Metabolic Effects on Mouse Embryonic Stem Cells and the Canonical Mammalian Target of Rapamycin Pathway
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments to authors,
This manuscript reported that the removal of the leucine and arginine from the culture medium of naïve mESCs could induce features of a paused pluripotent state, and mTi did indeed provoke a paused-like state this was distinct, and less pronounced. The removal of the leucine and arginine from the culture medium of naïve mESC performed a more effective in downregulating pluripotent stem cell proliferation than mTi, authors also analyzed the expression of GLUT1/3, the mass of the mitochondrial and the GLUT1 cellular distribution. This manuscript is interesting; however, it still needs some minor revisions shown as below:
1 Author only treated the cell with 100 nM INK28, how didn’t authors decide the INK28 concentration? Authors should treat the cell with different concentrations and make the conclusion.
2 The discussion should be rather organized around arguments avoiding simply describing details without providing much meaning. A real discussion should also link the findings of the study to theory and/or literature.
Comments on the Quality of English Language
The writing english only needs minor editing, overall, good job!
Author Response
Thank you for your comments on our manuscript. We value your interest in this paper and believe the alterations made will improve our work. We have performed the alterations and revised the text considering you suggestions. We also strived to answer the questions you presented.
Comments to authors,
This manuscript reported that the removal of the leucine and arginine from the culture medium of naïve mESCs could induce features of a paused pluripotent state, and mTi did indeed provoke a paused-like state this was distinct, and less pronounced. The removal of the leucine and arginine from the culture medium of naïve mESC performed a more effective in downregulating pluripotent stem cell proliferation than mTi, authors also analyzed the expression of GLUT1/3, the mass of the mitochondrial and the GLUT1 cellular distribution. This manuscript is interesting; however, it still needs some minor revisions shown as below:
1 Author only treated the cell with 100 nM INK28, how didn’t authors decide the INK28 concentration? Authors should treat the cell with different concentrations and make the conclusion.
In other work from our group (reference 9), we did treat the cells with different concentrations of INK128. We tested 100 nM and a 200 nM and 300 nM, and observed the same effects regardless the concentration used. Thus, for the purpose of this work (which was to more comprehensively study what INK128 did, or did not do) we focused on just this concentration. We have added this information.
2 The discussion should be rather organized around arguments avoiding simply describing details without providing much meaning. A real discussion should also link the findings of the study to theory and/or literature.
We appreciate your suggestion and performed changes that we feel are appropriate in this context.
Reviewer 2 Report
Comments and Suggestions for AuthorsCorreira et al describe the effect of leucine and arginine depletion on mESCs. The manuscript is well written, however several main concerns are present: 1. it is not clear which is the novelty of the study compared to the prevoius one (Correira et al, 2022), 2. effects of mTOR inhibition on mESC cells are reported, but the phosphorylation of the main substrates of mTOR are not detected. This discrepancy should be explained, 3. in the whole manuscript, authors justify experiments (metabolic data, glut expression and localization) linking them with mTOR pathway, even if they assess the mechanism is mTOR independent. 4. in Fig 2g, 4ebp1 is decreased in the last 3 conditions, which is in contrast with what reported in the text.
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Thank you for your comments on our manuscript. We appreciate the effort and the attention you gave to our paper.
Correira et al describe the effect of leucine and arginine depletion on mESCs. The manuscript is well written, however several main concerns are present:
- it is not clear which is the novelty of the study compared to the prevoius one (Correira et al, 2022),
In our previous study (reference 9) we focused on the effects of Leucine and Arginine withdrawal on naïve mESCs, while in this study we compare these effects with the effect of INK128, given that it was logical to assume that in both cases the results would be mTOR driven. According to the similarity of the features observed in naïve mESCs, when Leucine and Arginine are absent, to those reported in a paused-pluripotency report (https://doi.org/10.1038/nature20578), we used INK128 as a positive control to understand if the effects were in fact similar or not and how would it differ from the effects of INK128. Moreover, we find it necessary to share the results we obtained with INK128 on 2i/LIF cultured cells, as, in our hands, those results are not completely in line with the literature, although the practical effects are, suggesting that there may be alternative targets to mTOR in this system.
- effects of mTOR inhibition on mESC cells are reported, but the phosphorylation of the main substrates of mTOR are not detected. This discrepancy should be explained,
This is exactly what is stated above. We did try (many times) to confirm this data using several phosphorylation targets, but were unable to do so, and these were not technical difficulties (positive controls worked fine), as stated in the manuscript.
- in the whole manuscript, authors justify experiments (metabolic data, glut expression and localization) linking them with mTOR pathway, even if they assess the mechanism is mTOR independent.
As stated above: it was logical to believe that the effects observed in mESCs were related to the mTOR pathway. Therefore, all the experiments were designed with this in mind. However, the data show that INK128, in our cells and under these conditions, does not affect the downstream targets related to protein translation, as it is reported in the literature. The same was true for the absence of Leucine and Arginine, although these effects were stronger. We believe this information to be useful, basically stating that mTOR inhibition needs to be validated in all cases, and that alternative mechanisms may be at stake.
- in Fig 2g, 4ebp1 is decreased in the last 3 conditions, which is in contrast with what reported in the text
Unfortunately, that is the best membrane image we have to include. We understand the point, however, the error bars represented in fig 2f shows the high variability of the data. This will be clear to any interested reader.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this paper, Correia et al compared the paused pluripotency between Leucine and Arginine withdrawal and mTOR inhibition. Based on their results, they concluded that Leucine and Arginine removal induces the paused pluripotency independent of mTOR. While these results are potentially interesting, the work suffers from a lack of scientific rigor and in-depth understanding as to the underlying mechanisms. It would be important to uncover the unknown pathways through Leucine and Arginine removal. I will suggest a major revision.
Major comments
1. LINE 276 ‘Interestingly the mRNA levels of RAPTOR, an indispensable part of mTORC1; mTOR, 276
4EBP1 and S6K1 were not affected by any of the experimental condition (Figure 2a).’
This conclusion contradicts the results from fig 1 a, which showed an upregulation of those genes.
2. LINE 317 ‘Interestingly, glycolysis under basal conditions, ECAR of xAA, xAAxLIF and mTi cells were remarkably lower than those observed for cells in 2i/LIF, xLIF (Figure 3a)’.
The data did not match the conclusion mentioned here. Based on Figure 3a, the mTi condition showed similar or even higher ECAR level compared to 2i/LIF, xLIF controls, indicating a similar or higher glycolysis upon mTi.
3. LINE 328 ‘To determine mitochondrial mass we evaluated the protein expression of TOM20 and TFAM which is similarly ex-pressed in all conditions (Figure 3c, d), also confirmed using the fluorescence-based Mito-tracker green assay (Figure e)’.
This assertation was not consistent with the data from fig 3. Fig 3c showed a decrease (p-value is needed) in TOM20 protein level in xAA and the decrease seemed to be more obvious in the representative WB result in fig 3d, suggesting that the Leu/Arg starvation affected mitochondrial mass. Also, fig 3e showed decreased fluorescence intensity (p-value is needed) in both xAA and mTi, suggesting that the Leu/Arg starvation affected mitochondrial mass.
4. LINE 331 ‘Protein expression of GLUT1 and GLUT3 did not change (Figure 3c, d), however GLUT1 dotted staining can be observed surrounding cell nuclei in xAA and mTi cells, suggesting internalization, while the staining in 2i/LIF suggests a membrane location (Figure 3f).’
First, the GLUT3 protein level was decreased in xAA condition according to fig 3c and 3d (p-value is needed). Second, to compare the localization of GLUT1, the authors need to show the co-IF using both GLUT1 and nuclear membrane/ cell membrane markers, and at single cell level instead of the whole colony. And the GLUT1 signal is too weak to tell its localization in xAA cells.
Minor comments
1. In fig 1b and 1c, include Leu/Arg- condition. In addition, label the percentage of each quadrant.
2. In fig 1e, does different colony size indicate cell growth variances or related to other conditions? If treatments affected colony size, also compare the sizes quantitively.
3. Add p-value to fig 1b, 1f, 2a, 2b, 2f, 3c and 3e.
4. Correct the typos (i.e. LINE 265: two figure 1e in the legend; LINE 331 ‘Figure e’) and grammatical issues across the manuscript.
5. Make different groups more distinguishable using a larger symbol size and different colors in fig. 1a, 3a and 3b. Also, in fig 3a and 3b, use unified symbols.
6. Scale bars are needed for the IF results.
Comments on the Quality of English LanguageCorrect the typos (i.e. LINE 265: two figure 1e in the legend; LINE 331 ‘Figure e’) and grammatical issues across the manuscript.
Author Response
See attached pdf
Author Response File: 
Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe manuscript from Correia et.al. reports their study on the effects of arginine and leucine deficiency leading to pluripotent-paused cells. The study also investigates if these effects are mTOR-driven.
Overall, the study is well-designed to address a specific question that the authors are trying to address. The presentation is good, and the manuscript is well-written.
My only concern is for some of the data in this manuscript (specifically Figure 1 Panel A, C, D, E), I find a striking similarity to data reported in the previous paper by the same authors- Leucine and Arginine Availability Modulate Mouse Embryonic Stem Cell Proliferation and Metabolism Int. J. Mol. Sci. 2022, 23(22), 14286; https://doi.org/10.3390/ijms232214286 - Figure 2 and Figure 3.
Can the authors clarify if they are using some of their older data directly for this study or if they performed these experiments again?
Some minor comments:
· Y-axis labels are missing for Figure1 panel C and D
· Page5 line 212 “rule out” instead of “out rule”
Author Response
The manuscript from Correia et.al. reports their study on the effects of arginine and leucine deficiency leading to pluripotent-paused cells. The study also investigates if these effects are mTOR-driven.
Overall, the study is well-designed to address a specific question that the authors are trying to address. The presentation is good, and the manuscript is well-written.
My only concern is for some of the data in this manuscript (specifically Figure 1 Panel A, C, D, E), I find a striking similarity to data reported in the previous paper by the same authors- Leucine and Arginine Availability Modulate Mouse Embryonic Stem Cell Proliferation and Metabolism Int. J. Mol. Sci. 2022, 23(22), 14286; https://doi.org/10.3390/ijms232214286 - Figure 2 and Figure 3.
Can the authors clarify if they are using some of their older data directly for this study or if they performed these experiments again?
In figure 1a, 1c and 1d the data was obtained from the same experiments used in the previous paper, and were used as controls for the novel data presented here, which aimed, not to determine these effects, which was already known, but to try to determine if they were mediated by the mTOR pathway, which is a different issue.
The images in figure 1e is the same experiment reported in the previous paper, for the same reason (as a control).
Some minor comments:
- Y-axis labels are missing for Figure1 panel C and D
Relevant changes were performed
- Page5 line 212 “rule out” instead of “out rule”
Relevant changes were performed
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsEven if the authors replied to the comments, I still have some concerns about the scientific soundness of the study and the main take home message of the the study.
Reviewer 3 Report
Comments and Suggestions for AuthorsN/A
Comments on the Quality of English LanguageN/A
