EZ2121h 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.

EZ102Unfed 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.

EZ2131h 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.

EZ2434h 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.

EZ1424h 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.

EZ103Unfed 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.

EZ1111h 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.

Apolipoprotein-B (ApoB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular damage. Here we report the LipoGlo system, which uses a luciferase enzyme (NanoLuc) fused to ApoB to monitor several key determinants of lipoprotein atherogenicity including particle abundance, size, and localization. Using LipoGlo, we comprehensively characterize the lipoprotein profile of individual larval zebrafish and collect images of atherogenic lipoprotein localization in an intact organism. We report multiple extravascular lipoprotein localization patterns, as well as identify Pla2g12b as a potent regulator of lipoprotein size. ApoB-fusion proteins thus represent a sensitive and specific approach to study atherogenic lipoproteins and their genetic and small molecule modifiers.

Difficulty in imaging the vertebrate intestine in vivo has hindered our ability to model nutrient and protein trafficking from both the lumenal and basolateral aspects of enterocytes. Our goal was to use live confocal imaging to increase understanding of intestinal trafficking of dietary cholesterol and apolipoprotein A-I (APOA-I), the main structural component of high-density lipoproteins. We developed a novel assay to visualize live dietary cholesterol trafficking in the zebrafish intestine by feeding TopFluor-cholesterol (TF-cholesterol), a fluorescent cholesterol analog, in a lipid-rich, chicken egg yolk feed. Quantitative microscopy of transgenic zebrafish expressing fluorescently tagged protein markers of early, recycling, and late endosomes/lysosomes provided the first evidence, to our knowledge, of cholesterol transport in the intestinal endosomal-lysosomal trafficking system. To study APOA-I dynamics, transgenic zebrafish expressing an APOA-I fluorescent fusion protein (APOA-I-mCherry) from tissue-specific promoters were created. These zebrafish demonstrated that APOA-I-mCherry derived from the intestine accumulated in the liver and vice versa. Additionally, intracellular APOA-I-mCherry localized to endosomes and lysosomes in the intestine and liver. Moreover, live imaging demonstrated that APOA-I-mCherry colocalized with dietary TF-cholesterol in enterocytes, and this colocalization increased with feeding time. This study provides a new set of tools for the study of cellular lipid biology and elucidates a key role for endosomal-lysosomal trafficking of intestinal cholesterol and APOA-I.

Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. Whereas much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is un-known. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an independent regulator of HIF. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of the HIFalpha subunit of these heterodimeric transcription factors comparable with hypoxia with activation of downstream target genes. The addition of fatty acids to culture medium suppressed this signal, which required an intact mitochondrial respiratory chain. Mechanistically, fatty acids and oxygen are distinct signals integrated to control HIF activity. Finally, we observed lipid signaling to HIF and changes in target gene expression in developing zebrafish and adult mice, and this pathway operates in cancer cells from a range of tissues. This study identifies fatty acids as a physiological modulator of HIF, defining a mechanism for lipoprotein regulation that functions in parallel to oxygen.