Up-regulation of Neutral Glycosphingolipid Synthesis upon Long Term Inhibition of Ceramide Synthesis by Fumonisin B1

Abstract

In a previous study we observed that long term (5 days) incubation with fumonisin B1 (FB1), an inhibitor of acylation of sphingoid long chain bases to (dihydro)ceramide, resulted in morphological and biochemical changes in 3T3 fibroblasts (Meivar-Levy, I., Sabanay, H., Bershadsky, A. D., and Futerman, A. H. (1997) J. Biol. Chem. 272, 1558-1564). Among these were changes in the profile of synthesis of sphingolipids (SLs) and glycosphingolipids (GSLs). Whereas [3H]globotriaosylceramide ([3H]Gb3) comprised 1.9% of the total [3H]SLs and [3H]GSLs synthesized in control cells, it comprised 16. 5% in FB1-treated cells. We now demonstrate by in vitro analysis that inhibition of ceramide synthesis by FB1 for 5 days results in up-regulation of the activities of three enzymes in the pathway of Gb3 synthesis, namely glucosylceramide, lactosylceramide, and Gb3 synthases; up-regulation is due to an increase in Vmax, with no change in Km values toward lipid substrates. Moreover, molecular analysis (reverse transcriptase-polymerase chain reaction) of glucosylceramide synthase indicated that this enzyme is up-regulated at the transcriptional level. No changes in either the Vmax or Km values of sphingomyelin or of GM3 synthase were detected after FB1 treatment. Analysis of SL and GSL synthesis in cultured cells using [4,5-3H]sphinganine as a metabolic precursor demonstrated that at low substrate concentrations, Gb3 synthesis is favored over GM3 synthesis and glucosylceramide synthesis is favored over sphingomyelin synthesis, whereas the opposite is true at high substrate concentrations. These data demonstrate that GSL synthesis and in particular Gb3 synthesis are tightly regulated in fibroblasts, presumably so as to maintain constant levels of Gb3 on the cell surface.