Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress

Abstract

Background: Eukaryotic cells have evolved various response mechanisms to counteract the deleterious consequences of oxidative stress. Among these processes, metabolic alterations seem to play an important role.

Results: We recently discovered that yeast cells with reduced activity of the key glycolytic enzyme triosephosphate isomerase exhibit an increased resistance to the thiol-oxidizing reagent diamide. Here we show that this phenotype is conserved in Caenorhabditis elegans and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H) pool. Remarkably, another key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to be inactivated in response to various oxidant treatments, and we show that this provokes a similar redirection of the metabolic flux.

Conclusion: The naturally occurring inactivation of GAPDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract oxidative stress. As a consequence, altering the homoeostasis of cytoplasmic metabolites is a fundamental mechanism for balancing the redox state of eukaryotic cells under stress conditions.

Figure 1

Reduced triosephosphate isomerase (TPI) activity increases oxidant resistance of S. cerevisiae and C. elegans. (a) The left panel shows a Western blot analysis of yeast cells expressing wild-type human TPI under the control of promoters of different strengths: GPD1 (GPD_pr), TEF1 (TEF_pr), and CYC1 (CYC_pr). Yeast cells expressing human TPI_Ile170Val or yeast TPI under the control of the strong GPD1 promoter were used as controls. Equal loading of the lysates was controlled by visualizing G6PDH. The right panel shows yeast cells expressing yeast TPI and human TPIIle170Val controlled by the GPD1 promoter or yeast expressing wild-type human TPI controlled by the GPD1, TEF1 or CYC1 promoters, respectively. Yeast were spotted as fivefold serial dilutions on SC medium supplemented with different concentrations of diamide. Plates were incubated at 30°C for 3 days. (b) The left panel shows western blot analysis of cell extracts prepared from adult C. elegans that were fed with E. coli producing double-stranded RNA of the C. elegans tpi-1 gene (Y17G7B.7) (tpi-1 RNAi) or harboring the empty plasmid L4440 (control). The right panel shows the effects of the oxidants juglone and diamide on these worms. After feeding with E. coli as described above, worms were placed on agar plates supplemented with juglone or diamide. Multi-resistant daf-2 (e1370) mutant worms were included in every experiment as controls.

Figure 2

Reduced TPI activity protects against diamide by increasing the metabolic flux through the PPP. (a) Schematic illustration of a subset of biochemical reactions of the glycolytic pathway (left) and the associated pentose phosphate pathway (right). Solid lines represent direct, one-step biochemical reactions, and indirect, multi-step reactions are represented as dotted lines. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (b) Yeast deletion strains Δtpi1Δzwf1, Δtpi1Δsol3, and Δtpi1Δsol4 expressing wild-type human TPI or TPI_Ile170Val were spotted as fivefold serial dilutions on synthetic media supplemented with different concentrations of diamide, and plates were incubated at 30°C.

Figure 3

Reduced TPI activity protects against diamide by increasing NADPH. (a) S. cerevisiae strains MR101 and MR105 were grown in duplicate to mid-log phase, pyridine nucleotides were extracted, and LC-MS/MS measurements were performed in triplicate. MR105 cells expressing TPI_Ile170Val had a higher overall NADPH/NADP⁺ ratio compared with MR101 cells expressing wild-type TPI. (b) S. cerevisiae strain BY4741 was transformed with an empty 2μ plasmid or with a 2μ plasmid encoding K. lactis GDP1 (p1696). Afterwards, single transformants were selected, grown overnight and the same number of cells were spotted as fivefold serial dilutions on agar plates supplemented with different concentrations of diamide. Growth was monitored after plates were incubated at 30°C for 3 days. (c) The isogenic yeast strains MR101 expressing wild-type human TPI or MR105 expressing human TPI_Ile170Val were transformed with plasmids for expression of K. lactis GDP1 and processed as described in (b).

Figure 4

TPI and GAPDH inactivation increases the concentration of PPP metabolites. (a) For quality control of the metabolite quantifications and for analyzing the technical reproducibility, each metabolite was measured in duplicate (top panel). For analyzing the biological reproducibility, the metabolite concentrations were measured from cultures grown in parallel (bottom panel). Please note that for the purpose of illustration values greater than 10 are not shown. The complete plots are presented in Additional data file 3. (b) Upper panel, changes in metabolite levels in yeast strains with differing TPI activity. Lysates of yeast strains BY4741 (100% TPI activity), MR101 (70% TPI activity) and MR105 (20% TPI activity) were prepared and metabolites were quantified by LC-MS/MS. The absolute metabolite concentrations of MR101 and MR105 yeast were normalized and plotted as change given in percent relative to the wild-type (BY4741) strain. Middle panel, changes in metabolite levels in yeast with GAPDH inactivation. Cultures of strain BY4741 were treated with H₂O₂ or left untreated. The relative changes of the various metabolites of the H₂O₂-treated cells in comparison to untreated cells were plotted. Bottom panel, predicted qualitative changes in metabolite concentrations using the non-fitted metabolic model. Note that for technical reasons, the abbreviation g6p refers to the sum of glucose-6-phosphate and fructose-6-phosphate and x5p to the sum of xylulose-5-phosphate and ribulose-5-phosphate. (c) Upper panel, GAPDH activity in yeast cells treated with and without H₂O₂ as in (b). Lower panel, effect of H₂O₂ on wild-type yeast cells transformed with the 2μ plasmids p423GPD, p423GPD-EcoGAP encoding E. coli GAPDH, or p423GPD-TDH3 encoding the yeast GAPDH Tdh3p. Transformants were selected, grown overnight and the same number of cells were spotted as fivefold serial dilutions on SC^-his-ade media supplemented with H₂O₂ as indicated.

Figure 5

In silico model for the interplay of glycolysis and the pentose phosphate pathway in response to GAPDH or TPI inactivation. (a) Predicted changes of the cytoplasmic NADPH/NADP⁺ ratio of the unfitted model. The NADPH/NADP⁺ ratio increases in correlation with the rate of TPI (blue) or GAPDH (red) inactivation. (b) Quantitative accuracy of the metabolic model before and after parameter fitting. Upper panel, 21 measured metabolite concentrations (seven metabolites under three conditions) are plotted against the predicted values before fitting. Lower panel, data versus prediction after parameter fitting. (c) As (a), but after parameter fitting. (d) Comparison of quantitative predictions made with the parameter-fitted and the measured metabolite concentrations. Changes relative to the wild-type strain values are shown. Black and red bars correspond to yeast cells with inactivated GAPDH, green and yellow bars correspond to reduced TPI activity.

Figure 6

Lifespan analysis of S. cerevisiae and C. elegans. (a) The median replicative lifespan of yeast strains BY4741, MR130, MR131, MR136, and MR137 was determined by counting surviving mother cells per generation. (b) For the lifespan analysis of C. elegans the parental and the F1 generation (left panel, 117 wild-type and 80 tpi-1 RNAi animals were analyzed) or the F1 generation only (right panel, 74 wild-type and 47 tpi-1 RNAi animals) were placed on the respective agar plates and survival of the worms was monitored every day.