Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake

Abstract

Improved understanding of lipoproteins, particles that transport lipids throughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome. Apolipoproteins are a key component of lipoproteins. Apolipoproteins are proteins that structure lipoproteins and regulate lipid metabolism through control of cellular lipid exchange. Constraints of cell culture and mouse models mean that there is a need for a complementary model that can replicate the complex in vivo milieu that regulates apolipoprotein and lipoprotein biology. Here, we further establish the utility of the genetically tractable and optically clear larval zebrafish as a model of apolipoprotein biology. Gene ancestry analyses were implemented to determine the closest human orthologs of the zebrafish apolipoprotein A-I (apoA-I), apoB, apoE and apoA-IV genes and therefore ensure that they have been correctly named. Their expression patterns throughout development were also analyzed, by whole-mount mRNA in situ hybridization (ISH). The ISH results emphasized the importance of apolipoproteins in transporting yolk and dietary lipids: mRNA expression of all apolipoproteins was observed in the yolk syncytial layer, and intestinal and liver expression was observed from 4–6 days post-fertilization (dpf). Furthermore, real-time PCR confirmed that transcription of three of the four zebrafish apoA-IV genes was increased 4 hours after the onset of a 1-hour high-fat feed. Therefore, we tested the hypothesis that zebrafish ApoA-IV performs a conserved role to that in rat in the regulation of food intake by transiently overexpressing ApoA-IVb.1 in transgenic larvae and quantifying ingestion of co-fed fluorescently labeled fatty acid during a high-fat meal as an indicator of food intake. Indeed, ApoA-IVb.1 overexpression decreased food intake by approximately one-third. This study comprehensively describes the expression and function of eleven zebrafish apolipoproteins and serves as a springboard for future investigations to elucidate their roles in development and disease in the larval zebrafish model.

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

“…This assay was used to show that overexpression of Apolipoprotein A-IVb.1 (apoA-IVb.1) decreases food intake. Previously unfed Tg(hsp70:apoA-IVb.1:mCherry) larvae fed 10% egg yolk with 6.4 μM fluorescent C 16 analog for 4 hr at 7 dpf consumed approximately 30% fewer lipids than wild type larvae (Figure 4) 20 .…”

“…Moreover, fluorescent lipid analogs provided in the egg yolk visualize transport and accumulation of dietary lipids 18 , image cellular components including lipid droplets by acting both as vital dyes 3 and through covalent incorporation into complex lipids, investigate metabolism through thin layer chromatography (TLC) 19 and high performance liquid chromatography (HPLC) (S.A.F. unpublished data), and provide a quantitative assay for total food intake 20 .…”

Changes in Intestinal Gene Expression of Zebrafish (Danio rerio) Related to Sterol Uptake and Excretion upon β-Sitosterol Administration

“…Zebrafish is used as one of the model organisms to study the apolipoprotein trafficking mechanism in humans [18]. ApoA-I is an important component of high-density lipoprotein in mammals and a similar protein is also reported in the zebrafish.…”

“…Apolipoprotein A-I (ApoA-I) is responsible for the formation of pre-high density lipoprotein (HDL) particles in human, which then is associated with sterol transportation from the small intestine to the liver. In zebrafish, ApoA-I also contributes to sterol transporting system [18]. Excessive sterols are excreted out of the cells through the ATP-binding cassette sub-family G, member 5 and 8 (ABCG5 and ABCG8), back into the lumen in mammals [19].…”

Zebrafish Models for Dyslipidemia and Atherosclerosis Research

“…Finally, there are two zebrafish apob paralogs (two apob genes on different chromosomes). The contribution (expression and incorporation into chylomicrons and VLDL) of these Apob paralogs to circulating β-lipoproteins and atherogenesis is not known; however, their larval expression patterns are different, and their encoded proteins are structurally dissimilar, raising the possibility that they have unique functions (23). …”