The zebrafish larva is a powerful tool for the study of dietary triglyceride (TG) digestion and how fatty acids (FA) derived from dietary lipids are absorbed, metabolized and distributed to the body. While fluorescent FA analogues have enabled visualization of FA metabolism, methods for specifically assaying TG digestion are badly needed. Here we present a novel High Performance Liquid Chromatography (HPLC) method that quantitatively differentiates TG and phospholipid (PL) molecules with one or two fluorescent FA analogues. We show how this tool may be used to discriminate between undigested and digested TG or phosphatidylcholine (PC), and also the products of TG or PC that have been digested, absorbed and re-synthesized into new lipid molecules. Using this approach, we explored the dietary requirement of zebrafish larvae for phospholipids. Here we demonstrate that dietary TG is digested and absorbed in the intestinal epithelium, but without dietary PC, TG accumulates and is not transported out of the enterocytes. Consequently, intestinal ER stress increases and the ingested lipid is not available support the energy and metabolic needs of other tissues. In TG diets with PC, TG is readily transported from the intestine and subsequently metabolized.

EZ2111h low-fat fed 6.5 dpf zebrafish larvae (low-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.

EZ203Unfed 6.5 dpf zebrafish larvae (low-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.

EZ101Unfed 6.5 dpf zebrafish larvae (high-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.

Responding to a high-fat meal requires an interplay between multiple digestive tissues, sympathetic response pathways, and the gut microbiome. The epithelial enterocytes of the intestine are responsible for absorbing dietary nutrients and preparing them for circulation to distal tissues, which requires significant changes in cellular activity, including both morphological and transcriptional responses. Following a high-fat meal, we observe morphological changes in the enterocytes of larval zebrafish, including elongation of mitochondria, formation and expansion of lipid droplets, and the rapid and transient ruffling of the nuclear periphery. Dietary and pharmacological manipulation of zebrafish larvae demonstrated that these subcellular changes are specific to triglyceride absorption. The transcriptional changes that occur simultaneously with these morphological changes were determined using RNA sequencing, revealing a cohort of up-regulated genes associated with lipid droplet formation and lipid transport via lipoprotein particles. Using a microsomal triglyceride transfer protein (MTP) inhibitor to block -lipoprotein particle formation, we demonstrate that the transcriptional response to a high-fat meal is associated with the transfer of ER triglyceride to nascent -lipoproteins, possibly through the activation of Creb3l3/cyclic AMP-responsive element-binding protein. These data suggest that a transient increase in ER lipids is the likely mediator of the initial physiological response of intestinal enterocytes to dietary lipid.

EZ2424h low-fat fed 6.5 dpf zebrafish larvae (low-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.

EZ1414h high-fat fed 6.5 dpf zebrafish larvae (high-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.

EZ202Unfed 6.5 dpf zebrafish larvae (low-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.