Conversion of dihydroceramide into ceramide: involvement of a desaturase

Ceramide has been suggested to be a potent bioactive lipid involved in cell growth, differentiation and apoptosis. Its precursor, dihydroceramide, does not affect these processes. The truncated dihydroceramide analogues N-hexanoyl-[4,5-3H]-d-erythro-sphinganine and N-[1-14C]-hexanoyl-d-erythro-sphinganine were used to study the conversion of dihydroceramide into ceramide by rat hepatocytes. The formation of tritiated water after the addition of the tritiated substrate to intact and permeabilized rat hepatocytes was followed to measure enzyme activity. Desaturation was severely depressed in permeabilized hepatocytes, suggesting loss of cofactors. Of a variety of cofactors tested in the permeabilized cells, NADPH appeared to be stimulatory, pointing to the involvement of a desaturase. In agreement with this, the addition of inhibitors and redox effectors known to affect Delta9-stearoyl-CoA desaturase and Delta1-plasmanyl-ethanolamine desaturase to intact cells resulted in severe inhibition of the desaturation. When added to permeabilized cells fortified with NADPH, these compounds counteracted the NADPH stimulation. The enzyme system was further studied in broken cells. On cell fractionation, the activity was recovered in the microsomal fraction. The results indicate that the conversion of dihydroceramide into ceramide is ctalysed by a desaturase and not by a dehydrogenase or an oxidase as was generally believed.