complicationsrdquoMedical Clinics ofNorth America vol 99 no 1 pp 201ndash219 2015

[96] M Brownlee ldquoThe pathobiology of diabetic complications Aunifying mechanismrdquo Diabetes vol 54 no 6 pp 1615ndash16252005

[97] W T Cefalu ldquoAnimal models of type 2 diabetes clinicalpresentation and pathophysiological relevance to the humanconditionrdquo ILAR Journal vol 47 no 3 pp 186ndash198 2006

[98] S E Heinonen G Genove E Bengtsson et al ldquoAnimal modelsof diabetic macrovascular complications key players in thedevelopment of new therapeutic approachesrdquo Journal of Dia-betes Research vol 2015 Article ID 404085 14 pages 2015

[99] KMCapiotti RAntonioli LWKistMR BogoCD Bonanand R S Da Silva ldquoPersistent impaired glucosemetabolism in azebrafish hyperglycemia modelrdquoComparative Biochemistry andPhysiology Part B Biochemistry amp Molecular Biology vol 171no 1 pp 58ndash65 2014

[100] S Lenzen ldquoThe mechanisms of alloxan- and streptozotocin-induced diabetesrdquoDiabetologia vol 51 no 2 pp 216ndash226 2008

[101] Y Alvarez K Chen A L Reynolds N Waghorne J JOrsquoConnor and B N Kennedy ldquoPredominant cone photorecep-tor dysfunction in a hyperglycaemic model of non-proliferativediabetic retinopathyrdquo Disease Models amp Mechanisms vol 3 no3-4 pp 236ndash245 2010

[102] The Expert Committee on the Diagnosis and Classificationof Diabetes Mellitus ldquoReport of the expert committee on thediagnosis and classification of diabetes mellitusrdquoDiabetes Carevol 26 supplement 1 pp S5ndashS20 2002

[103] P G OrsquoMalley ldquoComparative effectiveness of anti-growth factortherapies for diabetic macular edema summary of primaryfindings and conclusionsrdquoArchives of InternalMedicine vol 172no 13 pp 1014ndash1015 2012

[104] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the AmericanHeart AssociationrdquoCirculation vol 100 pp1134ndash1146 1999

[105] J Liang Y Gui W Wang S Gao J Li and H Song ldquoElevatedglucose induces congenital heart defects by altering the expres-sion of tbx5 tbx20 and has2 in developing zebrafish embryosrdquoBirth Defects Research Part A Clinical andMolecular Teratologyvol 88 no 6 pp 480ndash486 2010

[106] E Ritz ldquoNephropathy in type 2 diabetesrdquo Journal of InternalMedicine vol 245 no 2 pp 111ndash126 1999

[107] J H Kim H D Shin B L Park et al ldquoSLC12A3 (solute carrierfamily 12 member [sodiumchloride] 3) polymorphisms are

associated with end-stage renal disease in diabetic nephropa-thyrdquo Diabetes vol 55 no 3 pp 843ndash848 2006

[108] N Abu Seman B He J R M Ojala et al ldquoGenetic and bio-logical effects of sodium-chloride cotransporter (SLC12A3) indiabetic nephropathyrdquo American Journal of Nephrology vol 40pp 408ndash416 2014

[109] D L Brown C D Kane S D Chernausek and D G Green-halgh ldquoDifferential expression and localization of insulin-likegrowth factors I and II in cutaneous wounds of diabetic andnondiabetic micerdquo The American Journal of Pathology vol 151no 3 pp 715ndash724 1997

[110] M P Czubryt ldquoCommon threads in cardiac fibrosis infarct scarformation and wound healingrdquo Fibrogenesis and Tissue Repairvol 5 no 1 article 19 2012

[111] H Brem and M Tomic-Canic ldquoCellular and molecular basis ofwound healing in diabetesrdquo Journal of Clinical Investigation vol117 no 5 pp 1219ndash1222 2007

[112] M C Mione and N S Trede ldquoThe zebrafish as a model forcomplex tissue regenerationrdquo Trends in Genetics vol 29 no 11pp 517ndash523 2013

[113] S-I Higashijima H Okamoto N Ueno Y Hotta and GEguchi ldquoHigh-frequency generation of transgenic zebrafishwhich reliably express GFP in whole muscles or the whole bodyby using promoters of zebrafish originrdquo Developmental Biologyvol 192 no 2 pp 289ndash299 1997

[114] S Bolen L Feldman J Vassy et al ldquoSystematic review com-parative effectiveness and safety of oral medications for type 2diabetes mellitusrdquo Annals of Internal Medicine vol 147 no 6pp 386ndash399 2007

[115] A Cahn R Miccoli A Dardano and S Del Prato ldquoNew formsof insulin and insulin therapies for the treatment of type 2diabetesrdquoThe Lancet Diabetes amp Endocrinology vol 3 no 8 pp638ndash652 2015

[116] P Royle N Waugh L McAuley L McIntyre S Thomas andN Waugh ldquoThe clinical effectiveness and cost-effectiveness ofinhaled insulin in diabetes mellitus a systematic review andeconomic evaluationrdquoHealth Technology Assessment vol 11 no33 pp 1ndash126 2007

[117] H-F Ji X-J Li and H-Y Zhang ldquoNatural products and drugdiscovery Can thousands of years of ancientmedical knowledgelead us to new and powerful drug combinations in the fightagainst cancer and dementiardquo EMBO Reports vol 10 no 3 pp194ndash200 2009

[118] C Nusslein-Volhard and R Dahm Zebrafish PracticalApproach Series OUP Oxford UK 2002

[119] K Jorgens J-L Hillebrands H-P Hammes and J KrollldquoZebrafish a model for understanding diabetic complicationsrdquoExperimental and Clinical Endocrinology amp Diabetes vol 120no 4 pp 186ndash187 2012

[120] R V Intine A S Olsen andM P Sarras Jr ldquoA zebrafish modelof diabetes mellitus and metabolic memoryrdquo Journal of Visual-ized Experiments vol 72 pp e50232ndashe50239 2013

[121] M P Sarras A A Leontovich andR V Intine ldquoUse of zebrafishas a model to investigate the role of epigenetics in propagatingthe secondary complications observed in diabetes mellitusrdquoComparative Biochemistry and Physiology Part C Toxicology ampPharmacology 2015

[122] P Kulkarni G H Chaudhari V Sripuram et al ldquoOral dosing inadult zebrafish proof-of-concept using pharmacokinetics andpharmacological evaluation of carbamazepinerdquo Pharmacologi-cal Reports vol 66 no 1 pp 179ndash183 2014


[123] L ZangDMorikane Y Shimada T Tanaka andNNishimuraldquoA novel protocol for the oral administration of test chemicalsto adult Zebrafishrdquo Zebrafish vol 8 no 4 pp 203ndash210 2011

[124] S-H Park H J Kim S-H Yim et al ldquoDelineation of the role ofglycosylation in the cytotoxic properties of quercetin usingnovel assays in living vertebratesrdquo Journal of Natural Productsvol 77 no 11 pp 2389ndash2396 2014

[125] J Appleton ldquoEvaluating the bioavailability of isoquercetinrdquoNatural Medicine Journal vol 2 pp 1ndash6 2010

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


of novel natural products also preclude their use in the drugdiscovery industry However natural products-based drugdiscovery continues to flourish in academia A recent exam-ple is the development of the anticancer drug ecteinascidin743 (from the sea squirt Ecteinascidia turbinate) (Figure 2)which obtained clinical approval to treat metastatic softtissue sarcoma [10] Therefore the development of powerfulhigh-content screening assays for natural products wouldcomplement their established advantages as candidates fordrug discovery

Over the past two decades major progress has beenachieved in the development of screening technologies suchas high throughput screening (HTS) [11] and chemical syn-thesis strategies such as diversity orientated synthesis [12]However the number of licensed compounds obtainedagainst novel drug targets is considerably low (just two tothree compounds per year) Moreover a single pharmaceu-tical company can spend m100ndash500million annually for inves-tigating 30ndash50 targets [13] and a new compound typicallyrequires 12 years to enter the drug market [14] There arenumerous reasons to account for this low number of newdrugs such as increasingly rigorous safety evaluations andthe reduced efficiency of target-based discovery (known asEroomrsquos Law) [12] Another major bottleneck in the drugdiscovery process is the failure of promising candidate com-pounds in animal tests due to issues related to absorptionsolubility metabolic stability distribution andor toxicityThese failures also arise because cell-based screening systemscannot recapitulate the physiological interactions that arecrucial in the evolution of some disorders such as metabolicdiseases [15] Another significant reason for the rejection ofcandidate drugs is that discovery methods rely mainly on theexistence of identifiable and ldquoscreenablerdquo targets [16] Thislate-stage attrition of drugs in development is highly costlyand time consuming for pharmaceutical companies Thesedrawbacks of target driven drug discovery have inducedresearchers to screen compounds including natural prod-ucts in whole animal systems to identify new targets andultimately successful drugs In the next section of this reviewwe compare conventional screening systems with animal-based approaches and introduce the zebrafish as an idealmodel system for in vivo drug discovery applications

2 Development of Animal-Based ScreeningSystems for Drug Discovery

The mouse (Mus musculus) is the most widely used animalmodel in biomedical research and can be considered as theldquowork horserdquo model organism [28] It possesses numerousadvantages for research purposes such as sharing 90genome homologywith humans in addition tomany geneticphysiological and organ anatomical similarities Anotherimportant advantage is that genetic manipulation in themouse can be used to model the action of potential drugs viathe knockdown of candidate target genes Thus the mouse isan invaluable model for the discovery of new targets for ther-apeutic interventions [29] However from the perspectiveof drug screening the mouse has significant disadvantages

restricting its use in the pharmaceutical field The majorproblem is the expense involved in maintaining large mousecolonies for compound screening There are also biologicalissues For example transgenic animals that are used tomodel disease may not be suitable for screening becauseof the potential initiation of compensatory gene expressionmechanisms during development This could be problematicfor discovering compounds that should be active in humans[16] Consequently alternative animal-based platforms forscreening have been investigated Two prominent examplesare invertebratemodels the fruit flyDrosophilamelanogasterand the roundworm Caenorhabditis elegans [30 31] Despiteoffering certain advantages such as applicability to HTSand the preservation of some physiological contexts thatare similar to humans these invertebrate models have notbecome widely used for drug screeningThis is because theseinvertebrates have markedly reduced genetic homology withhumans compared to the mouse In addition some of themajor organ systems present in humans such as respiratoryand intravascular circulatory systems are not present ininvertebrates [32] Another issue is the thick cuticle whichaffects drug penetration and complicates the interpretationof screening data (this is especially relevant for C elegans)[33 34] Thus the clinical potential of novel compoundsdiscovered in invertebrates may not be sufficient to justifyscreening in these animal systems

3 Zebrafish Swimming into Placein the Drug Discovery Field

These above-described limitations on mammalian and inver-tebrate models for compound screening provided impetusto develop alternative animal model systems for studyingdrug responses Consequently the zebrafish (Danio rerio) hasrisen to prominence The zebrafish is a small fresh waterteleost that was developed as an animal model in the 1980sfor the study of developmental biology and embryogenesis[36 37] A set of landmark studies published together ina single issue of the journal Development (1996) helpedto establish this animal model for characterizing vertebratedevelopmental pathways Further studies and the productionof gene targeting techniques such as anitisense morpholinosand the generation of transgenic fish enabled zebrafish tobecome an attractive model for studying human disease [3839] Critically the logistical advantages of using zebrafishhave also convinced the research community to adopt thisanimal model These advantages are listed as follows

(1) In contrast to rodents zebrafish embryos are opticallytransparent and zebrafish development is externalwhich permits direct observation of embryonic organsystems under a wide variety of laboratory conditions[40] For its size zebrafish embryos are relativelylarge which assists their handling in the laboratory

(2) Embryonic development is very rapid compared tomammals The entire body plan is established by24 hours after fertilization (hpf) and most majororgan systems such as the digestive tract and thecardiovascular system are developed 2 days after




































3 cm

(a)

EyeOtolith (ear)

HeartLiver

Intestinaltract

Ventralfin

Cloaca


Muscle

Tailfin

(b)






(4) Assessing

effects


diseasesmodels


(c)





NHO

HO

S

NO

O

OOO

N

OH

HOO

Ecteinascidin 743

NN

F

NSO

O

HO

OHOHO

Rosuvastatin

N NN

O

NN

Cl

Trazodone

OH HNHO

HO

Isoprenaline

NN

O

Cl

PK 11195

N

N

Cl

O

ClRo5-4864

HO

HOH

H

HO

OH

Maslinic acid

OHO O

N

O

OO

Ampkinone

OF

HHO

H

OOH

OH

Dexamethasone

HNNH

O SO O

O

N

N

Glipizide

NH

OOHO

O

O

Fraxidin

OHOHO

OH

OH O N NO

N

GB2-Cy3

N+

O

Trigonelline

N+

Ominus

OHO

Retinoic acid

H3CCH3CH3

CH3

CH3OHN

NO

N

HO

HO

OHOH

2-NBDG

NO2

N

NH NH

Metformin

NH

NH2

PP-II-A03

Cl

HN NNN

NH

NH

HN

OO

ONH2

N SS

S N

S

Disulfiram

H3CH3C

OH O

O

OH

OH

Emodin

CH3

NNN

N

OHO

HO

HN O


NH2

F

F

O

NH

HN

OO

O

Ph

DAPT

O

O

OHOHOH

HO

HO

Chlorogenic acid

CO2H

O

O

HO

HO

OH H

H

OHH

H

HO

cpp532

CH2OHNN N

NO

O

Caffeine

H3CCH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3













MTZ(ablation)


50 80 128 (hpf)

(a)

(b)

Num

ber o

f bet

a cel

ls


lowast

lowast16

14

12

10

8

6

4

2

0

(c)







UP423 UP521 UP32 UP33 UP523


fraction


UP hexanefraction

0

20

40

60

80

100


Hexane

Fluo

resc

ence

inte

nsity

Fraction tested


020406080

100120140


Fluo

resc

ence

inte

nsity

UP32(fraxidin)


Compound treatment


(a)

(b)





GB2 alone

Ampkinone + GB2

Rosiglitazone + GB2


(a)

(b)

(c)

lowast

lowast

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Control1 DMSO

Ampkinone10120583M


Drug treatment

90

80

70

60

50

40

30

20

10

0

20

19

18

17

16

15

14

13

12

11

10Control

1 DMSOAmpkinone


Drug treatment

lowastlowast













4 Conclusion








Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































3 cm

(a)

EyeOtolith (ear)

HeartLiver

Intestinaltract

Ventralfin

Cloaca


Muscle

Tailfin

(b)






(4) Assessing

effects


diseasesmodels


(c)





NHO

HO

S

NO

O

OOO

N

OH

HOO

Ecteinascidin 743

NN

F

NSO

O

HO

OHOHO

Rosuvastatin

N NN

O

NN

Cl

Trazodone

OH HNHO

HO

Isoprenaline

NN

O

Cl

PK 11195

N

N

Cl

O

ClRo5-4864

HO

HOH

H

HO

OH

Maslinic acid

OHO O

N

O

OO

Ampkinone

OF

HHO

H

OOH

OH

Dexamethasone

HNNH

O SO O

O

N

N

Glipizide

NH

OOHO

O

O

Fraxidin

OHOHO

OH

OH O N NO

N

GB2-Cy3

N+

O

Trigonelline

N+

Ominus

OHO

Retinoic acid

H3CCH3CH3

CH3

CH3OHN

NO

N

HO

HO

OHOH

2-NBDG

NO2

N

NH NH

Metformin

NH

NH2

PP-II-A03

Cl

HN NNN

NH

NH

HN

OO

ONH2

N SS

S N

S

Disulfiram

H3CH3C

OH O

O

OH

OH

Emodin

CH3

NNN

N

OHO

HO

HN O


NH2

F

F

O

NH

HN

OO

O

Ph

DAPT

O

O

OHOHOH

HO

HO

Chlorogenic acid

CO2H

O

O

HO

HO

OH H

H

OHH

H

HO

cpp532

CH2OHNN N

NO

O

Caffeine

H3CCH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3













MTZ(ablation)


50 80 128 (hpf)

(a)

(b)

Num

ber o

f bet

a cel

ls


lowast

lowast16

14

12

10

8

6

4

2

0

(c)







UP423 UP521 UP32 UP33 UP523


fraction


UP hexanefraction

0

20

40

60

80

100


Hexane

Fluo

resc

ence

inte

nsity

Fraction tested


020406080

100120140


Fluo

resc

ence

inte

nsity

UP32(fraxidin)


Compound treatment


(a)

(b)





GB2 alone

Ampkinone + GB2

Rosiglitazone + GB2


(a)

(b)

(c)

lowast

lowast

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Control1 DMSO

Ampkinone10120583M


Drug treatment

90

80

70

60

50

40

30

20

10

0

20

19

18

17

16

15

14

13

12

11

10Control

1 DMSOAmpkinone


Drug treatment

lowastlowast













4 Conclusion








Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NHO

HO

S

NO

O

OOO

N

OH

HOO

Ecteinascidin 743

NN

F

NSO

O

HO

OHOHO

Rosuvastatin

N NN

O

NN

Cl

Trazodone

OH HNHO

HO

Isoprenaline

NN

O

Cl

PK 11195

N

N

Cl

O

ClRo5-4864

HO

HOH

H

HO

OH

Maslinic acid

OHO O

N

O

OO

Ampkinone

OF

HHO

H

OOH

OH

Dexamethasone

HNNH

O SO O

O

N

N

Glipizide

NH

OOHO

O

O

Fraxidin

OHOHO

OH

OH O N NO

N

GB2-Cy3

N+

O

Trigonelline

N+

Ominus

OHO

Retinoic acid

H3CCH3CH3

CH3

CH3OHN

NO

N

HO

HO

OHOH

2-NBDG

NO2

N

NH NH

Metformin

NH

NH2

PP-II-A03

Cl

HN NNN

NH

NH

HN

OO

ONH2

N SS

S N

S

Disulfiram

H3CH3C

OH O

O

OH

OH

Emodin

CH3

NNN

N

OHO

HO

HN O


NH2

F

F

O

NH

HN

OO

O

Ph

DAPT

O

O

OHOHOH

HO

HO

Chlorogenic acid

CO2H

O

O

HO

HO

OH H

H

OHH

H

HO

cpp532

CH2OHNN N

NO

O

Caffeine

H3CCH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3













MTZ(ablation)


50 80 128 (hpf)

(a)

(b)

Num

ber o

f bet

a cel

ls


lowast

lowast16

14

12

10

8

6

4

2

0

(c)







UP423 UP521 UP32 UP33 UP523


fraction


UP hexanefraction

0

20

40

60

80

100


Hexane

Fluo

resc

ence

inte

nsity

Fraction tested


020406080

100120140


Fluo

resc

ence

inte

nsity

UP32(fraxidin)


Compound treatment


(a)

(b)





GB2 alone

Ampkinone + GB2

Rosiglitazone + GB2


(a)

(b)

(c)

lowast

lowast

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Control1 DMSO

Ampkinone10120583M


Drug treatment

90

80

70

60

50

40

30

20

10

0

20

19

18

17

16

15

14

13

12

11

10Control

1 DMSOAmpkinone


Drug treatment

lowastlowast













4 Conclusion








Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























MTZ(ablation)


50 80 128 (hpf)

(a)

(b)

Num

ber o

f bet

a cel

ls


lowast

lowast16

14

12

10

8

6

4

2

0

(c)







UP423 UP521 UP32 UP33 UP523


fraction


UP hexanefraction

0

20

40

60

80

100


Hexane

Fluo

resc

ence

inte

nsity

Fraction tested


020406080

100120140


Fluo

resc

ence

inte

nsity

UP32(fraxidin)


Compound treatment


(a)

(b)





GB2 alone

Ampkinone + GB2

Rosiglitazone + GB2


(a)

(b)

(c)

lowast

lowast

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Control1 DMSO

Ampkinone10120583M


Drug treatment

90

80

70

60

50

40

30

20

10

0

20

19

18

17

16

15

14

13

12

11

10Control

1 DMSOAmpkinone


Drug treatment

lowastlowast













4 Conclusion








Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















UP423 UP521 UP32 UP33 UP523


fraction


UP hexanefraction

0

20

40

60

80

100


Hexane

Fluo

resc

ence

inte

nsity

Fraction tested


020406080

100120140


Fluo

resc

ence

inte

nsity

UP32(fraxidin)


Compound treatment


(a)

(b)





GB2 alone

Ampkinone + GB2

Rosiglitazone + GB2


(a)

(b)

(c)

lowast

lowast

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Control1 DMSO

Ampkinone10120583M


Drug treatment

90

80

70

60

50

40

30

20

10

0

20

19

18

17

16

15

14

13

12

11

10Control

1 DMSOAmpkinone


Drug treatment

lowastlowast













4 Conclusion








Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GB2 alone

Ampkinone + GB2

Rosiglitazone + GB2


(a)

(b)

(c)

lowast

lowast

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Control1 DMSO

Ampkinone10120583M


Drug treatment

90

80

70

60

50

40

30

20

10

0

20

19

18

17

16

15

14

13

12

11

10Control

1 DMSOAmpkinone


Drug treatment

lowastlowast













4 Conclusion








Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























4 Conclusion








Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Abbreviations









Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References











































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article fishing for nature s hits: establishment of...

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