Induction of glucosylceramide synthase by synthase inhibitors and ceramide

Glucosylceramide (GlcCer) synthase acts on the sphingolipid, ceramide, to transer a glucose moiety from UDP-glc, thus forming the first member of a large family of glucosphingolipids. Two inhibitors of the enzyme, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) and N-butyldeoxynojirimycin (NBDN), have been found to induce an elevated level of the synthase in MDCK cells. In cells treated with 20 muM PDMP, then assayed for synthase activity under conditions in which the absorbed PDMP was partially diluted out, the assay showed that the enzyme's specific activity had risen considerably in only 1 h and reached a maximum of about three times the control activity within 6 h. Both cycloheximide and actinomycin D, inhibitors of translational and transcriptional protein synthesis, caused much of the synthase activity to disappear in 6 h, presumably because of normal catabolic destruction. However, simultaneous inclusion of PDMP or NBDN in the cell medium slowed the rate of synthase disappearance. L-Cycloserine, which blocked the synthesis of ceramide, nevertheless allowed PDMP to elevate the synthase activity. Thus the inductive effect appears to be due, in part at least, to resistance of the enzyme-inhibitor complex to the normal process of enzyme degradation. Two other inhibitors of GlcCer synthase, more active than PDMP, did not produce detectable induction because they could not be dissociated from the enzyme during the cell washing and diluting steps. Agents that produced a large increase in endogenous cell ceramide level (DL-erythro-PDMP,N-acetylsphingosine, and bacterial sphingomyelinase) also induced an elevated level of GlcCer synthase. The latter two agents did not protect the synthase from catabolism in the presence of cycloheximide. These findings suggest the existence of a second mechanism of enzyme induction, enhanced synthesis of the enzyme due to the increased availability of the enzyme's lipoidal substrate. The possibility is raised that events involving ceramide in cell signalling may be mediated in part by changes in glucosphingolipid levels.