Different rates of flux through the biosynthetic pathway for long-chain versus very-long-chain sphingolipids

摘要

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions. The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions. Sphingolipids (SLs) are important membrane components that have become the focus of great interest over the past couple of decades due to their roles in membrane structure, cellular signaling, and human disease (1Futerman, A. H., Sphingolipids In Biochemistry of Lipids, Lipoproteins and Membranes. 6th edition. N. D., Ridgway and R. S., McLeod, editors. Elsevier, Boston. 297–326.Google Scholar). In human disease, SL levels are altered either as the cause of the disease (such as in the SL storage diseases) or as a secondary response in diseases as diverse as cancer (2Saddoughi S.A. Ogretmen B. Diverse functions of ceramide in cancer cell death and proliferation.Adv. Cancer Res. 2013; 117: 37-58Crossref PubMed Scopus (125) Google Scholar, 3Jensen S.A. Calvert A.E. Volpert G. Kouri F.M. Hurley L.A. Luciano J.P. Wu Y. Chalastanis A. Futerman A.H. Stegh A.H. Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.Proc. Natl. Acad. Sci. USA. 2014; 111: 5682-5687Crossref PubMed Scopus (65) Google Scholar), epilepsy (4Mosbech M-B. Olsen A.S.B. Neess D. Ben-David O. Klitten L.L. Larsen J. Sabers A. Vissing J. Nielsen J.E. Hasholt L. et