Glycerol suppresses glucose consumption in trypanosomes through metabolic contest
Fig 3
Glycerol down-regulates GK expression but does not affect preference for glycerol over glucose.
(A) Glycerol kinase (GK), hexokinase (HK), and malic enzyme (ME) activity determined in total cell extracts of EATRO1125.T7T procyclic trypanosomes grown in glucose-rich (Glc/−), glycerol-rich (−/Glyc) or glucose/glycerol-depleted (−/−) conditions. (B) Western blot analysis of procyclic cells grown in glucose-rich medium (lane 0), then in glycerol-rich medium (in the absence of glucose and in the presence of N-acetyl-D-glucosamine) for 48 h, before reintroducing glucose (without glycerol and N-acetyl-D-glucosamine) for 48 h. The immune sera used against GK (αGK), pyruvate phosphate dikinase (αPPDK), and glyceraldehyde-3-phosphate dehydrogenase (αGAPDH) are indicated on the left margin. The bottom panel is a quantitative analysis of the GK signal indicated by an arrow (n = 4). (C) Metabolic end products of PCF trypanosomes from metabolism of [U-13C]-glycerol (13C-Glyc) and/or glucose (Glc) measured by proton nuclear magnetic resonance spectrometry (the values are calculated from the data presented in S2 Table). (D) Expression of the recoded (GKrec) and native GK in the wild-type (WT), RNAiGKcst, and tetracycline-induced (.i) and uninduced (.ni) RNAiGKcst/OEGKrec cell lines monitored by Western blotting on total cell extracts using immune sera against GK (αGK) and paraflagellar rod (αPFR) as control (top panel), and GK activity assay normalized with malic enzyme activity and expressed as a percentage of activity in the WT cells (bottom panel, n = 2). Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.