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J. Biol. Chem., Vol. 281, Issue 31, 21869-21877, August 4, 2006

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Metabolic Profiling of Glycerophospholipid Synthesis in Fibroblasts Loaded with Free Cholesterol and Modified Low Density Lipoproteins^*

From the Institute of Clinical Chemistry, University of Regensburg, 93042 Regensburg, Germany

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.

Received for publication, March 30, 2006 , and in revised form, June 8, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft (Li 923/2-1 and SFB-TR 13/A3). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Inst. of Clinical Chemistry, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany. Tel.: 49-941-944-6201; Fax: 49-941-944-6202; E-mail: gerd.schmitz{at}klinik.uni-regensburg.de.

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