Binder Marion, Liebisch Gerhard, Langmann Thomas, Schmitz Gerd
Institute of Clinical Chemistry, University of Regensburg, 93042 Regensburg, Germany.
Institute of Clinical Chemistry, University of Regensburg, 93042 Regensburg, Germany.
J Biol Chem. 2006 Aug 4;281(31):21869-21877. doi: 10.1074/jbc.M603025200. Epub 2006 Jun 8.
Currently, the detailed regulation of major pathways of glycerophospholipid synthesis upon cholesterol loading is largely unknown. Therefore, a detailed lipid metabolic profiling using stable isotope-labeled choline, ethanolamine, and serine was performed by quantitative electrospray ionization tandem mass spectrometry (ESI-MS/MS) in free cholesterol (FC), oxidized (Ox-LDL) and enzymatically modified LDL (E-LDL)-loaded primary human skin fibroblasts. As previously described, an adaptive induction of phosphatidylcholine (PC) synthesis via CDP-choline was found upon FC loading. In contrast to PC, CDP-ethanolamine-mediated phosphatidylethanolamine (PE) synthesis was inhibited by FC incubation. Furthermore, FC induced a shift toward polyunsaturated PE and PC species, which was mediated primarily by PE biosynthesis but not PE remodeling, whereas PC species were shifted mainly by fatty acid (FA) remodeling of existing PC. Modified lipoprotein incubation revealed rather different effects on glycerophospholipid synthesis. E-LDL greatly enhanced PC synthesis, whereas Ox-LDL did not change PC synthesis. Addition of different free FAs (FFA) with and without FC coincubation, as major components of E-LDL, clearly indicated an incorporation of FFA into newly synthesized PC and PE species as well as FFA as important driving force for PC synthesis. Because FC and FFA are known to affect lipid membrane properties including membrane curvature, these data support that CTP:phosphocholine cytidylyl-transferase activity and consequently PC synthesis are regulated by modulation of membrane characteristics at the cellular level. In conclusion, the application of high throughput metabolic profiling of major glycerophospholipid pathways by ESI-MS/MS is a powerful tool to unravel mechanisms underlying the regulation of cellular lipid metabolism.
目前，胆固醇负荷时甘油磷脂合成主要途径的详细调控情况很大程度上尚不清楚。因此，我们通过定量电喷雾电离串联质谱法（ESI-MS/MS），在游离胆固醇（FC）、氧化低密度脂蛋白（Ox-LDL）和酶修饰低密度脂蛋白（E-LDL）负载的原代人皮肤成纤维细胞中，使用稳定同位素标记的胆碱、乙醇胺和丝氨酸进行了详细的脂质代谢谱分析。如前所述，在FC负载时发现通过CDP-胆碱途径适应性诱导了磷脂酰胆碱（PC）的合成。与PC相反，FC孵育抑制了CDP-乙醇胺介导的磷脂酰乙醇胺（PE）合成。此外，FC诱导向多不饱和PE和PC种类转变，这主要由PE生物合成介导而非PE重塑，而PC种类的转变主要通过现有PC的脂肪酸（FA）重塑。修饰脂蛋白孵育对甘油磷脂合成显示出相当不同的影响。E-LDL极大地增强了PC合成，而Ox-LDL未改变PC合成。作为E-LDL的主要成分，添加有无FC共孵育的不同游离脂肪酸（FFA），清楚地表明FFA掺入新合成的PC和PE种类中，以及FFA是PC合成的重要驱动力。由于已知FC和FFA会影响脂质膜特性包括膜曲率，这些数据支持CTP：磷酸胆碱胞苷转移酶活性以及因此的PC合成是通过细胞水平膜特性的调节来调控的。总之，通过ESI-MS/MS对主要甘油磷脂途径进行高通量代谢谱分析是揭示细胞脂质代谢调控机制的有力工具。
