PONE-D-24-27587Effects of rapamycin administration on skeletal muscle function and metabolism in young adult rats.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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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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: This is an interesting study of the effects of rapamycin on skeletal muscle. All experiments are carefully conducted and contain enough numbers of rats. But, please address some minor concerns.

Abstract

RAPA notation is used in the abstract, but RAP notation is used in the results, so it is recommended to unify them.

Line 21

Please add the citation into "Skeletal muscle loss occurs under several conditions, including aging, cancer cachexia, and type 2 diabetes".

Line 54

What is PF, and CON? Please correct the abbreviation because Methods is behind results section.

Are Figure 2H-I quantified CBB above?

Figure 2G

Please provide a description of which band is which myosin next to the photo.

Figure 5E.

If P=0.1, isn't it significantly?

Figure 3.

Coomassie's photo is small, is the photo being shrunk? Or is it showing the result of dyeing the local membrane?

Line 373

Riki Ogasawara is not the corresponding author in this manuscript.

Reviewer #2: This is an important paper that presents new information from a well-designed study. The study aimed to compare the effects of chronic rapamycin treatment and calorie restriction on skeletal muscle. I have a few comments as follows:

1. Title (and throughout the manuscript): The word “metabolism” seems to be too broad. This is because, as mentioned in the results section, the authors only measured some of the “glucose metabolism regulators”. I suggest measuring some key indicators of glycolytic and oxidative markers if samples are still available.

2. Line 4: Is the term “mimetics” appropriate given that the impact of CR on mTOR signalling is clearly different from that of RAPA?

3. Line 13: Does the term “similar” correctly reflect the results? It does not appear to be consistent with the statements in the later discussion (Line 212-213).

4. Line 27: Please include an important reference from your own group on calorie restriction and skeletal muscle mitochondria (PMID: 27539498).

5. Line 203: As the RAPA group is not obese, this discussion may not be necessary.

6. Line 269: Please indicate what percentage of calories were restricted in this study.

7. Line 373: This may depend on the policy of the journal, but it would have to be someone else, as RO has already passed away.

8. Figures: Since the dots indicating statistical results and individual data are sometimes confusing, it would be a good idea to use color as this is an online journal.

Reviewer #3: Rationale for Significance Rating for Authors

The authors described the effects of chronic rapamycin administration compared to caloric restrictions on the muscle growth of early adult rats by analysing the longitudinal change of muscle mass and protein expressions associated with mTORC composition to suggest that rapamycin administration could reduce muscle mass and growth similar to reduced calorie intake by reducing mTORC signalling.

Reviewer Commentary

This manuscript by Ato et al. shows a similar reduction of muscle mass between rapamycin administration and caloric restrictions through alterations of mTOR inhibitor composition. The results show evident decrease in the weight of RAPA rats with corresponding reduction in food intake and serum insulin concentrations, suggesting reduced energy intake as the reasoning for this decrease. Muscle fiber quantification further show decreased fiber cross-sectional area and tetanic force from RAPA rats.

Gastrocnemius and soleus muscles were then analysed for protein expressions of mTORC components to show possible inhibition of mTORC-driven regulations that could affect skeletal muscle growth. While the gastrocnemius muscle did not show much effect among diet groups, the soleus muscle showed a significant reduction in the RAP rat model in mTORC2 components, Rictor, and phosphorylated mTORC1 residue, p70S6K Thr389. Furthermore, autophagy marker, LC3-I, was shown in reduced expression in RAP rat. The authors conclude that rapamycin administration reduces muscle-type-specific growth with varying age-dependent effects. With the majority of the manuscript supported by downstream events of the hypothesis, it may still require mechanistic insight to link up the cause (rapamycin or calorie restriction) and effect (weight loss, reduced muscle loss, and reduced protein expression).

Major Concerns

1. One of their conclusions is that the effect of rapamycin is variable among ages. The use of 10-week rats indicates their knowledge of their pre-adulthood growth and the factors that may affect the experiment, especially in skeletal muscle growth. Correspondingly, their results of weight changes (Fig. 1A) indicate that by 22-25 days post drug administration, the weights of CON and RAP rat decreases, showing an unprecedented trend in a young rat. Please explain the decision on the age choice of rat compared to a more stable age such as 5 or 6 months.

2. Many of their significant differences were seen in the soleus muscle, not the gastrocnemius muscle, yet the immunohistochemistry of fiber types and their CSA were from the gastrocnemius muscle. It would be beneficial for there to be similar data for the soleus muscle to connect the significant changes seen in the Western blots with changes in fiber area.

3. The use of Western blot to quantify proteins to suggest changes in the activity of mTOR is weak. They stated the relationship among autophagy of skeletal muscle cells, LC3-I and II expression, and rapamycin. This is strong supporting data if performed and will strengthen their hypothesis of the possible pathway resulting in the muscle reduction. Currently, they do not have any mechanistic nor proposed pathway of rapamycin’s effect on muscle growth.

4. According to Lines 203-206, they acknowledged the unprecedented obesity of their control group by suggesting that the calorie deficit “returned” the rat to normal weight, resulting in improved muscle function. If this is the case, then no conclusion nor comparisons could be drawn from any of their figures as the control group is no longer valid.

5. Their observations of the reduced mTORC components in RAP rats suggest alterations that may inhibit mTORC activity. What are the downstream effects of these changes? As mTOR is upstream of p70S6K and Rictor, why is mTOR unaffected, yet p70S6K and Rictor are significantly reduced in RAP rats? There are many holes that need to be filled to relate these events back to mTOR, and ultimately, muscle growth.

6. The curve for the PF group for Figure 1B is missing. Please add it back in.

Reviewer #4: Satoru et al. aimed to investigate effects of chronic rapamycin administration on skeletal muscle function and metabolism in young adult rats. The background for performing this study is associated to reported effects of rapamycin resembling those observed under calorie restriction. The authors found that rapamycin administration showed reduced skeletal muscle size with decreased mTORC1-p70S6K signaling, slightly negative impact on muscle contractile capacity and decreased Rictor expression in glycolytic and oxidative muscles and decreased mTORC2-Akt signalling and HK2 expression.

The paper presents some novel and interesting data by using a rat model, which might represent a valuable addition to the field. The experiments appear to be well-performed. Unfortunately, current manuscript gives a slightly unpolished overall impression, making it somewhat difficult to comprehend, but also to really assess the value of the presented data. My main advice would be to carefully go through all technical aspects of the manuscript, proof-read and make sure it is understandable. Here are my major concerns that need to be addressed by the authors for the paper to be considered for publication:

1. There are inconsistencies in the abbreviations used in the introduction part vs. the rest of the manuscript (RAPA vs. RAP; CR used in the abstract is not mentioned again in the text). Given the chronological order of the current text, it would be helpful for the reader if list of abbreviations was provided, or if the abbreviations would be explained first time they are used in the text.

2. The authors should also provide more details on the feeding protocol under materials and methods (the composition of the food in the different groups).

3. How many rats were included in the present study? What is the number of replicates that the statistical analysis is based on? Please provide a more detailed information under materials and methods, and this should also be specified in each figure legend.

4. In figure 1B, results for PF group are not presented. The authors need to clarify why these data are not presented.

A general comment to figure legends is that they need to be more detailed than they are in the submitted manuscript. A figure legend should be self-explanatory, providing sufficient amount of information for figure to be able to stand independently and yet be understood.

5. Figure 2: please provide description of the presented immunoblot. I assume this is a representative blot, but a detailed description should be included in the figure legend.

6. In several figures, the authors present “ME”, which is abbreviation for “Main effect”. Please specify the meaning of this term in each case. Also, in figure 5B, the authors use “ME muscle type and ME group”. Please specify the meaning of these terms.

7. In several figure legends, the authors write “different character indicates statistically significant difference”. I strongly encourage the authors to avoid this kind of unspecific terms and in stead specify each specific character that is used to label statistical differences.

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

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Rapamycin administration causes a decrease in muscle contractile function and systemic glucose intolerance concomitant with reduced skeletal muscle Rictor, the mTORC2 component, expression independent of energy intake in young rats.

PONE-D-24-27587R1

Dear Dr. Ato,

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.

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Kind regards,

Kai-Hei Tse

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: 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 #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: (No Response)

Reviewer #3: Yes

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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 #1: Yes

Reviewer #2: (No Response)

Reviewer #3: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Responses provided by the authors for comments made by reviewers were properly explained. There are no further comments from this side.

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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 #1: Yes: Keisuke Hitachi

Reviewer #2: No

Reviewer #3: No

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