Differential roles of de novo sphingolipid biosynthesis and turnover in the "burst" of free sphingosine and sphinganine, and their 1-phosphates and N-acyl-derivatives, that occurs upon changing the medium of cells in culture.
Long-chain (sphingoid) bases are highly bioactive intermediates for sphingolipid metabolism, yet relatively little is known about how the amounts of these compounds are regulated. This study used J774A.1 cells to characterize the "burst" of sphinganine and sphingosine, or the transient increase of up to 10-fold in long-chain base mass, that occurs when cells in culture are changed to fresh medium. The increase in sphinganine was attributable to de novo sphingolipid biosynthesis because: 1) there is increased incorporation of [3H]serine and [3H]palmitate into sphinganine; 2) the incorporation of [3H]serine was equivalent to the increase in sphinganine mass; 3) beta-F-alanine, an inhibitor of serine palmitoyltransferase, blocked the sphinganine burst; 4) the magnitude of the burst depended on the concentration of serine in the medium, which is known to affect long-chain base biosynthesis; and 5) the appearance of sphinganine was relatively unaffected by lyso-osmotrophic agents (NH4Cl and chloroquine) that blocked sphingolipid hydrolysis in these cells. In contrast, the sphingosine burst arose mainly from turnover of complex sphingolipids because no incorporation of [3H]serine or [3H]palmitate into sphingosine was detected; sphingosine mass was not affected by beta-F-alanine or the serine concentration; and, the burst could be followed by the release of sphingosine and ceramide from complex sphingolipids (especially sphingomyelin) in a process that was inhibited by NH4Cl and chloroquine. Additionally, the fate of these long-chain bases differed: sphinganine was mostly (80-85%) acylated and incorporated into dihydroceramide and complex sphingolipids, whereas most of the sphingosine (70%) was phosphorylated and degraded, with incorporation of the resulting ethanolamine phosphate into phosphatidylethanolamine. Sphinganine, however, could be diverted toward degradation by adding an inhibitor of N-acylation (fumonisin B1). In accounting for the elevation in sphingosine and sphinganine after cells are changed to new medium, these studies have provided fundamental information about long-chain base metabolism. The existence of differential changes in sphinganine and sphingosine, as well as their 1-phosphates and N-acyl-derivatives, should be considered when evaluating the roles of sphingolipid metabolites in cell regulation.
Smith
E R
ER
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Merrill
A H
AH
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003470:Culture Media; D051379:Mice; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000096:biosynthesis; D012694:Serine / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*
D003470:Culture Media; D010169:Palmitic Acids; D010714:Phosphatidylethanolamines; D013107:Sphingolipids; D000643:Ammonium Chloride; D019308:Palmitic Acid; D012694:Serine; D002738:Chloroquine; D013110:Sphingosine; C005682:safingol