PONE-D-20-23160

Protein fractional synthesis rates within tissues of high- and low-active mice

PLOS ONE

Dear Dr. Cross,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In particular, both reviewers were concerned that important methods were excluded from this manuscript which, made the reported methods somewhat confusing and limited the interpretation of the results. The premise of the study is also confusing. Why do the authors feel that FSR drives physical activity and not the other way around and can this premise actually be tested with the current experimental design? In addition, many questions still remain on the determination of FSR in this study including the methods used, the natural differences between the two inbred strains, and why the plasma alanine was so high. Finally, the authors need to be sure that the format carefully follows the journals specific guidelines. Please address these and the other comments brought up by both reviewers. 

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Randy Wayne Bryner, Ed.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript Cross et al. report the fractional synthesis rate of different tissues in two inbred mouse strains with different levels of physical activity. The manuscript is poorly written and does not follow the journal’s formatting guidelines. I find the premise that FSR drives physical activity puzzling. It would be easier (and testable) to argue that physical activity impacts FSR. In addition to physical activity, the two inbred strains differ in other traits which may confound the interpretation of the results. It was unclear why FSR was determined after surgery until it was mentioned in limitations that these results were part of another study. The feed removal, the anesthesia and the surgical procedure may have affected FSR without offering any advantage to its determination. In addition the administration of fluids during surgery likely diluted the D2O enrichment. Furthermore, the D2O enrichment reported in table 2 (0.2416) is outside the 95% CI of the values reported in the text. In fact, this value seems rather high if the authors successfully achieved a 5% D2O enrichment. Some tables and figures repeat the same data (e.g., Table 4 and Fig. 2) and some other are redundant and unnecessary (e.g., Table 3 and Fig. 3).

Reviewer #2: Summary.

Experts in the application of tracer methods in studies of protein kinetics have examined whether physical activity is associated with fundamental differences in protein synthesis. Mice were studied using D2O and protein synthesis was measured in multiple tissues over a 24-hour period. Overall, there were no major differences except for kidney and a suggestion in lung. Although the data are largely negative (i.e. virtually no differences between groups), the study is generally well done but it seems like several choices in the protocol design should be better explained.

Major comments.

If words permit, you might add a sentence or 2 in the ABSTRACT regarding a high-level view of the data. For example, values of protein synthesis in different tissues follow an expected pattern.

The need for a surgery seems odd. It looks like you only collected blood? Is this necessary? Also, there’s a peculiar statement in the 1st sentence under LIMITATIONS – you note that “…a pulse of stable isotope tracers …” was given ~ 1 hour before samples were collected. It looks like you had more going on in this study than simply giving D2O. It would be helpful to clarify the true protocol. What pulse of tracers?

Table 1 shows values for “total-body water” – the values are < 2 ml. What does this represent – intake? Please clarify.

It’s unfortunate that you did not directly measure body water labeling. I can appreciate your attention towards the labeling of alanine in plasma vs respective tissues, indeed, alanine is the true precursor. That said, the plasma alanine labeling seems too high given the dose of tracer you’ve administered. For example, we might expect ~ 5% water enrichment which limits the alanine to ~ 18.5 or 20% (3.7 or 4 times), however, the plasma is ~ 24%. This seems too high, in fact, tissue enrichments are very much in line with the expectation of ~ 3.7-4x equilibration between water and alanine labeling. You might want to elaborate on this point. It’s also odd that the suppl data show plasma alanine ~ 20% in each group – how does each group reach ~ 20% in suppl data but the combined data in Table 2 seem to reach ~ 24%?

The legend for Table 2 and 3 should more clearly state that you combined data for all animals. It might be better to change the order of Figure 2 and 3. In Figure 3 you get at more general trends in data / differences across tissues (comparable to what you show in Table 2 and 3) and then break data into active vs inactive groups (e.g. Table 4 comes last, why not make the data in Figure 2 become Figure 3)?

Minor comments.

You might explain whether you think studying the animals during activity would have been better? For example, animals were effectively sedentary for ~ 2 weeks prior to the study. Why not study subgroups who are regularly running vs those who are not? Can you comment on food intake when animals are exposed to running? It looks like there would be more marked effects if one group normally runs ~ 0.6 km/day vs another runs ~ 9.5 km/day. Not asking to do this experiment, just bring readers along with the rationale. As well, you chose to study all animals in a fed state. Would it have been advantageous to randomize each group to overnight fed vs fasted protocols and look for phenotypes that way? Again, not asking for more studies, just outline your choices. You are addressing a very important problem (i.e. the interplay between lifestyle and metabolic regulation), the question/comment here is … would it be of value to probe this with a different experimental paradigm?

You might want to explain why different collision energies are used for M0 (-68 V) vs M1 (-29 V)alanine.

It looks like low resolution figures are used here, the graphics are “fuzzy”. Presumably you will need to modify in finalized versions.

It does not seem like you need suppl data, I would suggest you consider adding those data to the main paper.

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Reviewer #1: No

Reviewer #2: No

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Protein fractional synthesis rates within tissues of high- and low-active mice

PONE-D-20-23160R1

Dear Dr. Cross,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Randy Wayne Bryner, Ed.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Thanks for taking the extra time to better explain the logic regarding the experimental design and for adding specific details regarding the protocol. The work should be easier for others to follow.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No