Directly Measuring Atherogenic Lipoprotein Kinetics in Zebrafish with the Photoconvertible LipoTimer Reporter.

Lipoprotein kinetics are a crucial factor in understanding lipoprotein metabolism since a prolonged time in circulation can contribute to the atherogenic character of apolipoprotein-B (ApoB)-containing lipoproteins (B-lps). Here, we report a method to directly measure lipoprotein kinetics in live developing animals. We developed a zebrafish geneticly encoded reporter, LipoTimer, in which endogenous ApoBb.1 is fused to the photoconvertible fluorophore Dendra2 which shift its emission profile from green to red upon UV exposure. By quantifying the red population of ApoB-Dendra2 over time, we found that B-lp turnover in wild-type larvae becomes faster as development proceeds. Mutants with impaired B-lp uptake or lipolysis present with increased B-lp levels and half-life. In contrast, mutants with impaired B-lp triglyceride loading display slightly fewer and smaller-B-lps, which have a significantly shorter B-lp half-life. Further, we showed that chronic high-cholesterol feeding is associated with a longer B-lp half-life in wild-type juveniles but does not lead to changes in B-lp half-life in lipolysis deficient apoC2 mutants. These data support the hypothesis that B-lp lipolysis is suppressed by the flood of intestinal-derived B-lps that follow a high-fat meal.

Pla2g12b drives expansion of triglyceride-rich lipoproteins.

Vertebrates transport hydrophobic triglycerides through the circulatory system by packaging them within amphipathic particles called Triglyceride-Rich Lipoproteins. Yet, it remains largely unknown how triglycerides are loaded onto these particles. Mutations in Phospholipase A2 group 12B (PLA2G12B) are known to disrupt lipoprotein homeostasis, but its mechanistic role in this process remains unclear. Here we report that PLA2G12B channels lipids within the lumen of the endoplasmic reticulum into nascent lipoproteins. This activity promotes efficient lipid secretion while preventing excess accumulation of intracellular lipids. We characterize the functional domains, subcellular localization, and interacting partners of PLA2G12B, demonstrating that PLA2G12B is calcium-dependent and tightly associated with the membrane of the endoplasmic reticulum. We also detect profound resistance to atherosclerosis in PLA2G12B mutant mice, suggesting an evolutionary tradeoff between triglyceride transport and cardiovascular disease risk. Here we identify PLA2G12B as a key driver of triglyceride incorporation into vertebrate lipoproteins.

Zebrafish ApoB-Containing Lipoprotein Metabolism: A Closer Look.

Zebrafish have become a powerful model of mammalian lipoprotein metabolism and lipid cell biology. Most key proteins involved in lipid metabolism, including cholesteryl ester transfer protein, are conserved in zebrafish. Consequently, zebrafish exhibit a human-like lipoprotein profile. Zebrafish with mutations in genes linked to human metabolic diseases often mimic the human phenotype. Zebrafish larvae develop rapidly and externally around the maternally deposited yolk. Recent work revealed that any disturbance of lipoprotein formation leads to the accumulation of cytoplasmic lipid droplets and an opaque yolk, providing a visible phenotype to investigate disturbances of the lipoprotein pathway, already leading to discoveries in MTTP (microsomal triglyceride transfer protein) and ApoB (apolipoprotein B). By 5 days of development, the digestive system is functional, making it possible to study fluorescently labeled lipid uptake in the transparent larvae. These and other approaches enabled the first in vivo description of the STAB (stabilin) receptors, showing lipoprotein uptake in endothelial cells. Various zebrafish models have been developed to mimic human diseases by mutating genes known to influence lipoproteins (eg, ldlra, apoC2). This review aims to discuss the most recent research in the zebrafish ApoB-containing lipoprotein and lipid metabolism field. We also summarize new insights into lipid processing within the yolk cell and how changes in lipid flux alter yolk opacity. This curious new finding, coupled with the development of several techniques, can be deployed to identify new players in lipoprotein research directly relevant to human disease.

Fish Playpens: Method for Raising Individual Juvenile Zebrafish on a Recirculating System for Studies Requiring Repeated Measures.

Even though many experimental approaches benefit from tracking individual juvenile animals, there is yet to be a commercial zebrafish rack system designed to accomplish this task. Thus, we invented playpens, an acrylic, and screen container, to raise 12 individual zebrafish juveniles per standard 10 L tank on an existing recirculating fish system. During a week-long experiment, fish raised in playpens grow to the same size as conventionally raised juveniles.

Fish Playpens - Method for raising individual juvenile zebrafish on a recirculating system for studies requiring repeated measures.

Even though many experimental approaches benefit from tracking individual larval animals, there is yet to be a commercial zebrafish rack system designed to accomplish this task. Thus, we invented playpens, an acrylic and screen container, to raise 12 individual zebrafish juveniles per standard 10 L tank on an existing recirculating fish system. During a week-long experiment, fish raised in playpens grow to the same size as conventionally raised juveniles.