Model Organisms in Drug Discovery

208 LIPID METABOLISM AND SIGNALING IN ZEBRAFISH
a fluorescent BODIPY acyl chain; this event is organ-specific (Hendrickson
et al., 1999). We utilized PED6 to visualize PLA 2 activity in zebrafish larvae 5
days post-fertilization. As shown in Figure 8.1B, the intestine and the gall
bladder are labeled by cleaved PED6 metabolites. Based on this observation
and time-course studies we hypothesized that quenched PED6 is cleaved by
PLA 2 in the intestine following PED6 ingestion, and the cleaved products –
unquenched green fluorescent PED6 metabolites – are rapidly transported to
the liver. These fluorescent metabolites are then secreted into newly formed
bile and stored in the gall bladder. Following extrusion from the gall bladder,
the fluorescent bile enters the intestine, where it is easily visualized.
To test our hypothesis, another fluorescent lipid reporter, BODIPY FR-PC
(Figure 8.2A), was generated (Farber et al., 2001). This fluorophore has two
BODIPY acyl chains that exhibit fluorescence resonance energy transfer
(FRET) to emit different spectra upon PLA 2 cleavage. When excited (505 nm),
the intact substrate emits orange (568 nm). Upon PLA 2 cleavage, the same
excitation results in a green emission (515 nm). Such a molecule can be used to
localize PLA 2 activity. As shown in Figure 8.2B, only green fluorescence
(cleaved product of PLA2) was observed in the gall bladder and liver, where
intact substrate (orange fluorescence) was located only in the intestinal
epithelium. In conclusion, these studies suggest that lipid digestion and
absorption systems in zebrafish larvae are similar to those in mammals.
We have initiated a physiological genetic screen in vivo with ENU
mutagenized zebrafish using these biosensors because they provide a rapid
readout of lipid metabolism and digestive organ morphology in living
zebrafish larvae. So far, we have identified eight mutants. Among the mutants
is one recessive lethal mutant, fat-free, that fails to accumulate fluorescently
labeled lipids in the gall bladder following PED6 and NBD-cholesterol (22-[N-
(7-nitronbenz-2-oxa-1,3-diazol-4-yl) amino]-23,24-bisnor-5-cholen-3-ol) ingestion,
but its digestive system appears morphologically normal. Phenotypic
analysis of this mutant indicated that the PLA 2 activity and swallowing are
normal (Farber et al., 2001). In contrast, fat-free had nearly normal
fluorescence in the digestive organ after BODIPY FL-C5 ingestion. Because
BODIPY FL-C5 is a short-chain fatty acid analog that is less hydrophobic
and more soluble in aqueous solution, emulsifiers (such as bile) are not critical
for its absorption. Instead, PED6 and NBD-cholesterol, the more hydrophobic
molecules, require biliary emulsification in order to be processed and
absorbed. Because the absorption of short-chain fatty acids is nearly normal
in fat-free, we hypothesized that the fat-free mutation may attenuate bile
synthesis or secretion.
Additional evidence that the fat-free mutant might be a potential animal
model to study biliary synthesis or secretion are the results of a statin drug
treatment study. As we have shown previously, when wild-type zebrafish
larvae are treated with the statin drug atorvastatin (Lipitor), PED6 processing