PONE-D-25-15201A New Investigation of Nonalcoholic Fatty Liver Disease: Effects of Hypoxia on Mitochondrial Function and Lipid Droplet AutophagyPLOS ONE

Dear Dr. Yanyan,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Md. Wasim Khan, Ph.D.

Academic Editor

PLOS ONE

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 [This study was financially supported by the Qinghai University Research Ability Enhancement Project, 2025 (2025KTSQ05)].

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Additional Editor Comments (if provided):

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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: Yes

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: Yes

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 paper, the study revealed that hypoxic conditions enhanced lipid droplet autophagy, reduced glycolipid accumulation, and alleviated mitochondrial damage in NAFLD mice. However, I have some questions:

1. What is the percentage of fat in a HFD in an animal model? Reference� Whether the 10% lard meet the HFD requirements?

2. It was mentioned in this paper that abnormal lipid metabolism is related to oxidative stress. Does hypoxic environment have any effect on oxidative stress in mice? Further discussion is needed.

Reviewer #2: This paper investigated the Effects of Hypoxia on Mitochondrial Function and Lipid Droplet Autophagy in NAFLD. The research methods are rich and the article has good innovation. Here are my major concerns.

1. The authors described in section 3.2“The levels of the alanine aminotransferase (ALT) in the liver tissue of the HFD-4500 m group were lower than those in the HFD-2200 m group (Figure 2F)”�

However, there was no significant difference between the two groups in Figure 2F. Besides, at the same altitude, High-fat diet seems to have no significant effect on ALT levels, and even tends to reduce them. How the authors interpret this result.

2. The discussion part of this paper mainly focuses on the experimental results, but the comparison and integration with the existing studies are not in-depth. It is suggested that the authors systematically comb the similarities and differences between the results of this study and those of previous studies, and deeply analyze the theoretical contribution of this study in the pathogenesis of NAFLD. The limitations of the study, such as the limitations of experimental conditions and unconsidered confounding factors, should be comprehensively analyzed, as well as the potential impact of these limitations on the study results and future research directions.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-25-15201R1A New Investigation of Nonalcoholic Fatty Liver Disease: Effects of Hypoxia on Mitochondrial Function and Lipid Droplet AutophagyPLOS ONE

Dear Dr. Yanyan,

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.

Please submit your revised manuscript by Jul 07 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Tatsuo Kanda, M.D.; Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

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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: Partly

Reviewer #2: (No Response)

Reviewer #3: Partly

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: N/A

Reviewer #4: 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: No

Reviewer #4: 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: (No Response)

Reviewer #3: Yes

Reviewer #4: 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: The authors investigated whether altitude differences influence the development of steatotic liver disease, with a focus on mitochondrial function and lipid droplet autophagy under hypoxic conditions. While the concept is interesting and potentially impactful, the manuscript raises several concerns that should be addressed to strengthen the scientific rigor and clarity of the work.

1. Figure Legends Require Improvement

The current figure legends are overly brief and provide minimal explanatory detail. Legends should include information on experimental groups, sample sizes (n), statistical methods, and any quantification performed. This is essential for interpreting the results without referring back to the main text.

2. Figure 1A Layout and Interpretation

The arrangement of Figure 1A is unclear, making it difficult to visually discern differences in subcutaneous or visceral fat between the altitude groups. Consider improving the layout (e.g., side-by-side panels with consistent scaling) and adding quantitative bar graphs comparing subcutaneous or visceral fat content between the 2200 m and 4500 m groups.

3. Oil Red O Staining Interpretation

In the presented images, it appears that the 4500 m group may exhibit more lipid accumulation, which seems to contradict the authors’ conclusion. This discrepancy should be clarified either through quantification of lipid content or by providing more representative images. Statistical validation would help resolve this point.

4. Sample Size Representation

While the manuscript mentions the use of mice, the exact number per group should be clearly indicated, either in the figure legends or with dot plots in the graphs. This transparency is critical for evaluating the reliability and reproducibility of the data.

5. Lack of Oxidative Stress Analysis

The role of oxidative stress is briefly discussed but not experimentally evaluated. Since oxidative stress is closely tied to mitochondrial function and NAFLD progression, direct measurement (e.g., ROS assays, lipid peroxidation markers) would significantly enhance the mechanistic insight of the study.

6. Experimental Design Suggestion – Altitude Transition

To better demonstrate causality between altitude and hepatic changes, the authors could consider a dynamic experimental model in which mice are transferred between altitudes. For example, transferring NAFLD-induced mice from 2200 m to 4500 m (or vice versa) could help establish whether hypoxia contributes to either improvement or worsening of disease parameters over time.

7. In real-world settings, dietary habits often vary significantly across different altitude regions due to cultural, agricultural, and socioeconomic factors. These differences in nutrition — such as total caloric intake, fat composition, and micronutrient availability — could independently influence the development or prevention of steatotic liver disease. While the current study uses a controlled experimental design, it would strengthen the translational relevance of the findings if the authors could briefly acknowledge this limitation and discuss how dietary variability in high- vs. low-altitude populations might impact NAFLD incidence in real-world contexts.

Reviewer #4: In this article, the authors indicated that hypoxic conditions enhanced lipid droplet autophagy, reduced glycolipid accumulation, and mitigated mitochondrial damage in NAFLD mice.

The result that hypoxia improves fatty liver is contrary to the established theory, but why it differs from other previous papers should be more deeply discussed.

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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 #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-25-15201R2A New Investigation of Nonalcoholic Fatty Liver Disease: Effects of Hypoxia on Mitochondrial Function and Lipid Droplet AutophagyPLOS ONE

Dear Dr. Yanyan,

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.

===================

Authors should revise the manuscript accordingly.

===================

Please submit your revised manuscript by Jul 15 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Tatsuo Kanda, M.D.; Ph.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

A New Investigation of Nonalcoholic Fatty Liver Disease: Effects of Hypoxia on Mitochondrial Function and Lipid Droplet Autophagy

PONE-D-25-15201R3

Dear Dr. Yanyan,

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,

Vahideh Behrouz

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 #5: 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 #5: Partly

**********

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

Reviewer #5: 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 #5: No

**********

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 #5: Yes

**********

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 #5: The authors have thoroughly addressed the reviewers' suggestions and incorporated valuable additions to the manuscript. I have no further concerns,

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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 #5: No

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