Fig. 4.

Inhibition of GlcCer synthesis promotes G. lamblia lipid turnover. Isolated parasites were labeled with [³H]palmitic acid or [³H]glucose for 3 h in presence of solvent (cntl) or 10 μM PPMP. [³H] incorporation was measured in both whole cell (TOT) and lipid extracts by liquid scintillation and normalized by protein content. A: Incorporation is expressed as percentage of untreated samples (cntl, dashed line); data are average ± SE (n = 9) of three experiments done in triplicate. Note that PPMP treatment increased [³H]palmitic acid incorporation both in whole cells and in the lipid fraction, while only in the lipid fraction in the case of [³H]glucose. B: [³H] incorporation in the lipid fraction is expressed as percentage of whole cell-associated precursors. Note the readily incorporation into lipids of [³H]palmitic acid, compared with [³H]glucose and the increased [³H]glucose incorporation upon PPMP treatment. C: Comparison of [³H]palmitic acid incorporation in G. lamblia extracts as described in panel A upon treatment with solvent (cntl), 10 μM PPMP, 50 μM fumonisin B1 (FB1), 20 μM myriocin (Myr), 500 μM L-cycloserine (L-cyc), 400 μM NB-DNJ, and 60 μM tunicamycin (TM). Incorporation is expressed as percentage of untreated samples (cntl, dashed line); data are average ± SE (n = 9) of three experiments done in triplicate. D: Isolated parasites were labeled with 4 μCi/ml of [³H]palmitic acid for 3 h in presence of solvent (cntl), 10 μM PPMP (PP) or 500 μM C6 ceramide (C6). Lipid aliquots corresponding to equal protein amount were separated using the solvent system D. (a) Representative 1D-HPTLC. DAG, diacylglycerols, Ori, origin. (b) Quantification of lipid levels expressed as percentage of untreated samples (cntl); data are average ± SE (n = 6) of two experiments done in triplicate. (c) Ratio of 1,2 and 1,3 DAG upon C6 ceramide treatment. Data are average ± SE (n = 6) of two experiments done in triplicate. Note the increase in 1,2 DAG level in PPMP and C6 ceramide treated samples.