The Role of ATP Synthase Subunit e (ATP5I) in Mediating the Metabolic and Antiproliferative Effects of Biguanides

Reviewer #1 (Public review):

Summary:

In the manuscript entitled 'The Role of ATP Synthase Subunit e (ATP5I) in 1 Mediating the Metabolic and Antiproliferative 2 Effects of Biguanides', Lefrancois G et al. identifies ATP5I, a subunit of F1Fo-ATP synthase, as a key target of medicinal biguanides. ATP5I stabilizes F1Fo-ATP synthase dimers, essential for cristae morphology, but its role in cancer metabolism is understudied. The research shows ATP5I interacts with a biguanide analogue, and its knockout in pancreatic cancer cells mimics biguanide treatment effects, including altered mitochondria, reduced OXPHOS, and increased glycolysis. ATP5I knockout cells resist biguanide-induced antiproliferative effects, but reintroducing ATP5I restores the effects of metformin and phenformin. These findings highlight ATP5I as a promising mitochondrial target for cancer therapies. The manuscript is well written.

Strengths:

Demonstrated the experiments in systematic and well-accepted methods.

Weaknesses:

The significance of the target molecule and mechanisms may help in understanding the molecular mechanisms of metformin.

Reviewer #2 (Public review):

Summary:

The mechanism(s) by which the therapeutic drug metformin lowers blood glucose in type 2 diabetes and inhibits cell proliferation at higher concentrations remain contentious. Inhibition of complex 1 of the mitochondrial respiratory chain with consequent changes in cellular metabolites which favour allosteric activation of phosphofructokinase-1, allosteric inhibition of fructose bisphosphatase-1 and cAMP signalling and activation of AMPK which phosphorylates transcription factors are candidate mechanisms. The current manuscript proposes the e-subunit of ATP-synthase as a putative binding protein of biguanides and demonstrates that it regulates the expressivity of the Complex 1 protein NDUFB8.

Strengths:

(1) The metformin conjugate and metformin show comparable efficacy on inhibition of cell proliferation in the millimolar range.

(2) Demonstration of compromised expression of the Complex I protein NDUFB8 by the ATP5I knockout and its reversal by ATP5I expression is an important strength of the study. This shows that the decreased "sensitivity" to metformin in the ATP5I knock-out cells could be due to various proteins.

(3) Demonstration of converse effects of ATP5I KO and re-expression ATP5I on the NAD/NADH ratio.

Weaknesses:

(1) The interpretation of the cellular co-localization of the biotin-biguanide conjugate with TOMM20 (Figure 1-D) as mitochondrial "accumulation" of the conjugate is overstated because it cannot exclude binding of the conjugate to the mitochondrial membrane. It would have been more convincing if additional incubations with the biotin-biguanide conjugate in combination with metformin had shown that metformin is competitive with the biotin-conjugate.

(2) The manuscript reports the identification of 69 proteins by mass spectrometry of the pull-down assay of which 31 proteins were eluted by metformin. However, no Mass Spectrometry data is presented of the peptides identified. The methodology does not state the minimum number of peptides (1, 2?) that were used for the identification of the 31/69 proteins.

(3) The validation of ATP5I was based on the use of recombinant protein (which was 90% pure) for the SPR and the use of a single antibody to ATP5I. The validity of the immunoblotting rests on the assumption that there is no "non-specific" immunoactivity in the relevant mol wt range. Information on the validation of the antibody would be helpful.

(4) Knock-out of ATP5I markedly compromised the NAD/NADH ratio (Fig.3A) and cell proliferation (Figure 3D). These effects may be associated with decreased mitochondrial membrane potential which could explain the low efficacy of metformin (and most of the data in Figures 3-5). This possibility should be discussed. Effects of [metformin] on the NAD/NADH ratio in control cells and ATP5I-KO would have been helpful because the metformin data on cell growth is normalized as fold change relative to control, whereas the NAD/NADH ratio would represent a direct absolute measurement enabling comparison of the absolute effect in control cells with ATP5I KO.

(5) Figure-6 CRISPR/Cas9 KO at 16mM metformin in comparison with 70nM rotenone and 2 micromolar oligomycin (in serum-containing medium). The rationale for the use of such a high concentration of metformin has not been explained. In liver cells metformin concentrations above 1mM cause severe ATP depletion, whereas therapeutic (micromolar) concentrations have minimal effects on cellular ATP status. The 16mM concentration is ~2 orders of magnitude higher than therapeutic concentrations and likely linked to compromised energy status. The stronger inhibition of cell proliferation by 16mM metformin compared with rotenone or oligomycin raises the issue of whether the changes in gene expression may be linked to the greater inhibition of mitochondrial metabolism. Validation of the cellular ATP status and NAD/NADH with metformin as compared with the two inhibitors could help the interpretation of this data.

Reviewer #3 (Public review):

Most of the data are based on measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured by the Seahorse analyser in control and ATP5l KO cells. However, these measurements are conducted by a single injection of a biguanide, followed over time and presented as fold change. By doing so, the individual information on the effect of metformin and derivate on control and KO cells are lost. In addition, the usual measurement of OCR is coupled with certain inhibitors and uncouplers, such as oligomycin, FCCP, and Antimycin A/rotenone, to understand the contribution of individual complexes to respiration. Since biguanides and ATP5l KO affect protein levels of components of complex I and IV, it would be informative to measure their individual contributions/effects in the Seahorse. To further strengthen the data, it would be helpful to obtain measurements of actual ATP levels in these cells, as this would explain the activation of AMPK.

The authors report on alterations in mitochondrial morphology upon ATP5l KO, which is measured by subjective quantifications of filamentous versus puncta structures. Fiji offers great tools to quantify the mitochondrial network unbiasedly and with more accuracy using deconvolution and skeletonization of the mitochondria, providing the opportunity to measure length, shape, and number quantitatively. This will help to understand better, whether mitochondria are really fragmented upon ATP5l KO and rescued by its re-introduction.

Finally, the authors report in the last part of the paper a genetic CRISPR/Cas9 KO screen in NALM-6 cells cultured with high amounts of metformin to identify potential new mediators of metformin action. It is difficult to connect that to the rest of the paper because a) different concentrations of metformin are used and b) the metabolic effects on energy consumption are not defined. They argue about the molecular function of the obtained hits based on literature and on a comparison of the pattern of genetic alterations based on treatments with known inhibitors such as oligomycin and rotenone. However, a direct connection is not provided, thus the interpretation at the end of the results that "the OMA1-DEL1-HRI pathway mediates the antiproliferative activity of both biguanides and the F1ATPase inhibitor oligomycin" while increasing glycolysis, needs to be toned down. This is an interesting observation, but no causality is provided. In general, this part stands alone and needs to be better connected to the rest of the paper.

Author response:

Reviewer #1 (Public review):

The significance of the target molecule and mechanisms may help in understanding the molecular mechanisms of metformin.

We greatly appreciate the reviewer’s insightful comment regarding the significance of the target molecule and its mechanisms in understanding the molecular actions of metformin. ATP5I is responsible for the dimerization of the F₁F₀-ATPase(1-3). Hence, we propose conducting BN-PAGE followed by a western blot using the β-subunit of the F1 domain of F1F0-ATP synthase to investigate whether metformin affects its dimerization. This will provide a more direct evidence of the on target action of metformin on ATP5I. Due to the high abundance of F₁F₀-ATP synthase in cells and the slow ability of metformin to enter mitochondria, we plan to perform long-term treatments (3 and 6 days) with high concentrations of metformin (10 mM) to enhance the likelihood of detecting subtle yet biologically relevant shifts in the monomer and dimer populations. Prolonged exposure is expected to reveal the cumulative effects of metformin on F₁F₀-ATP synthase dimers/monomers ratio. We do not expect that metformin will totally mimic the cumulative effect of the dimerization as in ATP5I KO cells but we think it will be important to report to what extent this ratio is affected.

Reviewer #2 (Public review):

(1) The interpretation of the cellular co-localization of the biotin-biguanide conjugate with TOMM20 (Figure 1-D) as mitochondrial "accumulation" of the conjugate is overstated because it cannot exclude binding of the conjugate to the mitochondrial membrane. It would have been more convincing if additional incubations with the biotin-biguanide conjugate in combination with metformin had shown that metformin is competitive with the biotin-conjugate.

We appreciate the reviewer’s insightful comment and agree that the resolution provided by fluorescence microscopy makes it challenging to pinpoint the specific mitochondrial compartment where the biotin-biguanide conjugate localizes, even with additional markers such as TOMM20 antibodies for the inner mitochondrial membrane. While it remains a possibility that the conjugate binds to the mitochondrial surface, another plausible explanation is that the biotin moiety may facilitate entry into mitochondria through a biotin-specific transporter, adding further mechanistic intricacies. Furthermore, while a competition assay with metformin might help investigate interactions with mitochondrial targets and transporters (OCT family), it would not compete for biotin-mediated transport. Thus, while we acknowledge the reviewer’s suggestion, we believe such an experiment may not provide conclusive evidence regarding the conjugate’s mitochondrial localization or mechanism of entry. Instead, we will revise the manuscript to more accurately describe the findings as "mitochondrial association" rather than "mitochondrial accumulation," ensuring that our interpretation remains consistent with the resolution and limitations of the data presented.

(2) The manuscript reports the identification of 69 proteins by mass spectrometry of the pull-down assay of which 31 proteins were eluted by metformin. However, no Mass Spectrometry data is presented of the peptides identified. The methodology does not state the minimum number of peptides (1, 2?) that were used for the identification of the 31/69 proteins.

Concerning the mass spectrometry results, our intention was to provide a comprehensive table summarizing these findings in a separate data sheet, as part of the data availability section. To address the reviewer’s comment and ensure full transparency, we will include this table as supplementary material in the revised manuscript. Additionally, we will update the methodology section to explicitly state these criteria and ensure clarity regarding the identification process.

(3) The validation of ATP5I was based on the use of recombinant protein (which was 90% pure) for the SPR and the use of a single antibody to ATP5I. The validity of the immunoblotting rests on the assumption that there is no "non-specific" immunoactivity in the relevant mol wt range. Information on the validation of the antibody would be helpful.

Regarding the recombinant protein used for SPR, its purity was evaluated using a Coomassie-stained gel. For the antibody used in immunoblotting, its specificity was validated through knockout cell lines, ensuring minimal concerns about non-specific immunoactivity within the relevant molecular weight range. Unfortunately, the KO data comes in the paper after the first immunoblots are presented. In the revised manuscript, we will clearly outline these validation steps in the methods section and additional manufacturer documentation for the antibody we used.

(4) Knock-out of ATP5I markedly compromised the NAD/NADH ratio (Fig.3A) and cell proliferation (Figure 3D). These effects may be associated with decreased mitochondrial membrane potential which could explain the low efficacy of metformin (and most of the data in Figures 3-5). This possibility should be discussed. Effects of [metformin] on the NAD/NADH ratio in control cells and ATP5I-KO would have been helpful because the metformin data on cell growth is normalized as fold change relative to control, whereas the NAD/NADH ratio would represent a direct absolute measurement enabling comparison of the absolute effect in control cells with ATP5I KO.

The mitochondrial membrane potential depends on a functional electron transport chain which drives proton pumping from the matrix to the intermembrane space. Metformin can decrease the mitochondrial membrane potential and this usually explained as a consequence of complex I inhibition(4). It has been published the metformin requires this membrane potential to accumulate in mitochondria so the actions of metformin are self-limiting due to this requirement. The reviewer is right that ATP5I KO cells could be resistant to metformin because they may have a lower membrane potential. We do not believe this to be the case because the response to phenformin, another biguanide that can enter mitochondria through the membrane without the need of the OCT transporters(5), is also affected in ATP5IKO cells. Of note, compensatory mechanisms such as enhanced glycolysis, as observed in ATP5I-KO cells (elevated ECAR and increased sensitivity to 2-D-deoxyglucose), and the ATPase activity of F₁F₀-ATP synthase could potentially help maintain membrane potential suggesting that this might not be an issue in the ATP5I KO cells. We will discuss these possibilities in the revised manuscript.

Nevertheless, to experimentally address this point, we propose measuring mitochondrial membrane potential using tetramethylrhodamine methyl ester (TMRE) and ATP levels using luciferase-based assays (CellTiter-Glo) in ATP5I-KO cells.

Regarding the NAD+/NADH in both control and KO cells may not be very helpful because this ratio can be corrected by LDH which is induced as part of the glycolytic adaptation that occurs after inhibition of respiration. Since our KO cells have been propagated already for several passages, the extent of this adaptation is likely different from metformin-treated cells. As we mentioned in answering Reviewer 1, we will provide a more direct measurement of metformin acting on ATP5I: the levels of F1F0-ATPase dimers and monomers.

(5) Figure-6 CRISPR/Cas9 KO at 16mM metformin in comparison with 70nM rotenone and 2 micromolar oligomycin (in serum-containing medium). The rationale for the use of such a high concentration of metformin has not been explained. In liver cells metformin concentrations above 1mM cause severe ATP depletion, whereas therapeutic (micromolar) concentrations have minimal effects on cellular ATP status. The 16mM concentration is ~2 orders of magnitude higher than therapeutic concentrations and likely linked to compromised energy status. The stronger inhibition of cell proliferation by 16mM metformin compared with rotenone or oligomycin raises the issue of whether the changes in gene expression may be linked to the greater inhibition of mitochondrial metabolism. Validation of the cellular ATP status and NAD/NADH with metformin as compared with the two inhibitors could help the interpretation of this data.

To address the reviewer’s final comment, we would like to clarify the rationale behind our experimental approach. NALM-6 cells are very glycolytic, have low respiration rates, and weak dependence on ATP5I (DepMap score: -0.47)(6). The concentration of 16 mM metformin was chosen based on the IC50 for this cell line. This approach aligns with our focus on the anticancer mechanism of action rather than the antidiabetic effects of metformin. Both ATP status and NAD+/NADH ratios will depend on the extent of the compensatory glycolysis. On the other hand, our genetic screening evaluates cell proliferation as an integration of all metabolic activities required for the process. This unbiased screening revealed a common pathway affected by metformin and oligomycin different that the pathway affected by rotenone, which is consistent with the finding that metformin acts of the F₁F₀ATPase.

Reviewer #3 (Public review):

(1) Most of the data are based on measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured by the Seahorse analyser in control and ATP5l KO cells. However, these measurements are conducted by a single injection of a biguanide, followed over time and presented as fold change. By doing so, the individual information on the effect of metformin and derivate on control and KO cells are lost. In addition, the usual measurement of OCR is coupled with certain inhibitors and uncouplers, such as oligomycin, FCCP, and Antimycin A/rotenone, to understand the contribution of individual complexes to respiration. Since biguanides and ATP5l KO affect protein levels of components of complex I and IV, it would be informative to measure their individual contributions/effects in the Seahorse. To further strengthen the data, it would be helpful to obtain measurements of actual ATP levels in these cells, as this would explain the activation of AMPK.

We appreciate the reviewer’s observations regarding the Seahorse measurements and acknowledge the potential limitations of presenting the data as fold change. Due to experimental challenges in maintaining KP-4 and ATP5I-KO cells with sufficient nutrients, caused by their rapid glucose uptake and subsequent lactate production, it was more practical to present the Seahorse results in this format. Using inhibitors at each time point during the Seahorse experiment was not feasible, as the delay between inhibitor injections and the corresponding changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) would introduce variability and complicate the interpretation of dynamic responses. Nevertheless, we recognize the importance of understanding the contributions of specific respiratory complexes to OCR and ECAR. To address this, we will include a representative figure showcasing a typical Seahorse analysis, highlighting ATP turnover and proton leak after oligomycin addition, maximal respiration with FCCP, and disruption with rotenone and antimycin A. While these experiments are inherently complex due to the metabolic demands of ATP5I-KO cells, this approach will provide a clearer breakdown of mitochondrial activity. Furthermore, as mentioned in our response to Reviewer 2, we will measure ATP levels using a luciferase-based assay (CellTiter-Glo) in both control and ATP5I-KO cells to better explain AMPK activation. This will provide additional context to strengthen the interpretation of mitochondrial function and metabolic compensation mechanisms in these cells.

(2) The authors report on alterations in mitochondrial morphology upon ATP5l KO, which is measured by subjective quantifications of filamentous versus puncta structures. Fiji offers great tools to quantify the mitochondrial network unbiasedly and with more accuracy using deconvolution and skeletonization of the mitochondria, providing the opportunity to measure length, shape, and number quantitatively. This will help to understand better, whether mitochondria are really fragmented upon ATP5l KO and rescued by its re-introduction.

Concerning the analysis of mitochondrial morphology, we acknowledge the potential benefits of using Fiji and additional plugins such as MiNA for more accurate and unbiased quantification. Indeed, this approach could provide stronger evidence for mitochondrial fragmentation upon ATP5I-KO and its potential rescue by ATP5I reintroduction. We will consider integrating this methodology into our analysis to enhance the precision and robustness of our findings.

(3) Finally, the authors report in the last part of the paper a genetic CRISPR/Cas9 KO screen in NALM-6 cells cultured with high amounts of metformin to identify potential new mediators of metformin action. It is difficult to connect that to the rest of the paper because a) different concentrations of metformin are used and b) the metabolic effects on energy consumption are not defined. They argue about the molecular function of the obtained hits based on literature and on a comparison of the pattern of genetic alterations based on treatments with known inhibitors such as oligomycin and rotenone. However, a direct connection is not provided, thus the interpretation at the end of the results that "the OMA1-DEL1-HRI pathway mediates the antiproliferative activity of both biguanides and the F1ATPase inhibitor oligomycin" while increasing glycolysis, needs to be toned down. This is an interesting observation, but no causality is provided. In general, this part stands alone and needs to be better connected to the rest of the paper.

NALM-6 are very glycolytic, have low respiration rates, and weak dependence on ATP5I(6), forcing us to use higher concentrations of metformin to inhibit their growth. Recent results show that metformin targets PEN2 in the cytosol to increase AMPK activity, controlling both the glucose lowering and the life span extension abilities of metformin 7. This work raises the question whether the antiproliferative and anticancer effects of metformin are due to a mitochondrial activity or are controlled by this new pathway of AMPK activation. Hence, the genetic screening was performed to unbiasedly find how metformin works. The results provide compelling evidence for mitochondria and in particular the ATP synthase as potential targets of metformin and a foundation for future studies. We will revise the text and abstract to better reflect the exploratory nature of this finding and ensure clarity.

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(2) Paumard, P. et al. The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J 21, 221-230 (2002). https://doi.org/10.1093/emboj/21.3.221

(3) Habersetzer, J. et al. ATP synthase oligomerization: from the enzyme models to the mitochondrial morphology. Int J Biochem Cell Biol 45, 99-105 (2013). https://doi.org/10.1016/j.biocel.2012.05.017

(4) Xian, H. et al. Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation. Immunity 54, 1463-1477 e1411 (2021). https://doi.org/10.1016/j.immuni.2021.05.004

(5) Hawley, S. A. et al. Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation. Cell metabolism 11, 554-565 (2010). https://doi.org/10.1016/j.cmet.2010.04.001

(6) Hlozkova, K. et al. Metabolic profile of leukemia cells influences treatment efficacy of L-asparaginase. BMC Cancer 20, 526 (2020). https://doi.org/10.1186/s12885-020-07020-y

(7) Ma, T. et al. Low-dose metformin targets the lysosomal AMPK pathway through PEN2. Nature 603, 159-165 (2022). https://doi.org/10.1038/s41586-022-04431-8