Studies of compounds that enhance sphingolipid metabolism in human keratinocytes

Several products are known to inhibit the biosynthesis of ceramides and glucosylceramides, but very few stimulate this process. We studied the influence of a hydrolysate of potato proteins (Lipidessence) in vitro on the sphingolipid metabolism of normal human epidermal keratinocytes. By measuring growth with the thymidine uptake assay, it was seen that Lipidessence, added in the culture medium up to an 8% concentration, did not change significantly the proliferation rate of keratinocytes, but beyond this concentration a progressive dose-dependent inhibition of growth was noticeable. Following incubation of cells with the product at 5% and 10% concentrations for 2 days, the lipids were extracted. The different lipid classes were separated by fractionation on columns of aminopropyl silica gel and analyzed by high-performance thin-layer chromatography. When keratinocytes were cultivated in the presence of Lipidessence, the biosynthesis of cholesterol, phosphatidylcholine, phosphatidylserine and gangliosides was stimulated, and a major increase was noticeable in the biosynthesis of free fatty acids, free ceramides, glucosylceramide and sphingomyelin. Radioactive [(14)C]-serine was used as a precursor of sphingoid bases to study sphingolipid biosynthesis. After migration of lipid fractions on thin-layer plates, autoradiography showed that free ceramides and glucosylceramide were labeled, thus suggesting that de novo biosynthesis was accounting for the increased cellular content in sphingolipids.