Department of Cell Biology, University of Texas Southwestern Medical Center.
Department of Cell Biology, University of Texas Southwestern Medical Center;
J Vis Exp. 2021 Feb 2(168). doi: 10.3791/62201.
Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo synthesis of major NL species in yeast is presented. We apply C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.
中性脂质（NLs）是一类疏水性、不带电荷的生物分子，在能量和脂质稳态中发挥关键作用。NLs 是从头从乙酰辅酶 A 合成的，主要以三酰甘油（TGs）和固醇酯（SEs）的形式存在于真核生物中。负责 NLs 合成的酶在酿酒酵母（酵母）到人类之间高度保守，这使得酵母成为研究 NL 代谢酶功能和调节的有用模型生物。虽然人们已经了解了乙酰辅酶 A 如何转化为多种 NL 物种，以及调节 NL 代谢酶的机制，以及失调如何导致细胞病变，但仍在不断发现。几十年来的研究已经开发并使用了许多分离和表征 NL 物种的方法；然而，还没有讨论用于全面表征主要 NL 物种的定量和简单方案。在这里，提出了一种简单且适应性强的方法，用于定量测定酵母中主要 NL 物种的从头合成。我们应用 C-乙酸代谢标记与薄层色谱相结合，以分离和定量多种生理上重要的 NL。此外，这种方法可以很容易地应用于研究 NL 酶的体内反应速率或 NL 物种随时间的降解。
