Abstract
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.
Highlights
In contrast to native LDL, which contains about two-thirds of total cholesterol as CE, enzymatic modification of LDL with trypsin and cholesteryl esterase decreased the proportion of CE to about one-third
Established assays based on ESI-MS/MS for high throughput lipid quantification (24 –26) were used to study glycerophospholipid metabolism in fibroblasts loaded with free cholesterol (FC), enzymatic digestion of LDL (E-LDL), and oxidized LDL (Ox-LDL), respectively
As expected PE methylation was not observed, because a relevant contribution to PC synthesis so far only has been described for liver, retina, and brain [27,28,29]
Summary
The emerging non-aggregated and non-opsonized minimally oxidized LDL (Ox-LDL) is preferentially internalized via receptor-mediated uptake through clathrin-coated pits [4, 5] Another modification relates to the enzymatic digestion of LDL (E-LDL) retained in the vessel wall. Cholesterol loading induces sphingomyelin (SM) synthesis [15, 16] Another adaptive cellular response is a species shift upon FC loading for PC and phosphatidylethanolamine (PE) from saturated and monounsaturated toward polyunsaturated species [17]. This increases membrane fluidity because unsaturated species have a lower gel-to-liquid-crystalline phase transition temperature Tm compared with saturated species [18]. Because no precise information about the influence of cholesterol loading on glycerophospholipid metabolism are available, our aim was to perform a detailed metabolic profiling of the major glycerophospholipid metabolism pathways using electrospray ionization tandem mass spectrometry
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