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BioAssay: AID 686

Zebrafish Lipid Metabolism Assay---Primary Screen

The zebrafish processes lipids through its digestive system in a similar way to mammals. Thus it is a useful model organism that provides for in vivo measurement of lipid absorption and processing in a vertebrate organism. Zebrafish larvae are transparent, allowing for observation of lipid metabolism in the whole organism using fluorescent lipid substrates. Zebrafish therefore provide a screening platform to identify compounds that interfere with lipid metabolism and lower the high levels of cholesterol and triglycerides implicated as a major contributor to heart disease in humans. ..more
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 Tested Compounds
 Tested Compounds
All(3801)
 
 
Active(53)
 
 
Inactive(3748)
 
 
 Tested Substances
 Tested Substances
All(3806)
 
 
Active(53)
 
 
Inactive(3753)
 
 
 Related BioAssays
 Related BioAssays
AID: 686
Data Source: PCMD (ZEBRAFISH LIPID METABOLISM-PRIMARY SCREEN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2007-04-20

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 53
Description:
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Dr. Amy Rubenstein, Zygogen LLC, Atlanta, GA
MLSCN Grant: X01-MH077634-01

The zebrafish processes lipids through its digestive system in a similar way to mammals. Thus it is a useful model organism that provides for in vivo measurement of lipid absorption and processing in a vertebrate organism. Zebrafish larvae are transparent, allowing for observation of lipid metabolism in the whole organism using fluorescent lipid substrates. Zebrafish therefore provide a screening platform to identify compounds that interfere with lipid metabolism and lower the high levels of cholesterol and triglycerides implicated as a major contributor to heart disease in humans.

The screen reported here was designed to identify compounds that inhibit the cleavage of PED6, a quenched fluorescent lipid substrate for phospholipase A2 [PubMed ID 11359013--Farber SA, Pack M, H0 S-Y, et al., Science, 292, 1385-1388 (2001)]. Zebrafish larvae were fed PED6, which is swallowed, emulsified in the lumen of the intestine, and cleaved by phospholipases. Cleavage of PED6 releases fluorescent fatty acids that are absorbed through the intestinal epithelium, transported to the liver, incorporated into bile, and concentrated in the gall bladder. Fluorescence is observed in the gall bladder and also in the intestine following secretion of fluorescent bile.
Protocol
Materials

Zebrafish were cultivated in the laboratory of Dr. Michael Pack, School of Medicine, University of Pennsylvania. PED6 was purchased from Invitrogen (catalog no. D-23739). Clear flat-bottom and V-bottom polypropylene 96-well microtiter plates were purchased from Corning and Greiner, respectively.

Assay

Zebrafish larvae (5-7 days old) were dispensed into clear flat-bottom microtiter plates to give 5 fish per well in 90 uL of E3 media (5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate in deionized water). Test compounds were diluted from a 10-mM stock solution in DMSO into V-bottom polypropylene microtiter plates to give a final concentration of 200 uM in 2% aqueous DMSO. These aqueous compound solutions (10 uL) were then transferred by multichannel pipet into the zebrafish plates to give a final concentration of 20 uM. PED6 was added to a final concentration of 100 nM.

After overnight incubation with PED6 and test compounds, zebrafish larvae were viewed under an Olympus IX81 inverted fluorescent microscope. Each well was scored visually based on the fluorescence observed in the gall bladder and intestine of each fish.

Data analysis

Each well was assigned activity as follows:

ABSENCE _FLUOR_GB&INT
= Complete inhibition of fluorescence in gall bladder and intestine
ABSENCE _FLUOR_GB_ONLY
= Complete inhibition of fluorescence in gall bladder but no reduction of fluorescence of intestine
ABSENCE _FLUOR_INT_ONLY
= Complete inhibition of fluorescence in intestine but no reduction of fluorescence of gall bladder
WEAK_FLUOR_GB&INT
= Partial inhibition of fluorescence in gall bladder and intestine
WEAK _FLUOR_GB_ONLY
= Partial inhibition of fluorescence in gall bladder but no reduction of fluorescence of intestine
WEAK _FLUOR_INT_ONLY
= Partial inhibition of fluorescence in intestine but no reduction of fluorescence of gall bladder
TOXIC
= No movement
INACTIVE
= No reduction in fluorescence
Comment
Activity scoring

Scores were assigned as follows:

100: Compounds that gave complete inhibition of fluorescence in gall bladder and intestine.
75: Compounds that gave partial inhibition of fluorescence in gall bladder and intestine.

50: Compounds that gave complete inhibition of fluorescence in gall bladder or intestine.

25: Compounds that gave partial inhibition of fluorescence in gall bladder or intestine.

0: Compounds that gave no reduction in fluorescence or were toxic to the fish

Activity Outcome

Active = Compounds that gave complete or partial inhibition of fluorescence in gall bladder or intestine or both.
Inactive = Compounds that gave no reduction in fluorescence or were toxic to the fish

Contributors

Zebrafish were cultivated in the laboratory of Dr. Michael Pack, School of Medicine, Unversity of Pennsylvania. Compound plates were prepared by Edinson Lucumi and screening was conducted by David Justin. Data were submitted by Edinson Lucumi and Andrew Napper.

Correspondence

Please direct correspondence to Andrew Napper (napper@seas.upenn.edu).
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ActivityString

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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