Glycerol suppresses glucose consumption in trypanosomes through metabolic contest
Fig 1
Procyclic trypanosomes prefer glycerol to glucose.
(A) Schematic representation of glycerol (black) and glucose (blue) metabolism in procyclic form (PCF) trypanosomes. The metabolic end products are shown in rectangles, and metabolites analyzed by ion chromatography high-resolution mass spectrometry (IC-HRMS) are underlined and in italic (a–g). The ATP molecules consumed and produced by substrate-level phosphorylation are shown, as well as the enzymes hexokinase (HK) and glycerol kinase (GK). (B) Glucose and glycerol consumption by PCF trypanosomes incubated in glucose (2 mM), glycerol (2 mM) and glucose + glycerol (2 mM each) conditions. (C) Metabolic end products produced by PCF trypanosomes from [U-13C]-glycerol (13C-Glyc) and/or glucose (Glc), as measured by proton nuclear magnetic resonance (1H-NMR) spectroscopy (the values are calculated from the data presented in S1 Table). (D) IC-HRMS analyses of intracellular metabolites collected from PCF trypanosomes after incubation with 2 mM [U-13C]-labeled carbon sources in the presence or not of unlabeled carbon sources, as indicated on the right margin. The figure shows the proportion (%) of molecules having incorporated 0 to 6 13C atoms (m0 to m6, color code indicated on the left margin). G6P (a), glucose 6-phosphate; F6P (b), fructose 6-phosphate; M6P (c), mannose 6-phosphate; F1,6BP (d), fructose 1,6-bisphosphate; Gly3P (e), glycerol 3-phosphate; 2/3PG, 2- or 3-phosphoglycerate (which are not undistinguished by IC-HRMS); PEP (g), phosphoenolpyruvate. (E) Western blot analysis of total protein extracts from the parental (wild-type [WT]) and tetracycline-induced (.i) or uninduced (.ni) RNAiGK cell line probed with anti-GK (αGK) and anti-paraflagellar-rod (αPFR) immune sera. The table below the blots shows the relative levels of GK expression in 5 × 106 (1), 5 × 105 (/10) and 105 (/50) parental cells, and 5 × 106 RNAiGK.ni and RNAiGK.i cells, as well as the corresponding GK activity. ND, not detectable. (F and G) Glucose and glycerol consumption by the (F) tetracycline-induced RNAiGK (RNAiGK.i) and (G) uninduced RNAiGK (RNAiGK.ni) mutant cell lines incubated in glucose (2 mM), glycerol (2 mM) and glucose + glycerol (2 mM each) conditions. (H) Production of metabolic end products by the parental (WT), RNAiGK.ni, and RNAiGK.i cell lines from [U-13C]-glycerol (13C-Glyc) and/or glucose (Glc), as measured by 1H-NMR spectroscopy (the values are calculated from the data presented in S1 Table). Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.