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.

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.

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).

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.

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.

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.

Author response:

We sincerely thank the reviewers for their thoughtful, critical, and constructive comments, which will help us in further exploring the mechanisms by which LDH regulates glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation future studies. The following is our responses to the reviewers' comments.

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 appreciate the reviewer’s critical comments. The main argument is whether the inhibition of LDH induces a temporal perturbation in glycolysis, the TCA cycle, and OXPHOS, or if it leads to a shift to a new steady state. We argue that this shift represents a transition between two steady states; specifically, GNE-140 treatment drives metabolism from one steady state to another.

Before conducting the experiment, we performed a time course experiment, measuring glucose consumption and lactate production in cells treated with GNE-140. The results demonstrated a very good linearity, indicating that the glycolytic rate remained constant—thus confirming that glycolysis was at steady state. Given the tight coupling between glycolysis, the TCA cycle, and OXPHOS, we infer that the TCA cycle and OXPHOS were also at steady state. However, this ‘infer’ requires further confirmation.

Multiple published reports have shown that LDH inhibition in cancer cells causes a shift from fermentative ATP production to respiratory ATP production. This notion persists because it is often compared to the well-established Crabtree and Pasteur effects, where cells toggle between fermentation and respiration based on glucose and oxygen availability. However, in the Pasteur or Crabtree effects, the deprivation of oxygen—the terminal electron acceptor—drives the switch, which is fundamentally different from 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 ∆GPFK1 (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 Aug 8;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 TCAC intermediates by [13C6]glucose (Figure 5A-E). However, this does not necessarily imply an increase in glucose carbon flux into TCAC; rather, it indicates a reduction in both the flux of glucose carbon into TCAC and the flux of intermediates leaving TCAC. 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.

While the roles of lactate as a signaling metabolite and metabolic models are important areas of research, our work focuses on different aspects.

It is true that cell homogenates contain many enzymes that use NAD as a hydride acceptor or NADH as a hydride donor. However, in our assay system, the substrates are pyruvate and NADH, meaning only enzymes that catalyze the conversion of pyruvate + NADH to NAD + lactate can utilize NADH. Other enzymes do not interfere with this reaction. Although some enzymes may also catalyze this reaction, their catalytic efficiency is markedly lower than that of LDH, ensuring the validity of this assay.

Similarly, the assays for glycolytic intermediates are validated by the substrate specificity.

We have developed an LC-MS methodology for some glycolytic intermediates, but the accuracy of quantification remains unsatisfactory due to inherent limitations of this methodology.