Elucidating the kinetic and thermodynamic insight into regulation of glycolysis by lactate dehydrogenase and its impact on tricarboxylic acid cycle and oxidative phosphorylation in cancer cells

Siying Zeng
Yuqi Wang
Minfeng Ying
Chengmeng Jin
Chang Ying
Di Wang
Hao Wu author has email address
Xun Hu author has email address

Cancer Institute (Key Laboratory for Cancer Intervention and Prevention, China National Ministry of Education, Zhejiang Provincial Key Laboratory of Molecular Biology in Medical Sciences), The Second Affiliated Hospital, Zhejiang University School of Medicine, China
Cancer Center, Zhejiang University, China
Department of Gastroenterology, The Second Affiliated Hospital, Zhejiang University School of Medicine, China

https://doi.org/10.7554/eLife.99576.1

Open access
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Figures and data

The effects of LDHA or LDHB knockout on LDH activity, glycolysis, glucose carbon into TCAC and OXPHOS in HeLa cells.
(A) Representative Western Blot of LDHA and LDHB and relative LDH activity in the cell lysate of HeLa-Ctrl, HeLa-LDHAKO, and HeLa-LDHBKO. (B) Glucose consumption rate and lactate generation rate. Cells were cultured in complete RPMI-1640 medium in a CO₂ incubator for 6 hours, and then the medium concentrations of glucose and lactate were determined as described in Materials and Methods. (C-F) Tracing glucose carbon to pyruvate, lactate, alanine, serine, and glycine. Cells were cultured in complete RPMI-1640 medium containing 6 mM [¹³C₆]glc in a CO₂ incubator for 6 hours, and then the percentages of isotopologues of pyruvate, lactate, alanine, serine, and glycine in cells and in medium were determined by LC-MS/MS as described in Materials and Methods (Table supplement 13). (G) The concentrations of glucose, lactate, and the glycolytic intermediates in cells (Table supplement 14). (H) ΔG of the reactions in the glycolytic pathways (Table supplement 15). (I) Free NADH/NAD⁺ in cells represented by ratiometric SoNar. Representative fluorescent confocal microscopic images of cells transfected with SoNar (left) and the statistics (right). Scale bar: 10μm. (J) Tracing glucose carbon to TCAC intermediates (citrate, α-KG, succinate, fumarate, malate). Cells were cultured in complete RPMI-1640 medium containing 6 mM [¹³C₆]glc in a CO₂ incubator for 6 hours, and then the percentages of isotopologues of the TCAC intermediates in cells were determined by LC-MS/MS as described in Materials and Methods. (K) OCR, measured as described in Materials and Methods. Data are mean ± SD from 3 independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001.

The effect of GNE-140 on LDH activity and glycolysis in HeLa cells.
(A & B) Ki of GNE-140 toward LDHA and LDHB. The Ki values were determined as described in Materials and Methods. (C) The effect of GNE-140 on cellular glucose consumption and lactate generation. Cells were cultured in complete RPMI-1640 medium in a CO₂ incubator for 6 hours, and then the medium concentrations of glucose and lactate were determined as described in Materials and Methods. (D) The curves of LDH activity versus GNE-140 concentration, which is generated based on Ki values. Data are mean ± SD from 3 independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001.

The effect of GNE-140 on the glycolytic pathway in HeLa-LDHBKO cells.
(A) The effect of GNE-140 on the free NADH/NAD⁺. Representative fluorescent confocal microscopic images of cells transfected with SoNar (left) and the response of free NADH/NAD⁺ to GNE-140 (right). Scale bar: 10μm. (B) The relationship between the free NADH/NAD⁺ and glycolytic rate. (C) The effect of GNE-140 on the concentrations of glucose, lactate, and the glycolytic intermediates in cells (Table supplement 9). (D) The effect of GNE-140 on the ΔG of reactions in the glycolytic pathways (Table supplement 16). (E-I) Tracing glucose carbon to serine, glycine, pyruvate, lactate, and alanine. Cells were cultured in complete RPMI-1640 medium containing 6 mM [¹³C₆]glc with or without GNE-140 in a CO₂ incubator for 6 hours, and then the percentages of isotopologues were determined by LC-MS/MS as described in Materials and Methods (Table supplement 17). Data are mean ± SD from 3 independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001.

The effect of GNE-140 on the glycolytic pathway in HeLa-LDHBKO cells under hypoxia.
(A) The glucose consumption rate and lactate generation rate in cells under normoxia and hypoxia (1% oxygen) and the response to GNE-140. (B) The effect of GNE-140 on the concentrations of glucose, lactate, and the glycolytic intermediates in cells under hypoxia (Table supplement 10). (C) The effect of GNE-140 on the ΔG of the reactions in the glycolytic pathways under hypoxia (Table supplement 18). Data are mean ± SD from 3 independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001.

The effect GNE-140 on TCAC and OXPHOS in HeLa-LDHBKO cells.
(A-K) Tracing glucose carbon to TCAC intermediates (citrate, α-KG, succinate, fumarate, malate). Cells were cultured in complete RPMI-1640 medium containing 6 mM [¹³C₆]glc with or without GNE-140 in a CO₂ incubator for 6 hours, and then the percentages of isotopologues of the TCAC intermediates in cells were determined by LC-MS/MS as described in Materials and Methods. (A-E) The total ¹³C labeling of the TCAC intermediates. (F) A metabolic diagram of the isotope labeling of TCAC when [¹³C₆]glc was used as labeling substrate. (G-K) The isotope labeling pattern of the TCAC intermediates, including m2 isotopologues% and the sum of other isotopologues% (m1 + m3 + m4 + m5 + m6 for citrate, m1 + m3 + m4 + m5 for α-KG, m1 + m3 + m4 for succinate/fumarate/malate). (L-V) Tracing glutamine carbon to TCAC intermediates (citrate, α-KG, succinate, fumarate, malate). Cells were cultured in complete RPMI-1640 medium containing 2 mM [¹³C₅]gln with or without GNE-140 in a CO₂ incubator for 6 hours, and then the percentages of isotopologues of the TCAC intermediates in cells were determined by LC-MS/MS as described in Materials and Methods. (L) A metabolic diagram of the isotope labeling of TCAC when [¹³C₅]gln was used as labeling substrate. (M-Q) The total ¹³C labeling of the TCAC intermediates. (R-V) The isotope labeling pattern of the TCAC intermediates, including m5 α-KG%, m4 succinate%, m4 fumarate%, m4 malate%, m4 citrate%, m5 citrate%, and the sum of other isotopologues% (m1 + m2 + m3 + m4 for α-KG, m1 + m2 + m3 for succinate/fumarate/malate, m1 + m2 + m3 + m6 for citrate). (W) OCR with or without GNE-140, measured as described in Materials and Methods. Data are mean ± SD from 3 independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001.

The effect of GNE-140 on energy production, redox state, and cell survival in HeLa-LDHBKO cells under normoxia and hypoxia.
(A) ATP generation rate from glycolysis, OXPHOS, and substrate-level phosphorylation in TCAC, according to the methods described in Materials and Methods. Under hypoxia, we assume OXPHOS is negligible. (B) Cellular concentrations of ATP, ADP, and AMP (Table supplement 9 & 10). (C) Cellular concentrations of NAD⁺ and NADH (Table supplement 9 & 10). (D) Cellular concentrations of NADP⁺ and NADPH. (E) Cellular concentrations of GSH and GSSG. (F) Cell growth curves and cell death assays under normoxia. (G) Cell growth curves and cell death assays under hypoxia. Data are mean ± SD from 3 independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001.

Schematic diagrams to demonstrate the impact on glycolysis, TCAC, and OXPHOS via regulation of LDH.
(A) Tumor cells consume glucose through glycolysis. A significant portion of the pyruvate generated during glycolysis is converted into lactate, while a fraction enters the TCAC. This metabolic pathway aids in the production of electron donors such as NADH and FADH₂, subsequently participating in OXPHOS. (B) When LDH is inhibited, it triggers a series of biochemical changes in the glycolytic pathway, resulting in the inhibition of glycolysis: the concentration of free NADH increases while the concentration of free NAD⁺ decreases; the decreased concentration of free NAD⁺ perturbs the activity of GAPDH, disrupting the balance of glycolytic intermediates, causing a marked increase of the concentrations in FBP, DHAP, GA3P, and significant decrease of the concentration in 3PG, leading to significant changes of ΔGs of the reactions catalyzed by PFK1, aldolase, TPI, and PGAM in the glycolytic pathway, favoring the reverse direction of these reactions. As the overall rate of pyruvate production decreases without significant proportion of pyruvate redirecting to mitochondrial metabolism, the flux of glucose carbon into the TCAC decreases, and the anaplerosis of glutamine to TCAC intermediates also decreases. Consequently, the TCAC and OXPHOS are inhibited. (C) Inhibiting LDH causes energy crisis and redox imbalance, particularly accentuated under hypoxia versus normoxia. As a result, inhibition of LDH activity inhibits growth under normoxia while kills cancer cells under hypoxia.

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