Characterization of ceramide synthesis - A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine at the level of dihydroceramide

Ceramide (N-acylsphingosine) biosynthesis has been proposed to involve introduction of the 4,5-trans-double bond of sphingosine after synthesis of dihydroceramide (i.e. N-acylsphinganine). For the first time, the in vitro conversion of dihydroceramide to ceramide has been demonstrated using rat liver microsomes and N-[1-C-14]octanoyl-D-erythro-sphinganine (st-H,Cer) and either NADH or NADPH as co-substrate; the apparent K-m values for st-H,Cer and NADH were 340 and 120 mu M, respectively. Molecular oxygen is required for enzymatic activity, and cyanide, divalent copper, as well as antibodies raised against cytochrome b(5) are inhibitory, which suggests that this enzyme should be named dihydroceramide desaturase based on these similarities with the mechanism of Delta(9)-desaturase (stearoyl-CoA desaturase). Factors that influenced the activity of dihydroceramide desaturase include the alkyl chain length of the sphingoid base (in the order C-18 > C-12 > C-8) and fatty acid (C-8 > C-18); the stereochemistry of the sphingoid base (D-erythro- > L-threo-dihydroceramides); the nature of the headgroup, with the highest activity with dihydroceramide, but some (similar to 20%) activity with dihydrosphingomyelin (activity was not detected with dihydroglucosylceramide, however); and the ability to utilize alternative reductants (ascorbic acid could substitute for a reduced pyridine nucleotide, but was inhibitory at higher concentrations). Dihydroceramide desaturase was inhibited by dithiothreitol, which suggests that it might be possible to alter ceramide synthesis by varying the thiol status of hepatocytes, Consistent with this hypothesis, when rat hepatocytes were cultured in varying concentrations of N-acetylcysteine (5 and 10 mM), there was a decrease in the relative incorporation of [C-14]serine into [C-14]ceramide. These studies have conclusively established the pathway of ceramide synthesis via desaturation of dihydroceramide and have uncovered several properties of this reaction that warrant further consideration for their relevance to both sphingolipid metabolism and signaling.