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

Reviewer #1 (Public Review):

Summary:

Zeng et al. have investigated the impact of inhibiting lactate dehydrogenase (LDH) on glycolysis and the tricarboxylic acid cycle. LDH is the terminal enzyme of aerobic glycolysis or fermentation that converts pyruvate and NADH to lactate and NAD+ and is essential for the fermentation pathway as it recycles NAD+ needed by upstream glyceraldehyde-3-phosphate dehydrogenase. As the authors point out in the introduction, multiple published reports have shown that inhibition of LDH in cancer cells typically leads to a switch from fermentative ATP production to respiratory ATP production (i.e., glucose uptake and lactate secretion are decreased, and oxygen consumption is increased). The presumed logic of this metabolic rearrangement is that when glycolytic ATP production is inhibited due to LDH inhibition, the cell switches to producing more ATP using respiration. This observation is similar to the well-established Crabtree and Pasteur effects, where cells switch between fermentation and respiration due to the availability of glucose and oxygen. Unexpectedly, the authors observed that inhibition of LDH led to inhibition of respiration and not activation as previously observed. The authors perform rigorous measurements of glycolysis and TCA cycle activity, demonstrating that under their experimental conditions, respiration is indeed inhibited. Given the large body of work reporting the opposite result, it is difficult to reconcile the reasons for the discrepancy. In this reviewer's opinion, a reason for the discrepancy may be that the authors performed their measurements 6 hours after inhibiting LDH. Six hours is a very long time for assessing the direct impact of a perturbation on metabolic pathway activity, which is regulated on a timescale of seconds to minutes. The observed effects are likely the result of a combination of many downstream responses that happen within 6 hours of inhibiting LDH that causes a large decrease in ATP production, inhibition of cell proliferation, and likely a range of stress responses, including gene expression changes.

Strengths:

The regulation of metabolic pathways is incompletely understood, and more research is needed, such as the one conducted here. The authors performed an impressive set of measurements of metabolite levels in response to inhibition of LDH using a combination of rigorous approaches.

Weaknesses:

Glycolysis, TCA cycle, and respiration are regulated on a timescale of seconds to minutes. The main weakness of this study is the long drug treatment time of 6 hours, which was chosen for all the experiments. In this reviewer's opinion, if the goal was to investigate the direct impact of LDH inhibition on glycolysis and the TCA cycle, most of the experiments should have been performed immediately after or within minutes of LDH inhibition. After 6 hours of inhibiting LDH and ATP production, cells undergo a whole range of responses, and most of the observed effects are likely indirect due to the many downstream effects of LDH and ATP production inhibition, such as decreased cell proliferation, decreased energy demand, activation of stress response pathways, etc.

Comments on revisions:

Based on the response to comments that the authors have submitted, I do not think I need to make any changes to my review, as the time course experiment that could have explained the difference between reported results and extensive prior literature has not been performed.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Zeng et al. have investigated the impact of inhibiting lactate dehydrogenase (LDH) on glycolysis and the tricarboxylic acid cycle. LDH is the terminal enzyme of aerobic glycolysis or fermentation that converts pyruvate and NADH to lactate and NAD+ and is essential for the fermentation pathway as it recycles NAD+ needed by upstream glyceraldehyde-3-phosphate dehydrogenase. As the authors point out in the introduction, multiple published reports have shown that inhibition of LDH in cancer cells typically leads to a switch from fermentative ATP production to respiratory ATP production (i.e., glucose uptake and lactate secretion are decreased, and oxygen consumption is increased). The presumed logic of this metabolic rearrangement is that when glycolytic ATP production is inhibited due to LDH inhibition, the cell switches to producing more ATP using respiration. This observation is similar to the well-established Crabtree and Pasteur effects, where cells switch between fermentation and respiration due to the availability of glucose and oxygen. Unexpectedly, the authors observed that inhibition of LDH led to inhibition of respiration and not activation as previously observed. The authors perform rigorous measurements of glycolysis and TCA cycle activity, demonstrating that under their experimental conditions, respiration is indeed inhibited. Given the large body of work reporting the opposite result, it is difficult to reconcile the reasons for the discrepancy. In this reviewer's opinion, a reason for the discrepancy may be that the authors performed their measurements 6 hours after inhibiting LDH. Six hours is a very long time for assessing the direct impact of a perturbation on metabolic pathway activity, which is regulated on a timescale of seconds to minutes. The observed effects are likely the result of a combination of many downstream responses that happen within 6 hours of inhibiting LDH that causes a large decrease in ATP production, inhibition of cell proliferation, and likely a range of stress responses, including gene expression changes.

Strengths:

The regulation of metabolic pathways is incompletely understood, and more research is needed, such as the one conducted here. The authors performed an impressive set of measurements of metabolite levels in response to inhibition of LDH using a combination of rigorous approaches.

Weaknesses:

Glycolysis, TCA cycle, and respiration are regulated on a timescale of seconds to minutes. The main weakness of this study is the long drug treatment time of 6 hours, which was chosen for all the experiments. In this reviewer's opinion, if the goal was to investigate the direct impact of LDH inhibition on glycolysis and the TCA cycle, most of the experiments should have been performed immediately after or within minutes of LDH inhibition. After 6 hours of inhibiting LDH and ATP production, cells undergo a whole range of responses, and most of the observed effects are likely indirect due to the many downstream effects of LDH and ATP production inhibition, such as decreased cell proliferation, decreased energy demand, activation of stress response pathways, etc.

We thank reviewer for the careful reading of our manuscript, the accurate summary of the prevailing model, and the positive assessment of the rigor of our measurements. We agree that much prior literature reports increased oxygen consumption following LDH inhibition, and we recognize that our finding—coordinated suppression of glycolysis, the TCA cycle, and OXPHOS—differs from this prevailing interpretation. We address below the reviewer’s main concern regarding the 6-hour time point and clarify the conceptual scope of our study.

(1) Scope: steady-state metabolic regulation versus immediate transient effects

The reviewer raises an important point that many metabolic perturbations can trigger rapid, transient responses within seconds to minutes, whereas our measurements were performed after sustained LDH inhibition. We agree that very early time points would be required if the primary goal were to isolate the most immediate, proximal consequence of LDH inhibition before downstream propagation. However, the objective of our study is different: we aim to characterize the metabolic steady state re-established after sustained inhibition of LDH activity, because this adapted steady state is more relevant for understanding long-term metabolic consequences and therapeutic outcomes of LDH inhibition in cancer cells.

(2) Genetic LDHA/LDHB knockout: comparison of two steady states

A related point applies to the LDHA/LDHB knockout models. We fully agree that the knockout process necessarily involves a temporal perturbation during cell line generation and adaptation. Nevertheless, the experimental comparison in our study is explicitly between two steady states: the baseline steady state of control cells and the steady state achieved after stable genetic disruption of LDHA or LDHB. The observation that LDHA or LDHB knockout alone had minimal effects on glycolysis and respiration indicates that partial reduction of LDH activity can be compensated in a steady-state manner, consistent with the exceptionally high catalytic capacity of LDH in cancer cells relative to upstream rate-limiting enzymes.

(3) LDH-activity-dependent quantitative relationships support stable metabolic states

Importantly, our conclusions do not rely on a single inhibitor condition at a single time point. Rather, we established quantitative steady-state relationships between residual LDH activity and pathway behavior across a wide range of LDH inhibition. These LDH-activity-dependent data strongly support that the system resides in stable metabolic states at different degrees of LDH activity, rather than reflecting non-specific collapse due to prolonged stress.

Specifically, we observed that when LDH activity was reduced from 100% to approximately ~9% (e.g., by genetic perturbation and partial pharmacologic inhibition), glucose consumption and lactate production remained essentially unchanged, indicating maintenance of a steady-state glycolytic flux despite substantial LDH inhibition. Only when LDH activity was further reduced below this threshold did glycolytic flux decrease in a graded manner, consistent with a nonlinear control structure (Figure 8 A & B)).

Likewise, the isotope tracing results showed distinct LDH-activity-dependent transitions in TCA cycle labeling patterns. Over the range in which LDH activity decreased from 100% to ~9%, the [¹³C₆]glucose-derived labeling pattern of citrate remained largely unchanged, whereas deeper inhibition led to a decrease in m2 citrate with a compensatory rise in higher-order citrate isotopologues, consistent with altered flux entry versus cycling/retention in the TCA cycle (Figure 8C). Similarly, [¹³C₅]glutamine tracing revealed that deeper LDH inhibition reduced the direct m5 contribution, accompanied by corresponding shifts in other isotopologues (Figure 8D). These graded, quantitative transitions—rather than an abrupt global failure—support the interpretation of distinct metabolic steady states across LDH activity levels, linking LDH inhibition to changes in both glycolysis and mitochondrial metabolism.

(4) Reconciling discrepancies with prior studies

We agree that multiple prior studies have reported increased oxygen consumption or enhanced oxidative metabolism following LDH inhibition in cancer cells. However, we note that this prevailing notion often persists because LDH inhibition is frequently discussed by analogy to the classical Pasteur and Crabtree effects, in which cells toggle between fermentation and respiration depending on oxygen and glucose availability. We believe this analogy can be misleading.

In the Pasteur effect, the metabolic shift is primarily driven by oxygen limitation, i.e., restriction of the terminal electron acceptor for the mitochondrial electron transport chain, which enforces reliance on fermentation. In the Crabtree effect, high glucose availability suppresses respiration through regulatory mechanisms while glycolysis is strongly activated. Both phenomena are fundamentally controlled by oxygen availability and respiratory capacity, rather than by inhibition of a specific cytosolic enzyme.

By contrast, LDH inhibition is mechanistically distinct: it directly perturbs cytosolic redox recycling by limiting NADH-to-NAD⁺ regeneration and can therefore constrain upstream glycolytic flux (particularly at GAPDH) and reshape pathway thermodynamics. Under conditions where LDH inhibition sufficiently limits effective NAD⁺ availability and reduces glycolytic flux into pyruvate, the downstream consequence is reduced carbon input into the TCA cycle and suppressed OXPHOS—consistent with our experimental measurements. We therefore suggest that divergent outcomes reported across studies likely reflect differences in residual LDH activity, cell-type–specific metabolic wiring, and the extent to which glycolytic flux remains sustained versus becoming redox-limited upstream, rather than a universal Pasteur/Crabtree-like “switch” from fermentation to respiration. Accordingly, interpreting LDH inhibition as a Pasteur/Crabtree-like toggle may oversimplify the biochemical consequences of disrupting cytosolic NAD⁺ regeneration.

We have revised the Discussion to clarify this conceptual distinction and to avoid relying on comparisons that are not mechanistically equivalent to LDH inhibition.

Reviewer #2 (Public Review):

Summary:

Zeng et al. investigated the role of LDH in determining the metabolic fate of pyruvate in HeLa and 4T1 cells. To do this, three broad perturbations were applied: knockout of two LDH isoforms (LDH-A and LDH-B), titration with a non-competitive LDH inhibitor (GNE-140), and exposure to either normoxic (21% O2) or hypoxic (1% O2) conditions. They show that knockout of either LDH isoform alone, though reducing both protein level and enzyme activity, has virtually no effect on either the incorporation of a stable 13C-label from a 13C6-glucose into any glycolytic or TCA cycle intermediate, nor on the measured intracellular concentrations of any glycolytic intermediate (Figure 2). The only apparent exception to this was the NADH/NAD+ ratio, measured as the ratio of F420/F480 emitted from a fluorescent tag (SoNar).

The addition of a chemical inhibitor, on the other hand, did lead to changes in glycolytic flux, the concentrations of glycolytic intermediates, and in the NADH/NAD+ ratio (Figure 3). Notably, this was most evident in the LDH-B-knockout, in agreement with the increased sensitivity of LDH-A to GNE-140 (Figure 2). In the LDH-B-knockout, increasing concentrations of GNE-140 increased the NADH/NAD+ ratio, reduced glucose uptake, and lactate production, and led to an accumulation of glycolytic intermediates immediately upstream of GAPDH (GA3P, DHAP, and FBP) and a decrease in the product of GAPDH (3PG). They continue to show that this effect is even stronger in cells exposed to hypoxic conditions (Figure 4). They propose that a shift to thermodynamic unfavourability, initiated by an increased NADH/NAD+ ratio inhibiting GAPDH explains the cascade, calculating ΔG values that become progressively more endergonic at increasing inhibitor concentrations.

Then - in two separate experiments - the authors track the incorporation of 13C into the intermediates of the TCA cycle from a 13C6-glucose and a 13C5-glutamine. They use the proportion of labelled intermediates as a proxy for how much pyruvate enters the TCA cycle (Figure 5). They conclude that the inhibition of LDH decreases fermentation, but also the TCA cycle and OXPHOS flux - and hence the flux of pyruvate to all of those pathways. Finally, they characterise the production of ATP from respiratory or fermentative routes, the concentration of a number of cofactors (ATP, ADP, AMP, NAD(P)H, NAD(P)+, and GSH/GSSG), the cell count, and cell viability under four conditions: with and without the highest inhibitor concentration, and at norm- and hypoxia. From this, they conclude that the inhibition of LDH inhibits the glycolysis, the TCA cycle, and OXPHOS simultaneously (Figure 7).

Strengths:

The authors present an impressively detailed set of measurements under a variety of conditions. It is clear that a huge effort was made to characterise the steady-state properties (metabolite concentrations, fluxes) as well as the partitioning of pyruvate between fermentation as opposed to the TCA cycle and OXPHOS.

A couple of intermediary conclusions are well supported, with the hypothesis underlying the next measurement clearly following. For instance, the authors refer to literature reports that LDH activity is highly redundant in cancer cells (lines 108 - 144). They prove this point convincingly in Figure 1, showing that both the A- and B-isoforms of LDH can be knocked out without any noticeable changes in specific glucose consumption or lactate production flux, or, for that matter, in the rate at which any of the pathway intermediates are produced. Pyruvate incorporation into the TCA cycle and the oxygen consumption rate are also shown to be unaffected.

They checked the specificity of the inhibitor and found good agreement between the inhibitory capacity of GNE-140 on the two isoforms of LDH and the glycolytic flux (lines 229 - 243). The authors also provide a logical interpretation of the first couple of consequences following LDH inhibition: an increased NADH/NAD+ ratio leading to the inhibition of GAPDH, causing upstream accumulations and downstream metabolite decreases (lines 348 - 355).

Weaknesses:

Despite the inarguable comprehensiveness of the data set, a number of conceptual shortcomings afflict the manuscript. First and foremost, reasoning is often not pursued to a logical conclusion. For instance, the accumulation of intermediates upstream of GAPDH is proffered as an explanation for the decreased flux through glycolysis. However, in Figure 3C it is clear that there is no accumulation of the intermediates upstream of PFK. It is unclear, therefore, how this traffic jam is propagated back to a decrease in glucose uptake. A possible explanation might lie with hexokinase and the decrease in ATP (and constant ADP) demonstrated in Figure 6B, but this link is not made.

We appreciate the reviewer's critical comment. In Figure 3C, there is no accumulation of F6P or G6P, which are upstream of PFK1. This is because the PFK1-catalyzed reaction sets a significant thermodynamic barrier. Even with treatment using 30 μM GNE-140, the ∆G_PFK1 (Gibbs free energy of the PFK1-catalyzed reaction) remains -9.455 kJ/mol (Figure 3D), indicating that the reaction is still far from thermodynamic equilibrium, thereby preventing the accumulation of F6P and G6P.

We agree with the reviewer that hexokinase inhibition may play a role, this requires further investigation.

The obvious link between the NADH/NAD+ ratio and pyruvate dehydrogenase (PDH) is also never addressed, a mechanism that might explain how the pyruvate incorporation into the TCA cycle is impaired by the inhibition of LDH (the observation with which they start their discussion, lines 511 - 514).

We agree with the reviewer’s comment. In this study, we did not explore how the inhibition of LDH affects pyruvate incorporation into the TCA cycle. As this mechanism was not investigated, we have titled the study:

"Elucidating the Kinetic and Thermodynamic Insights into the Regulation of Glycolysis by Lactate Dehydrogenase and Its Impact on the Tricarboxylic Acid Cycle and Oxidative Phosphorylation in Cancer Cells."

It was furthermore puzzling how the ΔG, calculated with intracellular metabolite concentrations (Figures 3 and 4) could be endergonic (positive) for PGAM at all conditions (also normoxic and without inhibitor). This would mean that under the conditions assayed, glycolysis would never flow completely forward. How any lactate or pyruvate is produced from glucose, is then unexplained.

This issue also concerned me during the study. However, given the high reproducibility of the data, we consider it is true, but requires explanation. The PGAM-catalyzed reaction is tightly linked to both upstream and downstream reactions in the glycolytic pathway. In glycolysis, three key reactions catalyzed by HK2, PFK1, and PK are highly exergonic, providing the driving force for the conversion of glucose to pyruvate. The other reactions, including the one catalyzed by PGAM, operate near thermodynamic equilibrium and primarily serve to equilibrate glycolytic intermediates rather than control the overall direction of glycolysis, as previously described by us (J Biol Chem. 2024 Aug8;300(9):107648).

The endergonic nature of the PGAM-catalyzed reaction does not prevent it from proceeding in the forward direction. Instead, the directionality of the pathway is dictated by the exergonic reaction of PFK1 upstream, which pushes the flux forward, and by PK downstream, which pulls the flux through the pathway. The combined effects of PFK1 and PK may account for the observed endergonic state of the PGAM reaction.

However, if the PGAM-catalyzed reaction were isolated from the glycolytic pathway, it would tend toward equilibrium and never surpass it, as there would be no driving force to move the reaction forward.

Finally, the interpretation of the label incorporation data is rather unconvincing. The authors observe an increasing labelled fraction of TCA cycle intermediates as a function of increasing inhibitor concentration. Strangely, they conclude that less labelled pyruvate enters the TCA cycle while simultaneously less labelled intermediates exit the TCA cycle pool, leading to increased labelling of this pool. The reasoning that they present for this (decreased m2 fraction as a function of DHE-140 concentration) is by no means a consistent or striking feature of their titration data and comes across as rather unconvincing. Yet they treat this anomaly as resolved in the discussion that follows.

GNE-140 treatment increased the labeling of TCA cycle intermediates by [¹³C₆]glucose but decreased the OXPHOS rate, we consider the conflicting results as an 'anomaly' that warrants further explanation. To address this, we analyzed the labeling pattern of TCA cycle intermediates using both [¹³C₆]glucose and [¹³C₅]glutamine. Tracing the incorporation of glucose- and glutamine-derived carbons into the TCA cycle suggests that LDH inhibition leads to a reduced flux of glucose-derived acetyl-CoA into the TCA cycle, coupled with a decreased flux of glutamine-derived α-KG, and a reduction in the efflux of intermediates from the cycle. These results align with theoretical predictions. Under any condition, the reactions that distribute TCA cycle intermediates to other pathways must be balanced by those that replenish them. In the GNE-140 treatment group, the entry of glutamine-derived carbon into the TCA cycle was reduced, implying that glucose-derived carbon (as acetyl-CoA) entering the TCA cycle must also be reduced, or vice versa.

This step-by-step investigation is detailed under the subheading "The Effect of LDHB KO and GNE-140 on the Contribution of Glucose Carbon to the TCA Cycle and OXPHOS" in the Results section in the manuscript.

In the Discussion, we emphasize that caution should be exercised when interpreting isotope tracing data. In this study, treatment of cells with GNE-140 led to an increase labeling percentage of TCA cycle intermediates by [¹³C₆]glucose (Figure 5A-E). However, this does not necessarily imply an increase in glucose carbon flux into TCA cycle; rather, it indicates a reduction in both the flux of glucose carbon into TCA cycle and the flux of intermediates leaving TCA cycle. When interpreting the data, multiple factors must be considered, including the carbon-13 labeling pattern of the intermediates (m1, m2, m3, ---) (Figure 5G-K), replenishment of intermediates by glutamine (Figure 5M-V), and mitochondrial oxygen consumption rate (Figure 5W). All these factors should be taken into account to derive a proper interpretation of the data.

Reviewer #3 (Public Review):

Hu et al in their manuscript attempt to interrogate the interplay between glycolysis, TCA activity, and OXPHOS using LDHA/B knockouts as well as LDH-specific inhibitors. Before I discuss the specifics, I have a few issues with the overall manuscript. First of all, based on numerous previous studies it is well established that glycolysis inhibition or forcing pyruvate into the TCA cycle (studies with PDKs inhibitors) leads to upregulation of TCA cycle activity, and OXPHOS, activation of glutaminolysis, etc (in this work authors claim that lowered glycolysis leads to lower levels of TCA activity/OXPHOS). The authors in the current work completely ignore recent studies that suggest that lactate itself is an important signaling metabolite that can modulate metabolism (actual mechanistic insights were recently presented by at least two groups (Thompson, Chouchani labs). In addition, extensive effort was dedicated to understanding the crosstalk between glycolysis/TCA cycle/OXPHOS using metabolic models (Titov, Rabinowitz labs). I have several comments on how experiments were performed. In the Methods section, it is stated that both HeLa and 4T1 cells were grown in RPMI-1640 medium with regular serum - but under these conditions, pyruvate is certainly present in the medium - this can easily complicate/invalidate some findings presented in this manuscript. In LDH enzymatic assays as described with cell homogenates controls were not explained or presented (a lot of enzymes in the homogenate can react with NADH!). One of the major issues I have is that glycolytic intermediates were measured in multiple enzyme-coupled assays. Although one might think it is a good approach to have quantitative numbers for each metabolite, the way it was done is that cell homogenates (potentially with still traces of activity of multiple glycolytic enzymes) were incubated with various combinations of the SAME enzymes and substrates they were supposed to measure as a part of the enzyme-based cycling reaction. I would prefer to see a comparison between numbers obtained in enzyme-based assays with GC-MS/LC-MS experiments (using calibration curves for respective metabolites, of course). Correct measurements of these metabolites are crucial especially when thermodynamic parameters for respective reactions are calculated. Concentrations of multiple graphs (Figure 1g etc.) are in "mM", I do not think that this is correct.

We thank the reviewer’s comment and the following are clarification of the conceptual framework, the quantitative methodology, and the experimental basis supporting our conclusions.

(1) “It is well established that glycolysis inhibition or forcing pyruvate into the TCA cycle… leads to upregulation of TCA/OXPHOS… (authors claim lowered glycolysis leads to lower TCA/OXPHOS)”

This framing is not accurate in the context of our study. PDK inhibition and LDH inhibition are fundamentally different perturbations. PDK inhibition directly promotes mitochondrial pyruvate oxidation by enabling PDH flux, whereas LDH inhibition primarily perturbs cytosolic redox balance (free NADH/NAD⁺) and thereby constrains upstream glycolytic reactions, particularly the GAPDH step. Therefore, the metabolic outcomes of these interventions are not expected to be identical and should not be treated as interchangeable.

Importantly, we do not “ignore” prior studies proposing increased OXPHOS after LDH inhibition; we explicitly cite and summarize this prevailing interpretation in the Introduction. Our study was motivated precisely because this interpretation does not resolve key quantitative inconsistencies, including (i) the large mismatch between glycolytic flux and mitochondrial oxidative capacity, and (ii) the exceptionally high catalytic capacity of LDH relative to upstream rate-limiting glycolytic enzymes. These constraints raise a mechanistic question: how does LDH inhibition actually suppress glycolytic flux in intact cancer cells, and what are the consequences for TCA cycle and OXPHOS?

Our central contribution is the identification of a biochemical mechanism supported by integrated measurements of fluxes, metabolite concentrations, redox state, and reaction thermodynamics: LDH inhibition increases free NADH/NAD⁺, decreases free NAD⁺ availability, inhibits GAPDH, drives accumulation/depletion patterns in glycolytic intermediates, shifts Gibbs free energies of near-equilibrium reactions (PFK1–PGAM segment), suppresses pyruvate production, and consequently reduces carbon input into TCA cycle and OXPHOS. These analyses are not provided by most prior work and directly address the mechanistic gap.

(2) Lactate signaling (Thompson/Chouchani) and metabolic modeling (Titov/Rabinowitz)

These research directions are valuable, but they address questions that are different from the one investigated here. Our manuscript focuses on steady-state biochemical control of metabolic flux by LDH inhibition through redox-linked kinetics and pathway thermodynamics.

(3) Pyruvate in RPMI

Pyruvate in standard medium does not invalidate our conclusions. All experimental comparisons were performed under identical conditions across groups, and the major conclusions rely on orthogonal measurements including glycolytic flux (glucose consumption/lactate production), OCR profiling, and isotope tracing with [¹³C₆]glucose and [¹³C₅] glutamine, which directly quantify carbon entry into lactate and TCA cycle intermediates. These tracer-based results are not confounded by unlabeled extracellular pyruvate in a way that would reverse the mechanistic conclusions.

(4) LDH activity assay in homogenates and “many enzymes can react with NADH”

This concern is overstated. In the LDH assay, substrates are pyruvate + NADH, and the measured signal reflects NADH oxidation coupled to pyruvate reduction. In cell lysates, LDH is uniquely abundant and catalytically efficient for this reaction pair, and the inhibitor-response behavior matches the known LDHA/LDHB selectivity of GNE-140 and the cellular phenotypes. Thus, the assay is mechanistically specific in this context.

(5) Enzyme-coupled metabolite assays and request for LC–MS validation

The reviewer’s implication that enzyme-coupled assays are intrinsically unreliable is incorrect. Enzymatic cycling assays are a widely used quantitative approach when performed with proper specificity and calibration, and they are particularly useful for labile glycolytic intermediates that are challenging to quantify reproducibly by MS without specialized quenching, derivatization, and isotope dilution standards.

We agree that MS-based quantification is valuable, and we have developed LC–MS methods for selected metabolites. However, absolute quantification of these intermediates remains technically difficult due to the inherent limitation of this method and, in our hands, did not provide uniformly robust performance for all intermediates required for thermodynamic analysis.

(6) Units (“mM”)

The metabolite concentration units are correct.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

If the goal is to investigate the direct impact of LDH inhibition, then in my opinion, most of these experiments need to be repeated at a very early time point immediately after or a few minutes after LDH inhibition. I understand that this is a tremendous amount of work that the authors might not want to pursue. I do want to highlight that the quality of the experiments performed in this work is impressive. I hope the authors continue investigating this subject and look forward to reading their future manuscripts on this topic.

We thank the reviewer for this thoughtful and constructive comment and for the positive assessment of the experimental quality of our work.

We fully agree that measurements at very early time points after LDH inhibition would be required if the goal were to isolate an immediate, proximal molecular event occurring before downstream propagation. However, the primary objective of our study is not to dissect a single instantaneous biochemical consequence of LDH inhibition, but rather to characterize the metabolic steady state that is re-established after sustained suppression of LDH activity, which we believe is more relevant for understanding the long-term metabolic and therapeutic consequences of LDH inhibition in cancer cells.

(1) Scope: steady-state metabolic regulation versus immediate transient effects

The reviewer raises an important point that many metabolic perturbations can trigger rapid, transient responses within seconds to minutes, whereas our measurements were performed after sustained LDH inhibition. We agree that very early time points would be required if the primary goal were to isolate the most immediate, proximal consequence of LDH inhibition before downstream propagation. However, the objective of our study is different: we aim to characterize the metabolic steady state re-established after sustained inhibition of LDH activity, because this adapted steady state is more relevant for understanding long-term metabolic consequences and therapeutic outcomes of LDH inhibition in cancer cells.

(2) Genetic LDHA/LDHB knockout: comparison of two steady states

A related point applies to the LDHA/LDHB knockout models. We fully agree that the knockout process necessarily involves a temporal perturbation during cell line generation and adaptation. Nevertheless, the experimental comparison in our study is explicitly between two steady states: the baseline steady state of control cells and the steady state achieved after stable genetic disruption of LDHA or LDHB. The observation that LDHA or LDHB knockout alone had minimal effects on glycolysis and respiration indicates that partial reduction of LDH activity can be compensated in a steady-state manner, consistent with the exceptionally high catalytic capacity of LDH in cancer cells relative to upstream rate-limiting enzymes.

(3) LDH-activity-dependent quantitative relationships support stable metabolic states

Importantly, our conclusions do not rely on a single inhibitor condition at a single time point. Rather, we established quantitative steady-state relationships between residual LDH activity and pathway behavior across a wide range of LDH inhibition. These LDH-activity-dependent data strongly support that the system resides in stable metabolic states at different degrees of LDH activity, rather than reflecting non-specific collapse due to prolonged stress.

Specifically, we observed that when LDH activity was reduced from 100% to approximately ~9% (e.g., by genetic perturbation and partial pharmacologic inhibition), glucose consumption and lactate production remained essentially unchanged, indicating maintenance of a steady-state glycolytic flux despite substantial LDH inhibition. Only when LDH activity was further reduced below this threshold did glycolytic flux decrease in a graded manner, consistent with a nonlinear control structure.

Likewise, the isotope tracing results showed distinct LDH-activity-dependent transitions in TCA cycle labeling patterns. Over the range in which LDH activity decreased from 100% to ~9%, the [¹³C₆]glucose-derived labeling pattern of citrate remained largely unchanged, whereas deeper inhibition led to a decrease in m2 citrate with a compensatory rise in higher-order citrate isotopologues, consistent with altered flux entry versus cycling/retention in the TCA cycle. Similarly, [¹³C₅]glutamine tracing revealed that deeper LDH inhibition reduced the direct m5 contribution, accompanied by corresponding shifts in other isotopologues. These graded, quantitative transitions—rather than an abrupt global failure—support the interpretation of distinct metabolic steady states across LDH activity levels, linking LDH inhibition to changes in both glycolysis and mitochondrial metabolism.

Reviewer #2 (Recommendations For The Authors):

All in all, the authors would benefit from collaboration with a group more well-versed in quantitative aspects of metabolism (such as Metabolic Control Analysis) and modelling methods (such as flux analysis) to boost the interpretation and impact of their really nice data set.

We sincerely thank the reviewer for this insightful and constructive suggestion. We fully agree that collaboration with groups specializing in quantitative metabolic analysis, such as Metabolic Control Analysis and flux modeling, would further expand the interpretative depth and broader impact of this work.

The primary objective of the present work, however, was not to construct a global mathematical model, but to experimentally dissect the biochemical mechanism by which LDH inhibition coordinately suppresses glycolysis, the TCA cycle, and OXPHOS, integrating enzyme kinetics with thermodynamic constraints at steady state. Within this scope, we focused on experimentally demonstrable relationships between LDH activity, redox balance, GAPDH perturbation, thermodynamic shifts in near-equilibrium reactions, and emergent flux suppression.

We fully recognize the power of MCA and related modeling approaches in formalizing control coefficients and system-level sensitivities, and we view our dataset as particularly well suited to support such future analyses. We therefore see this work as providing a robust experimental platform upon which more comprehensive quantitative modeling can be built, either in future studies or through collaboration with specialists in metabolic modeling.

Reviewer #3 (Recommendations For The Authors):

We sincerely thank the reviewer for the important suggestions.

(1) I strongly disagree that "regulation of glycolytic flux".. "remained largely unexplored.”

Our original wording was meant to emphasize not the absence of prior work on glycolytic flux regulation, but rather that the specific biochemical mechanism by which LDH regulates glycolytic flux—particularly through the integrated effects of enzyme kinetics, redox balance, and thermodynamic constraints within the pathway—has not been fully elucidated.

To avoid any ambiguity or overstatement, we have revised the relevant text to more precisely reflect this intent. The revised wording now reads:

“This study elucidates a biochemical mechanism by which lactate dehydrogenase influences glycolytic flux in cancer cells, revealing a kinetic–thermodynamic interplay that contributes to metabolic regulation.”

We believe this revised phrasing more accurately acknowledges prior work while clearly defining the specific mechanistic contribution of the present study.

(2) Very confusing in the Introduction section: "If LDH is inhibited at the LDH step..”

We sincerely thank the reviewer for pointing out the potential confusion caused by the phrase “If LDH is inhibited at the LDH step” in the Introduction.

Our intention was to contrast two conceptual models of LDH inhibition. The first is the conventional view, in which the effect of LDH inhibition is assumed to be confined to the LDH-catalyzed reaction itself, leading primarily to local accumulation of pyruvate and its redirection toward mitochondrial metabolism. The second, which is supported by our data, is that LDH inhibition initiates a system-wide biochemical response, perturbing redox balance, upstream enzyme kinetics, and the thermodynamic state of the glycolytic pathway, ultimately resulting in coordinated suppression of glycolysis, the TCA cycle, and OXPHOS.

We agree that the original phrasing was ambiguous and potentially misleading. To improve clarity, we have revised the text as follows:

“If the effect of LDH inhibition were confined solely to its catalytic step…”

(3) The entire introduction part when the authors attempt to explain how decreased glycolysis will lead to decreased mitochondrial respiration is confusing.

We would like to clarify that the Introduction does not attempt to explain how decreased glycolysis leads to decreased mitochondrial respiration. Rather, the final paragraph of the Introduction is intended to highlight an unresolved conceptual inconsistency in the existing literature and to motivate the central question addressed in this study.

Specifically, we summarize the prevailing view that LDH inhibition redirects pyruvate toward mitochondrial metabolism and enhances oxidative phosphorylation, and then point out that this interpretation is difficult to reconcile with quantitative considerations, such as the large disparity between glycolytic and mitochondrial flux capacities and the excess catalytic activity of LDH relative to upstream glycolytic enzymes. These observations are presented to emphasize that the biochemical mechanism linking LDH inhibition to changes in glycolysis and mitochondrial respiration has not been fully resolved.

Importantly, the Introduction does not propose a mechanistic explanation for the observed suppression of mitochondrial respiration; rather, it poses this as an open question, which is then systematically addressed through experimental analysis in the Results section.

(4) Line 144: "which is 81(HeLa-LDHAKO) -297(HeLa-Ctrl) times"- here and in many other places wording is confusing to the reader.

Our intention was to emphasize the significant redundancy of LDH activity relative to hexokinase (HK), the first rate-limiting enzyme in the glycolysis pathway, in cancer cells.

Specifically, we wanted to express that in HeLa-Ctrl cells, the total LDH activity is 297 times that of HK activity; while in HeLa-LDHAKO cells, although the total LDH activity decreased, it was still 81 times that of HK activity. This data comes from supplement Table 1 in the paper and aims to provide quantitative evidence for "why knocking out LDHA or LDHB alone is insufficient to significantly affect glycolysis flux," because the remaining LDH activity is still far higher than the HK activity at the pathway entrance, sufficient to maintain flux.

Based on your suggestion, we rewrite it in the revised draft with a more specific statement: "...the total activity of LDH in HeLa cells is very high, which is 297-fold higher than the first rate-limiting enzyme HK activity in HeLa-Ctrl cells and 81-fold higher in HeLa-LDHAKO cells.”

(5) Line 153: "in the following four aspects:"- but what are these aspects, the text below has no corresponding subtitles, etc.

Our intention was to indicate that after LDHA or LDHB knockout alone failed to affect the glycolysis rate, we further explored its potential impact on the glycolytic pathway from four deeper perspectives: the glucose carbon to pyruvate and lactate, the glucose carbon to subsidiary branches of glycolysis, the concentration of glycolytic intermediates and the thermodynamic state of the pathway, and the redox state of cytosolic free NADH/NAD⁺.

Following your valuable suggestion, we have now added the aforementioned clear subtitles to these four aspects in the revised manuscript.

(6) Lines 193, another example of the very confusing statement: "The results suggested that the loss of total LDH concentration was compensated.."

The actual catalytic activity (reaction rate) of LDH is determined by both its enzyme concentration and substrate concentration (pyruvate and NADH). When the total LDH protein concentration (enzyme amount) in the cell is reduced through gene knockout, the reaction equilibrium is disrupted. To maintain sufficient lactate production flux to support a high glycolysis rate, the cell compensates by increasing the concentration of one of the substrates—free NADH (as shown in Figure 1I). This results in an increased substrate concentration, despite a reduction in the amount of enzyme, thus partially maintaining the overall reaction rate.

We have revised the original statement to more accurately describe this kinetic equilibrium process: "The decrease in total LDH concentration was counterbalanced by a concomitant increase in the concentration of its substrate, free NADH, thereby maintaining the reaction velocity.”

(7) Line 222-223: "did not or marginally significantly affect....”

Our intention is to reflect the complexity of the data in Figure 1. Specifically: Regarding "did not affect": This means that there were no statistically significant differences in most key parameters, such as glycolytic flux (glucose consumption rate, lactate production rate). Regarding "or marginally significantly affected": This means that in a few indicators, although statistical calculations showed p-values less than 0.05, the absolute value of the difference was very small, with limited biological significance.

To clarify this, we rewrite it as: "...did not significantly affect glucose-derived pyruvate entering into TCA cycle, neither significantly affect mitochondrial respiration, although statistically significant but minimal changes were observed in a few specific parameters (e.g., m3-pyruvate% in medium).”

(8) It is very confusing to use the same colors for three GNE-140 drug concentrations (Figure 2a-b) and for 3 different cell lines right next to each other (Figure 2c-d).

The figures have been revised accordingly.

(9) Lines 263-273: nothing is new here as oxidized NAD+ is required for run glycolysis and LDH inhibition/KO leads to a high NADH/NAD+ ratio; Also below it is well known that reductive stress blocks serine biosynthesis;

It is well established that oxidized NAD⁺ is required for glycolysis, that LDH inhibition or knockout increases the NADH/NAD⁺ ratio, and that reductive stress can suppress serine biosynthesis. We did not intend to present these observations as novel.

The key point of this section is not the qualitative requirement of NAD⁺ for GAPDH, but rather the mechanistic alignment between LDH inhibition, changes in free NAD⁺ availability, and the emergence of GAPDH as a flux-controlling step within the glycolytic pathway under steady-state conditions. Previous studies have largely treated the increase in NADH/NAD⁺ following LDH inhibition as a correlative or downstream effect, without directly demonstrating how this redox shift quantitatively propagates upstream to reorganize glycolytic flux distribution and thermodynamic driving forces.

In our study, we explicitly link LDH inhibition to (i) an increase in free NADH/NAD⁺ ratio, (ii) inhibition of GAPDH activity in intact cells, (iii) accumulation of upstream glycolytic intermediates, (iv) suppression of serine biosynthesis from 3-phosphoglycerate, and critically, (v) coordinated shifts in the Gibbs free energies of reactions between PFK1 and PGAM. This integrated kinetic–thermodynamic framework goes beyond the established qualitative understanding of NAD⁺ dependence and provides a pathway-level mechanism by which LDH activity controls glycolytic flux.

(10) Lines 368-370: "... we reached an alternative interpretation of the data.."- does not provide much confidence.

Our intention was to prudently emphasize that we proposed a new interpretation based on detailed data, differing from conventional views. Our interpretation is grounded in key and consistent evidence from dual isotope tracing experiments using [¹³C₆]glucose and [¹³C₅]glutamine: The [¹³C₆]glucose tracing data: the labeling pattern of citrate, the starting product of TCA cycle, showed a significant decrease in m+2 %. This directly reflects a reduction in the flux of newly generated acetyl-CoA from glucose entering the TCA cycle. Simultaneously, the sum of other isotopologues % (m+1/ m+3/ m+4/m+5/m+6) increased, indicating a longer retention time of the labeled carbon in the cycle, implying a simultaneous decrease in the flux of cycle intermediates effluxed for biosynthesis. [¹³C₅]Glutamine tracing data: the labeling pattern of α-ketoglutarate showed a decrease in m+5 %, indicating a reduction in glutamine replenishment flux. The pattern of change in the total percentage of other isotopologues % (m+1/ m+2/ m+3/m+4) also supports the conclusion of reduced intermediate product efflux.

These two sets of data corroborate each other, pointing to a unified conclusion: LDH inhibition not only reduces carbon source inflow into the TCA cycle but also decreases intermediate product efflux, leading to a decrease in overall cycle activity. Therefore, our "alternative interpretation" is a well-supported and more consistent explanation of our overall experimental results. We revise the original wording to: "Integrated analysis of dual isotope tracing data demonstrates that LDH inhibition reduces both influx and efflux of the TCA cycle..."

(11) Lines 418-421: This entire discussion on how TCA cycle activity is decreased upon LDH inhibition is very confusing. I also would like to see these tracer studies when ETC is inhibited with different inhibitors.

We would like to clarify that the mitochondrial respiration rate data presented in Figure 5W are based on studies using different ETC inhibitors, and the cell treatment conditions (including culture time, etc.) for these oxygen consumption measurements are consistent with the conditions for the [¹³C₆]glucose and [¹³C₅]glutamine isotope tracing experiments (Figure 5A-V). Therefore, the changes in TCA cycle flux revealed by the tracing data and the inhibition of OXPHOS rate shown by the respiration measurements are mutually corroborating evidence from the same experimental conditions.

(12) Figure 6F, G - very limited representation of growth curves, why not perform these experiments with all corresponding cell lines and over multiple days. Especially since proliferation arrest vs cell death was implicated.

We have provided the growth curves of the HeLa-Ctrl and HeLa-LDHAKO cell lines under the corresponding treatments in Figure 6—figure supplement 1, as a supplement to Figure 6F, G (HeLa-LDHBKO cells). The choice of 48 hours as the cutoff observation point is based on clear biological evidence: under the stress of hypoxia (1% O₂) combined with GNE-140 treatment, HeLa-LDHBKO cells experienced substantial death within 24 to 48 hours, at which point the differences in the growth curves were already very significant.

(13) Move most of the Supplementary tables into an Excel file - so values can be easily accessed.

We have compiled the tables into an Excel file and submitted it along with the revised manuscript as supplementary material.

(14) Consider changing colors to more appealing- especially jarring is a bright blue, red, black combination on many bar graphs.

We have adjusted the color scheme of the figures (especially the bar graphs) in the paper, and have submitted them with the revised manuscript.

(15) Double check y-axis on multiple graphs it says "mM".

We have checked y-axis, the unit (mM) is correct.

(16) Instead TCA cycle use the TCA cycle.

In the revised manuscript, TCA cycle is used.