Dear editor and reviewers:

Thank you very much for the valuable and constructive comments on our manuscript (PONE-D-25-28234: The Mechanism of Aerobic Exercise in Ameliorating Glycolipid Metabolic Disorders in Metabolic Syndrome Rats via the miR-27a-PPARγ Pathway

). We appreciate the opportunity to improve the paper and responses to reviewers are addressed below. All changes made in the revised manuscript were highlighted in

yellow to make them easily identified.

Journal Requirements

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, and (2) efforts to alleviate suffering.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

5. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

6. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files.

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

7. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Response:

Thank you for your comments. We have supplemented and revised the manuscript according to your journal’s additional requirements, including formatting, original gel blot images (See attached supplementary information - Original gel blot images), details regarding laboratory animals (Line 64-66, 79-82, 96-98�106-109), verification of the corresponding author’s ORCID iD, verification and standardization of the correct funding number (In the Additional Information section of the submission system, we did not see an option for funding disclosure. We entered the funding information in the Manuscript Data - Funding Information section, but the submitted version displayed “The author(s) received no specific funding for this work.” This has left us confused), and confirmation of the data availability statement. These changes are highlighted in yellow in the corresponding sections of the manuscript. We hope this round of revision will make you satisfied and progress to the process.

Overall Assessment

The study provides compelling evidence that aerobic exercise ameliorates metabolic syndrome (MS) via the miR-27a-PPARγ pathway, offering a novel mechanistic target for intervention. Well-designed experiments with appropriate controls, though some technical limitations weaken robustness. First to link exercise-induced metabolic benefits to miR-27a downregulation in MS, advancing the field beyond correlative studies.

Weaknesses & Limitations

Technical Concerns:

�Sample Size Issues: Underpowered analyses (e.g., LCAD mRNA trends, Page 19) risk Type II errors. Power analysis is missing.

�Blot Integrity: Cutting blots pre-hybridization (Page 12) is problematic; full blots should be supplemental.

�Viral Delivery: No data on knockdown efficiency over time (Page 39: “expression gradually decreased”).

Interpretational Gaps:

�miR-27a Regulation: How exercise suppresses miR-27a (transcriptional vs. post-transcriptional?) is unexplored.

�Human Relevance: No discussion of miR-27a/PPARγ conservation in human MS or exercise responses.

Presentation:

�Figure Clarity: Some panels (e.g., Fig 1A–B vs. C–H) are poorly labeled; HE stains lack scale bars (Fig 5).

�Repetition: Methods for PCR/WB are overly detailed; streamline or supplement.

Recommendations for Revision

1.Address Technical Gaps:

oJustify sample sizes with power calculations.

oInclude uncut blots in supplements.

oQuantify miR-27a knockdown efficiency longitudinally.

2.Expand Discussion:

oCompare findings to human miRNA studies (e.g., circulating miR-27a in MS patients).

oHypothesize mechanisms linking exercise to miR-27a downregulation (e.g., exosomal sorting?).

3.Improve Clarity:

oSimplify abstract for broader accessibility.

oStandardized figure labels (e.g., “Panel A” vs. “Fig 1A”).

Comparison to Field

�Advances Beyond Prior Work:

oExtends Yu et al. (2018) by showing PPARγ upregulation (not just IRS-1 inhibition) mediates exercise benefits.

oConfirms Chen T et al. (2019) in vivo but adds exercise as a physiological modulator.

�Unresolved Questions:

oDoes miR-27a suppression also improve inflammation/fibrosis in MS?

Response:

Thank you for your constructive comments and advice, which will significantly enhance the rigor and readability of this study. The revisions are outlined below:

1.Technical gaps:

• Power calculation: Thank you for your valuable comments. We fully recognize the importance of a priori power analysis. As this study did not involve pilot experiments, we were unable to perform such an analysis in advance, which we acknowledge as a limitation of our study design.

First, we supplemented the methodology section with the principles for determining the sample size for this study (Methods-Animals, diets and interventions. Lines 83-93) .

The revised part: The sample size in this study was determined based on the following principles: The sample size in this study was determined based on the following principles: (1) To account for individual variability and the risk of failure in establishing the high-fat and high-sugar diet-induced model, more animals were initially included in the modeling group to ensure that a sufficient number of successful model animals would be available for subsequent grouping; (2) In accordance with the ARRIVE 2.0 guidelines [18], when pilot data are unavailable, referencing existing literature is a valid approach for determining sample size. Studies in the field of metabolism commonly employ sample sizes of 5–8 animals per group, which has been proven effective in detecting statistically significant differences in key metabolic parameters [19, 20]; (3) Strict adherence to the 3R principles was maintained by controlling for factors such as animal strain, sex, age, and rearing conditions to minimize within-group variability and reduce animal usage. (Lines 83-93)

Besides, we conducted a post-hoc power analysis following your suggestion. The results showed that for primary positive outcomes, such as the expression level of the core indicator miR-27a, the effect size was large (Cohen’s d > 4.9), indicating that the current sample size is sufficient to support these conclusions. We openly acknowledge that for comparisons with smaller effect sizes or non-significant results, the statistical power may be insufficient (Power < 0.8), implying a risk of Type II errors. We have clearly stated this limitation in the “Discussion” sections of the manuscript, interpreted all results more cautiously, and highlighted the need for verification in larger future studies (Lines 738-747). The relatively small sample size was a result of balancing animal ethics (the 3R principles) and the practical challenges of the experimental complexity (attrition due to viral modeling and exercise interventions). We are confident that, under these constraints, the data obtained are robust enough to address the main scientific questions of this study.

The revised part: However, several limitations must be acknowledged. First, we did not examine whether miR-27a suppression improves inflammation or fibrosis in MS, which are critical to disease progression. Second, potential synergistic effects involving other miRNAs in the regulatory network cannot be ruled out. Third, although effect sizes were observed for primary outcomes, the sample size may limit power to detect smaller effects or complex interactions, increasing the risk of Type II errors. Consequently, caution is warranted when interpreting negative outcomes, and larger studies are needed to confirm these findings. Future research should use single-cell sequencing to clarify tissue-specific mechanisms and explore combined exercise-genetic strategies for improved clinical translation. (Lines 738-747)

• Uncut blots: The complete images for all western blots are provided in Supplementary information - Original gel blot images.

• Quantify miR-27a knockdown efficiency longitudinally: We appreciate the reviewer’s insightful comment regarding the longitudinal assessment of miR-27a knockdown efficiency. In the present study, due to technical limitations and animal welfare considerations (the 3R principle), we evaluated the knockdown efficacy at the experimental endpoint by measuring miR-27a expression in both skeletal muscle and liver tissues across all groups. While longitudinal measurements could provide additional kinetic information, our terminal assessment robustly confirmed significant miR-27a reduction in both silencing groups (SD-S-I and MS-S-I) compared with their respective null controls (SD-S-N and MS-S-N, P < 0.05), supporting the conclusion that effective and stable knockdown was achieved throughout the critical intervention period. Future studies employing longitudinal sampling or multiple time points would be valuable to further characterize the time-dependent dynamics of miR-27a suppression and its functional consequences.

2.Discussion expansion

• Human context and Mechanistic hypothesis: We thank the reviewer for this valuable suggestion. In response, we have expanded the Discussion section to include a comparison with human miRNA studies and have proposed a potential mechanism linking exercise to miR-27a downregulation. Specifically, we added clinical evidence showing elevated serum miR-27a levels in patients with metabolic syndrome and type 2 diabetes, and introduced exosome-mediated sorting and secretion as a plausible mechanism through which aerobic exercise may reduce miR-27a content (Lines 552-555, Lines 577-590).

3.Clarity improvements

We have simplified the abstract for enhanced readability (Lines 2-11) and standardized figure labels.

The revised part:

Abstract: This study investigated how aerobic exercise improves metabolic disorders in rats with metabolic syndrome (MS) and explored the role of miR-27a in this process. MS was induced by a high-fat and high-sugar diet. After eight weeks of treadmill training, aerobic exercise was found to reduce metabolic abnormalities and decrease elevated miR-27a levels, while increasing the expression of PPARγ and key downstream molecules involved in glycolipid metabolism. Downregulating miR-27a via a viral vector produced benefits similar to those of exercise, and combining miR-27a inhibition with exercise led to further improvement. These results suggested that aerobic exercise alleviates MS-related metabolic disorders partly through suppressing miR-27a and promoting PPARγ signaling, revealing a potential therapeutic target. (Lines 2-11)

4.Comparison to field & unresolved questions

Regarding this recommendation, we have added a section on strengths and limitations at the end of the discussion section (Lines 735-747).

The revised part: A key strength of this study is the translation of previous in vitro results to an in vivo setting, confirming PPARγ upregulation-beyond IRS-1 inhibition-as a central mechanism, as reported by Yu et al. [14], and validating the regulatory function of miR-27a under physiological conditions, consistent with Chen T et al. [34]. However, several limitations must be acknowledged. First, we did not examine whether miR-27a suppression improves inflammation or fibrosis in MS, which are critical to disease progression. Second, potential synergistic effects involving other miRNAs in the regulatory network cannot be ruled out. Third, although effect sizes were observed for primary outcomes, the sample size may limit power to detect smaller effects or complex interactions, increasing the risk of Type II errors. Consequently, caution is warranted when interpreting negative outcomes, and larger studies are needed to confirm these findings. Future research should use single-cell sequencing to clarify tissue-specific mechanisms and explore combined exercise-genetic strategies for improved clinical translation. (Lines 735-747)

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

Attachments

Attachment

Submitted filename: Response to Reviewers.docx